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Mammalian Near-Infrared Image Vision
through Injectable and Self-Powered

Retinal Nanoantennae Yuqian Ma & Jin
Bao & Yuanwei Zhang & Zhanjun Li &
Xiangyu Zhou & Changlin Wan & Ling
Huang & Yang Zhao & Gang Han & Tian
Xue
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Article
Mammalian Near-Infrared Image Vision through

Injectable and Self-Powered Retinal Nanoantennae
Graphical Abstract Authors
Yuqian Ma, Jin Bao, Yuanwei Zhang, ...,
Yang Zhao, Gang Han, Tian Xue
Correspondence
baojin@ustc.edu.cn (J.B.),
gang.han@umassmed.edu (G.H.),
xuetian@ustc.edu.cn (T.X.)
In Brief
Injectable photoreceptor-binding
nanoparticles with the ability to convert
photons from low-energy to high-energy
forms allow mice to develop infrared
vision without compromising their normal
vision and associated behavioral
responses.
Highlights
d We designed ocular injectable photoreceptor-binding
upconversion nanoparticles
d The nanoparticles are safe and enable NIR light sensation

and pattern vision
d This NIR pattern vision is compatible with native daylight

vision
d This method offers options for mammalian vision repair and

enhancement
Ma et al., 2019, Cell 177, 1–13

April 4, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.cell.2019.01.038
Please cite this article in press as: Ma et al., Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoanten-
nae, Cell (2019), https://doi.org/10.1016/j.cell.2019.01.038
Article
Mammalian Near-Infrared Image Vision

through Injectable and Self-Powered
Retinal Nanoantennae
Yuqian Ma,1,5 Jin Bao,1,2,5,* Yuanwei Zhang,3,5 Zhanjun Li,3 Xiangyu Zhou,1 Changlin Wan,1 Ling Huang,3 Yang Zhao,3
Gang Han,3,* and Tian Xue1,2,4,6,*
1Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, CAS Key Laboratory of
Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China,
Hefei, Anhui 230026, China
2Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
4Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
5These authors contributed equally
6Lead Contact
*Correspondence: baojin@ustc.edu.cn (J.B.), gang.han@umassmed.edu (G.H.), xuetian@ustc.edu.cn (T.X.)

https://doi.org/10.1016/j.cell.2019.01.038
SUMMARY lower energy photons, requires opsins (e.g., human red cone op-
sins) to have much lower energy barriers. Consequently, this re-
Mammals cannot see light over 700 nm in wave- sults in unendurable high thermal noise, thus making NIR visual
length. This limitation is due to the physical thermo- pigments impractical (Ala-Laurila et al., 2003; Baylor et al., 1980;
dynamic properties of the photon-detecting opsins. Luo et al., 2011; St George, 1952). This physical limitation means
However, the detection of naturally invisible near- that no mammalian photoreceptor can effectively detect NIR
infrared (NIR) light is a desirable ability. To break light that exceeds 700 nm, and mammals are unable to see
NIR light and to project a NIR image to the brain.
this limitation, we developed ocular injectable
To this end, the successful integration of nanoparticles with
photoreceptor-binding upconversion nanoparticles biological systems has accelerated basic scientific discoveries
(pbUCNPs). These nanoparticles anchored on retinal and their translation into biomedical applications (Desai, 2012;
photoreceptors as miniature NIR light transducers to Mitragotri et al., 2015). To develop abilities that do not exist natu-
create NIR light image vision with negligible side ef- rally, miniature nanoscale devices and sensors designed to inti-
fects. Based on single-photoreceptor recordings, mately interface with mammals including humans are of growing
electroretinograms, cortical recordings, and visual interest. Here, we report on an ocular injectable, self-powered,
behavioral tests, we demonstrated that mice with built-in NIR light nanoantenna that can extend the mammalian vi-
these nanoantennae could not only perceive NIR sual spectrum to the NIR range. These retinal photoreceptor-
light, but also see NIR light patterns. Excitingly, the binding upconversion nanoparticles (pbUCNPs) act as miniature
injected mice were also able to differentiate sophisti- energy transducers that can transform mammalian invisible NIR
light in vivo into short wavelength visible emissions (Liu et al.,
cated NIR shape patterns. Moreover, the NIR light
2017; Wu et al., 2009). As sub-retinal injections are a commonly
pattern vision was ambient-daylight compatible and
used ophthalmological practice in animals and humans (Haus-
existed in parallel with native daylight vision. This wirth et al., 2008; Peng et al., 2017), our pbUCNPs were dis-
new method will provide unmatched opportunities solved in PBS and then injected into the sub-retinal space in
for a wide variety of emerging bio-integrated nanode- the eyes of mice. These nanoparticles were then anchored and
vice designs and applications. bound to the photoreceptors in the mouse retina.
Through in vivo electroretinograms (ERGs) and visually evoked
INTRODUCTION potential (VEP) recordings in the visual cortex, we showed that
the retina and visual cortex of the pbUCNP-injected mice were
Vision is an essential sensory modality for humans. Our visual both activated by NIR light. From animal behavioral tests, we
system detects light between 400 and 700 nm (Dubois, 2009; further demonstrated that the pbUCNP-injected mice acquired
Wyszecki and Stiles, 1982; Schnapf et al., 1988), so called visible NIR light sensation and unique ambient daylight-compatible
light. In mammalian photoreceptor cells, light absorbing pig- NIR light image vision. As a result, the built-in NIR nanoantennae
ments, consisting of opsins and their covalently linked retinals, allowed the mammalian visual spectrum to extend into the NIR
are known as intrinsic photon detectors. However, the detection realm effectively without obvious side effects. Excitingly, we
of longer wavelength light, such as near-infrared (NIR) light, found that pbUCNP-injected animals perceived both NIR and
though a desirable ability, is a formidable challenge for mam- visible light patterns simultaneously. They also differentiated be-
mals. This is because detecting longer wavelength light, with tween sophisticated NIR light shape patterns (such as triangles
Cell 177, 1–13, April 4, 2019 ª 2019 Elsevier Inc. 1

(legend on next page)
2 Cell 177, 1–13, April 4, 2019

and circles). Importantly, this nanoscale device activated the spectrum (Figure S1B). To confirm the glyosidic bonds between
photoreceptors by an exceptionally low power NIR light-emitting ConA and glycoproteins, we added b-cyclodextrin, which pos-
diode (LED) light (1.62 mW/cm2), which was attributed to the sesses a similar glucosyl unit as that found on the photoreceptor
proximity between the nanoantennae and photoreceptors in outer segment, to the pbUCNP solution. Characteristic ConA-
the eye. Moreover, we comprehensively examined the biocom- b-cyclodextrin aggregation thus occurred, as seen in the trans-
patibility of the pbUCNPs and found negligible side effects. mission electron microscope (TEM) images (Figure 1G) and
Therefore, these novel photoreceptor-binding NIR light nano- dynamic light scattering (DLS) spectrum (Figure S1C). This result
antennae provide an injectable, self-powered, biocompatible, suggests that the pbUCNPs can bind to glycoproteins on
and NIR-visible light compatible solution to extend the mamma- the photoreceptor outer segment. In contrast, the paaUCNPs
lian visual spectrum into the NIR range. This concept-proving without ConA remained monodispersed when b-cyclodextrin
research should guide future studies with respect to extending was added (Figures 1H and S1D). After injecting these pbUCNPs
human and non-human vision without the need for any external into the mouse sub-retinal spaces (Figures 1F and S1E), we
device or genetic manipulation. Endowing mammals with NIR observed that, through the glyosidic bond, these pbUCNPs
vision capacity could also pave the way for critical civilian and self-anchored and remained tightly bound to the inner and outer
military applications. segments of both rods and cones (Figures 1J–1L) forming a layer
of built-in nanoantennae with the characteristic upconversion
RESULTS spectrum (Figures 1I, left, S1F, and S1G). In contrast, the in-
jected paaUCNPs were loosely bound and easily removed
The Design of pbUCNPs from the photoreceptors with gentle washing (Figure 1I, right).
The human eye is most sensitive to visible light at an electromag- We then evaluated the biocompatibility and potential side ef-
netic wavelength of 550 nm under photopic conditions (Bieber fects of the pbUCNPs in vivo. We found that the pbUCNP injec-
et al., 1995; Boynton, 1996). To convert NIR light to this tion did not cause a higher rate of adverse reactions compared
wavelength, we generated core-shell-structured upconversion with the control PBS injection. All common minor or transient
nanoparticles (UCNPs) (i.e., 38 ± 2 nm b-NaYF4:20%Yb, 2% side effects (e.g., cataracts, corneal opacity) generally associ-
Er@b-NaYF4) (Figures 1A and 1B), which exhibited an excitation ated with sub-retinal injection (Qi et al., 2015; Zhao et al., 2011)
spectrum peak at 980 nm and emission peak at 535 nm upon disappeared completely 2 weeks after the injections (Table
980-nm light irradiation (Mai et al., 2006; Wu et al., 2015) (Figures S1). In addition, we evaluated possible retinal degeneration by
1C and 1D). To design water-soluble pbUCNPs, we further con- counting the number of photoreceptors in the retinal outer nu-
jugated concanavalin A protein (ConA) with poly acrylic acid- clear layer (ONL), a standard and widely used method in the field
coated UCNPs (paaUCNPs) (Figure 1E; STAR Methods). ConA of retinal research, as photoreceptors are sensitive and prone to
can bind to sugar residue and derivatives of the photoreceptor degenerate upon stress (Chen et al., 2006; Namekata et al.,
outer segment, forming glyosidic bonds (Bridges, 1981; Bridges 2013; Wang et al., 2013). As a result, we observed that the retinal
and Fong, 1980; Rutishauser and Sachs, 1975). Successful layer structure and the number of photoreceptor layers in the
ConA conjugation on the surface of the UCNPs was suggested retinal ONL were not changed, even with 50 mg of pbUCNPs in-
by the appearance of N-H bending peaks in the Fourier transform jected into each eye, up to 2 months after the injections (Figures
infrared (FT-IR) spectrum (Figure S1A) and by the 285 nm pro- 2A and 2B). This result clearly suggested that there was no
tein absorption on the ultraviolet-visible spectroscopy (UV/Vis) obvious retinal degeneration using this standard measure.
Figure 1. Characterzations of pbUCNPs

(A) Transmission electron microscopy (TEM) image of UCNPs (as-synthesized core-shell-structured b-NaYF4:20%Yb, 2%Er@b-NaYF4). Scale bar, 100 nm.
(B) Corresponding size distribution of UCNPs.
(C) Excitation spectrum of UCNPs measured as emission light intensity at 535 nm by 700–1,040 nm excitation.
(D) Emission spectrum of UCNPs upon 980-nm continuous wave (CW) laser irradiation. Inset displays photographs of UCNP solutions with (right) and without (left)
980-nm CW laser excitation.
(E) Schematic illustration of surface modification procedures for ConA-functionalized photoreceptor-binding UCNPs (pbUCNPs).
(F) Left: illustration of sub-retinal injection of pbUCNPs in mice. See also Figure S1. Right: illustration of pbUCNPs binding to the outer segments of photore-
ceptors and generation of green light upon near-infrared (NIR) light illumination.
(G) TEM images of pbUCNPs before (top) and after (bottom) addition of 200 nM b-cyclodextrin showing characteristic aggregation of pbUCNPs in the presence of
b-cyclodextrin. See also Figure S1. Scale bar, 2 mm.
(H) TEM images of UCNPs without ConA-conjugation (paaUCNPs) mixed with 200 nM b-cyclodextrin, showing no obvious aggregation. See also Figure S1. Scale
bar, 2 mm.
(I) Top: overlays of transmission and luminescence optical images (green: 980-nm excitation/535-nm emission) of retinal slices from pbUCNP-injected, PBS-
injected, and paaUCNP-injected mice. Bottom: emission spectrum recorded from retinal outer segment layers (OS) upon 980-nm light excitation. All retinal slices
were washed with PBS during fixation. Only pbUCNPs remained bound to the photoreceptor outer segments. RPE, retinal pigment epithelium; OS, outer segment
of photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar, 30 mm.
(J) Schematic illustration of distribution of pbUCNPs (green) in the retina. Rods are labeled with Nrl-GFP in pseudo color violet. Cones are labeled with Opn1LW-
Cre; Ai9-lsl-tdTomato in pseudo color red. OS, outer segment of photoreceptors; IS, inner segment of photoreceptors; OLM, outer limiting membrane; ONL, outer
nuclear layer.
(K and L) Overlaid green (pbUCNPs)/violet (rods) and green (pbUCNPs)/red (cones) channel fluorescence images of retina from PBS-injected mice (K) and
pbUCNP-injected mice (L). Examples of continuous inner and outer segments of a rod and a cone are shown in dashed contour lines. Scale bars, 10 mm.
Cell 177, 1–13, April 4, 2019 3

Figure 2. Biocompatibility of pbUCNPs

(A) H&E staining of retinal slices from non-injected, PBS-injected, 20 mg/eye and 50 mg/eye pbUCNP-injected mice. OS, outer segment of photoreceptors; ONL,
outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 mm.
(B) Number of cell layers in outer nuclear layer (ONL) of retinae. Data are mean ± SD (n = 4 retinae).
(C) Microglia marker Iba1 staining of retinal slices 3 days after injection. H2O2-injected mice (positive control) showed strong activation of microglia. Few basal
Iba1 signals were observed in the pbUCNP-injected retina, similar to that observed in PBS-injected retina (indicated by arrow heads). Red, Iba1; green, pbUCNP
emission upon excitation by NIR light; blue, DAPI (40 ,6-diamidino-2-phenylindole) signal indicating cell nuclei. Scale bar, 50 mm.
(D) Apoptosis detection by TUNEL staining 3 days after injection. Strong TUNEL signals were observed in the H2O2-injected mouse retinae (positive control), but
few were observed in PBS-injected or pbUCNP-injected mice (indicated by arrow heads). Red, TUNEL staining; green, pbUCNP emission upon excitation by NIR
light; blue, DAPI. Scale bar, 50 mm.
(E) Number of Iba1 and TUNEL stained cells per 0.04 mm2 averaged from four retinae for each condition. See also Figure S2. Data are mean ± SD (n = 4 retinae).
See also Table S1.
Moreover, we examined potential inflammation in the retinal injection did not cause obvious acute or long-term side effects.
through microglia marker Iba1 staining that is a widely used indi- In addition, the excitation and emission spectra of the pbUCNPs
cator of microglia accumulation (Krady et al., 2005). From this, in either fixed or fresh retinae were in good agreement with those
we observed negligible retinal inflammation at 3 days or 1, 2, 4, measured from pbUCNP solution, indicating that binding with
and 10 weeks after pbUCNP injection (Figures 2C, 2E, and the photoreceptors did not change the characteristics of the
S2A). We further examined retinal cell apoptosis after injection pbUCNPs (Figures S2C and S2D).
via terminal deoxynucleotidyl transferase deoxyuridine triphos-
phate (dUTP) nick-end labeling (TUNEL). We only found sparse NIR Light-Mediated Photoreceptor Activation
TUNEL signals 3 days after injection in both the PBS and Based on the biocompatibility noted above, we tested if the pho-
pbUCNP-injected retinae (Figure 2D), with the TUNEL signals toreceptors could be activated by NIR light with the help
being undetectable 1, 2, 4, and 10 weeks after pbUCNP injection of pbUCNPs. We performed single rod suction pipette record-
(Figures 2E and S2B). These results suggest that the pbUCNP ings on acutely dissected retinae from pbUCNP-injected and
4 Cell 177, 1–13, April 4, 2019

non-injected mice (Figure 3A). The action spectra of rods from agreed well with the experimental NIR light PLR response (Fig-
pbUCNP-injected and non-injected mice were identical in the ure S5C). Therefore, the non-linearity shown in the NIR light-
visible light range, with differences only appearing after induced behavior was attributed to the non-linearity of the
900 nm, where the action spectra of rods from pbUCNP-in- upconversion process.
jected mice matched the excitation spectra of pbUCNPs (Fig- In addition to the above sub-conscious light sensation PLR
ure S3A). The rods from pbUCNP-injected mice had normal behavior, we also explored whether pbUCNP-injected mice
visible light (535 nm)-elicited photocurrents compared with could consciously perceive NIR light. In this regard, we
that of non-injected mice (Figures 3B and 3D). The 980-nm light performed light-dark box experiments with visible and NIR
flash elicited rod photocurrents from pbUCNP-injected mice light (Figures 4C and 4D) as well as light-induced fear-condition-
(Figure 3E), whereas the rods from non-injected mice exhibited ing experiments (Figures 4E and 4F). In the conventional light-
no responses (Figure 3C). The amplitude and kinetics of the dark box experiments with visible light, mice instinctively
980-nm light-elicited photocurrents were identical to those preferred the dark box to the light box illuminated with visible
activated by 535-nm visible light (Figures 3F–3H). The similar light. In our study, we replaced conventionally used visible
time-to-peak values suggest that, compared to the visible light with 980-nm LED light, which delivered 8.1 3 107
light stimulation, there was no delay in the activation of the photons 3 mm2 3 s1 at the center of the light box, equal to
rods by NIR light. Furthermore, the pbUCNPs did not alter a power density of 1.62 mW/cm2. The pbUCNP-injected mice
the light adaptation or dark noise characteristics of the rods, exhibited a significant preference for the dark box, whereas
and rods adapted to visible and NIR light in the same manner the non-injected control mice could not distinguish between
following the Weber-Fechner relationship (Baylor et al., 1980; the NIR light (980 nm)-illuminated and dark boxes (Figure 4D;
Morshedian et al., 2018; Fu et al., 2008) (Figure S4). To Video S1). This suggests that mice with injected nanoantennae
determine whether the pbUCNPs can serve as NIR nanosen- perceived NIR light and exhibited innate light-sensing behavior.
sors in vivo, we recorded the population response of photore- To exclude the possibility of any visible light emission from the
ceptors activated by light via ERGs (Dalke et al., 2004) NIR LEDs, the emission spectra of the 980-nm LEDs were
(Figure 3I). Upon 980-nm NIR light illumination to the eye, the measured and no light emission below 900 nm was detected
ERG from pbUCNP-injected mice resembled that of visible (Figures S5D and S5E).
light-induced responses, whereas no such signal could be de- We then tested whether such NIR light perception can serve
tected from the non-injected control mice. Furthermore, we as a visual cue for learned behavior. Mice were trained to pair
performed ERG recordings on pbUCNP-injected rod-function- a 20-s 535-nm light pulse to a 2-s foot shock (Figure 4E) in order
less mice (Gnat1/) and demonstrated that, through the to acquire a conditioned freezing behavior. After acquisition of
pbUCNPs, 980-nm NIR light indeed activated cones in vivo such conditioning, mice received either NIR light at 980 nm
(Figure S3B). or visible light at 535 nm as conditional stimuli (CS) in the test tri-
als. The pbUCNP-injected mice showed significant freezing
NIR Light Sensation of pbUCNP-Injected Mice behavior in response to both wavelengths, whereas the non-in-
To reveal whether pbUCNP-injected mice could see NIR light, jected control mice exhibited freezing behavior only to visible
we first performed pupillary light reflex (PLR) experiments (Xue light stimuli (Figure 4F; Video S2). These results clearly demon-
et al., 2011). The pupils of the pbUCNP-injected mice showed strated that mice acquired NIR light sensation and were able to
strong constrictions upon 980-nm light illumination, whereas ‘‘see’’ NIR light with our ocular injectable photoreceptor target-
the non-injected control mice did not exhibit PLR with the ing nanoantennae.
same NIR illumination (Figure 4A). Moreover, we discovered
that the PLR of the pbUCNP-injected mice was two orders of NIR Light-Activated Imaging Visual Pathway of
magnitude more sensitive to NIR light than that of the non-photo- pbUCNP-Injected Mice
receptor-binding paaUCNP-injected mice (Figure 4B). This was In addition to the NIR light sensation, we were curious whether
attributed to the proximity between the pbUCNPs and the bound pbUCNP-injected mice had acquired NIR light image visual
photoreceptors. Photon upconversion was measured (Fig- ability. In general, visual image perception is associated with
ure S5A) and showed a non-linear light intensity relationship activation of the visual cortex. In order to record visually evoked
plotted at the log-log scale (Figure S5B). We fitted the power potential (VEPs), we placed recording electrodes in six different
relationship between emitted 535-nm light and 980-nm excita- locations of the visual cortex (No. 1, 2, 3, and 5 in the monocular
tion light and determined the power to be 1.6. Interestingly, we areas and No. 4 and 6 in the binocular areas) during contralateral
found that this non-linearity was also shown in the NIR light- eye illumination (Cooke et al., 2015; Smith and Trachtenberg,
induced behavior. The light dose-response curves of the PLR 2007) (Figure 5A). When the visible 535-nm light pulse was
(normalized pupil area versus light intensity) were fitted to the applied, VEPs were detected at all locations in both the non-in-
Hill function. The NIR light-induced PLR dose-response curve jected controls and pbUCNP-injected mice (Figures 5B and
was steeper than that of visible light, and the Hill coefficients 5D). In contrast, under 980-nm NIR light illumination, no VEPs
for the NIR and visible light PLR dose-responses were 1.10 were observed in the control mice, but were detected from the
and 0.78, respectively (Figure S5C). To obtain the theoretical binocular visual cortical areas in pbUCNP-injected mice (Figures
NIR light PLR dose-response curve, the fitted upconversion 5C and 5E). This is topologically consistent with the pbUCNP in-
function was applied to the visible light PLR dose-response Hill jection site (temporal side, binocular projection area) in the
function. This theoretical NIR light PLR dose-response curve retina.
Cell 177, 1–13, April 4, 2019 5

6 Cell 177, 1–13, April 4, 2019

Figure 4. NIR Light Sensation of pbUCNP-

Injected Mice
(A) Images showing pupil constriction from non-in-
jected control and pbUCNP-injected mice under
980-nm light stimulation (40 s). Intensity of 980-nm
light: 1.21 3 108 photons 3 mm2 3 s1.
(B) Dose-response curves of normalized pupil
constriction with 980-nm light stimulation (paaUCNP-
injected mice, n = 4; pbUCNP-injected mice, n = 5;
control mice, n = 4; data are mean ± SD).
(C) Light-dark box experiment diagram. Light box was
illuminated with an array of LED lights interlaced by
980-nm and 535-nm LEDs. Illumination protocol is
shown at the bottom. Each section contained four
episodes and each episode was 5 min long. The first
5-min episode was adaptation in the light-dark box
with ambient light followed by a 5-min episode in
complete darkness. The 980-nm and 535-nm LEDs
were then lit consecutively for the light box for 5 min
each.
(D) Preference index for dark box under three different
light box conditions (light off, 980 nm, and 535 nm).
Preference index = (time spent in dark box – in
light box) / (time spent in dark box + in light box). In-
tensities of the 980-nm and 535-nm lights at the
center of the box were 8.1 3 107 and 9.1 3 102 pho-
tons 3 mm2 3 s1, respectively (Control: n = 5,
pbUCNP-injected: n = 6; data are mean ± SD, two-
sided t test, ***p < 0.001; n.s., not significant).
(E) Fear-conditioning experiment diagram and pro-
tocol. A 535-nm light pulse was paired with a foot-
shock to form conditioning during training. Tests were
then carried out 24 h later with a 980-nm or 535-nm
light pulse alone.
(F) Percentages of freezing time during 20 s ‘‘Pre-CS,’’
980-nm, and 535-nm light stimulation (‘‘Pre-CS’’:
before conditional stimulation, a 20-s period of
adaptation right before light stimulation onset). In-
tensities of the 980-nm and 535-nm lights at the
center of the box were 1.07 3 108 and 1.47 3 103
photons 3 mm2 3 s1, respectively. Data are
mean ± SD (control: n = 6, pbUCNP-injected: n = 7;
two-sided t test; n.s., not significant; ***p < 0.001).
See also Figure S5 and Videos S1 and S2.
NIR Light Pattern Vision discriminate between different light patterns (Prusky et al.,
We next examined whether mice obtained NIR light pattern 2000) (Figure 6A). The mice were trained to find a hidden platform
vision. Accordingly, Y-shaped water maze behavioral experi- that was associated with one of two patterns. We designed
ments were conducted to determine whether mice could five different tasks to examine their NIR pattern vision ability
Figure 3. NIR Light-Mediated Photoreceptor Activation through pbUCNPs

(A) Illustration of rod outer segment suction pipette recordings from freshly isolated retinae. Stimulation light was either 980-nm or 535-nm through the imaging
objective.
(B–E) Photocurrents and intensity-response curves of rods from non-injected mice with 535-nm (B) (n = 5) or 980-nm (C) (n = 6) light stimulations and pbUCNP-
injected mice with 535-nm (D) (n = 5) or 980-nm (E) (n = 6) light stimulations. Tiny colored vertical bars on the x axis indicate time of light flashes. Photocurrent
traces were averaged from 5–7 sweeps. Intensity-response data are mean ± SD.
(F) Saturated photocurrent in (B)–(E).
(G) Time-to-peak, time from light stimulation to peak amplitude of dim light photocurrents in (B)–(E).
(H) Decay time constant of dim light photocurrent in (B)–(E). Data are mean ± SD; n.s., not significant; ***p < 0.001.
(I) Electroretinograms (ERGs) recorded from mice under 535-nm or 980-nm light stimulation. No response was observed in non-injected control mice under
980-nm light stimulation (gray). Light intensities: 535 nm, 8.26 3 103 photons 3 mm2; 980 nm, 9.83 3 108 photons 3 mm2.
See also Figures S3 and S4.
Cell 177, 1–13, April 4, 2019 7

Figure 5. NIR Light Activated the Imaging Visual Pathway of pbUCNP-Injected Mice
(A) Diagram of six recording sites for visually evoked potentials (VEPs) in the mouse visual cortex.
(B and C) VEPs of non-injected control (black traces) and pbUCNP-injected mice (gray traces) under 535-nm (B) and 980-nm (C) light illumination. Intensities of the
535-nm and 980-nm lights were 3.37 3 103 and 7.07 3 108 photons 3 mm2 3 s1, respectively. Recording sites 1, 2, 3, and 5 were monocular areas; 4 and 6
were binocular areas. Traces were averaged from six sweeps and presented as mean ± SD (shaded area).
(D and E) Peak VEPs triggered by 535-nm (D) or 980-nm (E) light at each recording site (mean ± SD, n = 4 for both, two-sided t test, **p < 0.01, ***p < 0.001).
regarding different pattern stimuli and various background light that the sub-retinal injection of pbUCNPs did not interfere with
conditions. Task 1 used light gratings as pattern stimuli (Figures visible light vision. With respect to NIR light gratings, the
6B and S6A). After training with 980-nm light gratings, the pbUCNP-injected mice detected a maximum of 0.14 ± 0.06 cy-
pbUCNP-injected mice were able to discriminate between the cles/degree. This decrease in spatial resolution in NIR light vision
two orientations (vertical or horizontal) of the NIR light gratings, may be due to the isotropic radiation and scattering of the in situ
whereas the non-injected control mice made such choices in a transduced visible light from the NIR light-excited pbUCNPs
random manner (Figure 6C; Video S3). In the parallel control (Figures 6D and S6D).
testing, when the mice were trained and tested with visible light In addition, to confirm if visible light background interfered
gratings, both the pbUCNP-injected and non-injected mice were with the NIR light pattern perception, we designed Task 2 using
able to find the associated platform (Figures 6C, S6B, and S6C). two LED boards with visible (535 nm) and NIR (980 nm) LED
We then measured the spatial resolution of the NIR image arrays arranged in a perpendicular manner on each board. These
perception. The pbUCNP-injected mice detected the visible light two boards appeared identical under an ambient visible light
gratings with a maximum spatial frequency of 0.31 ± 0.04 cycles/ background when all LEDs (visible and NIR) were turned off.
degree, which did not significantly differ from that of the non-in- The orientations for the 535-nm and 980-nm LED stripes be-
jected control mice (0.35 ± 0.02 cycles/degree). This indicates tween the two boards were 90 rotated respectively (Figure 6E).
8 Cell 177, 1–13, April 4, 2019

Cell 177, 1–13, April 4, 2019 9

Trainings were carried out under visible room light (196 lux) and the test trials, we presented one visible (535 nm) and one NIR
with only the 980-nm LEDs on. In the tests, only pbUCNP-in- (980 nm) light in a triangular-circular pattern at the left-right
jected mice learnt to locate the platform (Figure 6F), indicating ends of the water maze, shuffled in a random sequence (Fig-
that NIR light pattern vision persisted in the visible light-illumi- ure 6I). Only the pbUCNP-injected mice were able to discrimi-
nated environment. Interestingly, we subsequently tested these nate between the two patterns with different shapes and
mice with the 535-nm LEDs on and 980-nm LEDs off. Both wavelengths (Figures 6J, left, and S6I). To exclude the possibility
pbUCNP-injected and control mice could discriminate the visible that mice simply used either visible or NIR light patterns to guide
light gratings, again indicating that pbUCNP injection did not decision-making rather than seeing them simultaneously, we
affect normal visible light vision. Additionally, pbUCNP-injected calculated the correct choice rates separately for the visible
mice could discriminate the visible light gratings from the begin- and NIR light triangle patterns. In the subset of stimuli where
ning of the test, suggesting that pbUCNP-injected mice were the triangular patterns was in visible light (Figure 6J, middle),
able to implement the rule learnt from the NIR light pattern to control mice selected both sides randomly, indicating they did
visible light pattern discrimination, indicating that NIR light pat- not simply use the visible triangle to make decisions. When the
terns did not differ perceptually from visible light patterns for circular pattern was in visible light, control mice still picked the
pbUCNP-injected mice (Figure 6F; Video S4). side randomly, indicating that the mice did not use the strategy
To test more sophisticated pattern vision, we further prompted of avoiding circles to make decisions (Figure 6J, right). In
animals to discriminate triangular and circular patterns in Task 3 contrast, pbUCNP-injected mice made correct choices in both
(Figure 6G). We found that pbUCNP-injected mice were able to cases (Video S5), suggesting they used visible and NIR light pat-
discriminate NIR and visible light patterns in the dark environ- terns together to guide behavior. These results clearly indicate
ment, whereas non-injected control mice could only detect the that the built-in nanoantennae enabled mice to see visible and
visible light pattern (Figures 6H, S6E, and S6F; Video S5), indi- NIR light patterns simultaneously.
cating that pbUCNP-injected mice could perceive sophisticated
NIR light patterns. We subsequently speculated whether back- DISCUSSION
ground NIR light would interfere with the visible light pattern
vision of pbUCNP-injected mice. Thus, in Task 4, mice were In this study, we demonstrated the successful application of
tested to discriminate between visible light triangles and circles UCNPs as ocular injectable NIR light transducers, which
under a visible or NIR light background (Figure 6G). Same as extended mammalian vision into the NIR realm. These implanted
control mice, the pbUCNP-injected mice did not behave differ- nanoantennae were proven to be biocompatible and did not
ently regarding their ability to discriminate visible light patterns interfere with normal visible light vision. Importantly, animals
under dark, visible, or NIR light backgrounds (Figures 6H and were able to detect NIR and visible light images simultaneously.
S6F–S6H; Video S5). These results clearly suggest that back-
ground NIR light does not interfere with visible light pattern Extension of the Visual Spectrum into the NIR Range
perception. One way to obtain NIR light vision is to implement new machinery
Task 5 was designed to test whether pbUCNP-injected mice for NIR photon transduction, such as the thermal detection of
could see NIR and visible light patterns simultaneously. In gen- snakes (Gracheva et al., 2010). However, a more plausible
eral, saturation by visible light is a common problem for conven- method to achieve such NIR photon detection is the use of the
tionally used tools, such as optoelectronic night vision devices or endogenous visual system. The method we developed here uti-
IR cameras, as it prevents smooth detection between visible and lized the very first step of the visual image perception process
NIR light objects. To test if our built-in NIR light vision could over- through photoreceptor outer-segment binding NIR nanoanten-
come this problem and coexist with visible light vision, we de- nae. The NIR light image was projected to the retina through
signed the following experiments. Mice were first trained in a the optical part of the eyes, cornea, and lens, after which the
Y-shaped water maze with visible light triangles and circles to pbUCNPs upconverted NIR light into visible light and then acti-
learn that the platform was associated with triangles only. During vated the bound photoreceptors. Subsequently, the retinal
Figure 6. NIR Light Pattern Vision of pbUCNP-Injected Mice

(A) Diagram of Y-shaped water maze for Tasks 1–5.
(B) Stimuli of Task 1. Experiments were under dark background. See also Figure S6.
(C) Correct rates of Task 1 for light grating discrimination (pbUCNP-injected mice: n = 7; non-injected control mice: n = 6).
(D) Visual spatial resolutions of pbUCNP-injected and control mice for 535-nm and 980-nm light gratings.
(E) Diagram of visual stimuli in Task 2. Light grating stimulations were LED arrays with ambient room light as background.
(F) Correct rates of Task 2 with respect to discrimination of 980-nm (days 1–9) and 535-nm (days 15–22) light LED gratings under room light background
(pbUCNP-injected mice: n = 7 and control mice: n = 5).
(G) Visual stimuli of Tasks 3 and 4. Triangular and circular patterns were made of LEDs and presented at the end of the water maze.
(H) Correct rates of Tasks 3 and 4 in discriminating triangular and circular patterns under dark, visible light, or NIR light background (pbUCNP-injected: n = 5 and
control: n = 6).
(I) Diagram of four stimuli in Task 5. Four stimuli were mixed and shuffled randomly in position.
(J) Correct rates of Task 5 in discriminating NIR and visible light shape patterns simultaneously (left), with triangular pattern in visible light (middle) and in NIR light
(right) (pbUCNP-injected: n = 5 and Control: n = 6). All data are mean ± SD (two-sided t test, **p < 0.01, ***p < 0.001; n.s., not significant).
See also Videos S3, S4, and S5.
10 Cell 177, 1–13, April 4, 2019

circuit and cortical visual system generated perception of the not cause any separation between the photoreceptors and
NIR image. It is important to note that these injected nanoanten- retinal pigment epithelium, the latter of which is the supporting
nae did not interfere with natural visible light vision. The ability to layer for photoreceptors. As a result, neither inflammation nor
simultaneously detect visible and NIR light patterns suggests apoptosis occurred, which is in line with that of another reported
enhanced mammalian visual performance by extending the retinal application of rare earth nanoparticles (Chen et al., 2006).
native visual spectrum without genetic modification and avoiding The stability and compatibility of the pbUCNPs were also
the need for bulky external devices. This approach offers several demonstrated by successful detection of NIR light images,
advantages over the currently used optoelectronic devices, such even after 10 weeks without any repeated injections.
as no need for any external energy supply, and is compatible
with other human activities. Further Development of pbUCNPs
In the present study, we created NIR light vision while over-
Improved Efficiency through ConA Modification coming several key drawbacks that yet exist in currently used
of UCNPs man-made systems. It may also be possible to design NIR color
Regarding the practical applications of UCNPs, higher visual vision through multicolor NIR light-sensitive UCNPs that have
sensitivity and resolution are desirable. We modified the UCNPs multiple NIR light absorption peak wavelengths and correspond-
and generated photoreceptor-binding nanoparticles to increase ing multicolor visible light emissions. Further applications using
the proximity between the nanoparticles and photoreceptors. our strategy for visual repair and enhancement could also be
Thus, sensitivity to NIR light with respect to generating light- achieved by similar nanoparticles with tailored light absorptions.
induced behaviors was improved by two orders of magnitude. In addition, combined with a drug delivery system, these photo-
Therefore, it is now possible to use biocompatible low-power receptor-binding nanoparticles could be modified to release
NIR LEDs to elicit visual behavior in animals, rather than the small molecules locally upon light stimulation.
more invasive high-power NIR lasers inevitably used in conven- In summary, these nanoparticles not only provide the potential
tional UCNP biomedical applications (Chen et al., 2018; He et al., for close integration within the human body to extend the visual
2015). In the Y-shaped water maze experiment, we estimated spectrum, but also open new opportunities to explore a wide
that the 980-nm LED light was transduced to 535-nm light by variety of animal vision-related behaviors. Furthermore, they
the pbUCNPs at 293 photons 3 mm2 3 s1 intensity at the exhibit considerable potential with respect to the development
retina. The rod and cone-mediated visual behavior thresholds of bio-integrated nanodevices in civilian encryption, security,
of mice are 0.012 and 200 photons 3 mm2 3 s1 at the cornea, military operations, and human-machine interfaces, which
respectively (Sampath et al., 2005), equal to 0.003 and 50 pho- require NIR light image detection that goes beyond the normal
tons 3 mm2 3 s1 at the retina (Do et al., 2009). Therefore, in functions of mammals, including human beings. Moreover, in
our system, the 293 photons 3 mm2 3 s1 at the retina was addition to visual ability enhancement, this nanodevice can serve
adequate to activate both rod and cone photoreceptors, and in as an integrated and light-controlled system in medicine, which
practice, this NIR visual system was able to detect NIR light could be useful in the repair of visual function as well as in drug
that was of several magnitudes lower intensity than currently delivery for ocular diseases.
applied. Compared to rods, cones encode several orders of
magnitude higher intensity light and are more important for hu- STAR+METHODS
man high acuity vision. Thus, pbUCNP-bound cones may
mediate high-resolution NIR image pattern vision. Retinae also Detailed methods are provided in the online version of this paper
possess intrinsic photosensitive retinal ganglion cells (ipRGCs), and include the following:
which mediate non-image forming visual functions, such as pho-
toentrainment of the circadian rhythm (Do and Yau, 2010). Under d KEY RESOURCES TABLE
the intensity used in our behavioral experiment, NIR light did not d CONTACT FOR REAGENT AND RESOURCE SHARING
activate ipRGCs (Figure S5F), which was likely due to the longer d EXPERIMENTAL MODEL AND SUBJECT DETAILS
distance of ipRGCs to pbUCNPs and their low sensitivity (Do B Mice
et al., 2009). With respect to NIR image spatial resolution, d METHOD DETAILS
pbUCNP-injected mice had good NIR eye sight (0.14 ± 0.06 cy- B Synthesis of pbUCNPs
cles/degree, half of the visible image resolution), allowing them B Sub-retinal injection
to see sophisticated NIR light patterns. B Distribution and spectrum analysis
B Retinal histology
Biocompatible NIR Nanoantennae B TUNEL Apoptosis detection
Sub-retinal injection in humans is a common practice in ophthal- B Microglia staining
mological treatment (Hauswirth et al., 2008; Peng et al., 2017). B Single cell electrophysiology
The implantation of microscale sub-retinal devices is a potential B Electroretinography
method of repairing vision following retinal photoreceptor B Pupillary light reflex
degeneration, though current devices can lead to biocompati- B Light-dark box
bility issues, such as retinal detachment, fibrosis, and inflamma- B Light induced fear conditioning
tion (Zrenner, 2013). Yet, this did not occur in our system, as the B Visually evoked potential
intimate contact between the pbUCNPs and photoreceptors did B Y shaped water maze
Cell 177, 1–13, April 4, 2019 11

d QUANTIFICATION AND STATISTICAL ANALYSIS Boynton, R.M. (1996). Frederic Ives Medal paper. History and current status of
B Imaging quantifications a physiologically based system of photometry and colorimetry. J. Opt. Soc.
B Statistics Am. A Opt. Image Sci. Vis. 13, 1609–1621.
B Fitting procedures Bridges, C.D.B. (1981). Lectin receptors of rods and cones. Visualization by
fluorescent label. Invest. Ophthalmol. Vis. Sci. 20, 8–16.
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org/10.1016/j.cell.2019.01.038. Lemke, G. (2012). Genetic dissection of TAM receptor-ligand interaction in
A video abstract is available at https://doi.org/10.1016/j.cell.2019.01. retinal pigment epithelial cell phagocytosis. Neuron 76, 1123–1132.
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ACKNOWLEDGMENTS
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We thank members of the Neuroscience Pioneer Club for valuable discus-
sions. We thank Drs. Dangsheng Li and Zilong Qiu for reading this manuscript Chen, J., Patil, S., Seal, S., and McGinnis, J.F. (2006). Rare earth nanoparticles
and providing helpful comments. We thank Dr. Yang Xiang from University of prevent retinal degeneration induced by intracellular peroxides. Nat. Nano-
Massachusetts Medical School for helpful discussion. We thank Min Wei and technol. 1, 142–150.
Shouzhen Li for helpful discussions and Jiawei Shen and Dr. Huan Zhao for Chen, S., Weitemier, A.Z., Zeng, X., He, L., Wang, X., Tao, Y., Huang, A.J.Y.,
helping illustration drawing and videos clipping. We thank Dr. Kai Huang and Hashimotodani, Y., Kano, M., Iwasaki, H., et al. (2018). Near-infrared deep
Mr. Nuo Yu for helping characterizing nanoparticles. We thank Dr. Qiuping brain stimulation via upconversion nanoparticle-mediated optogenetics. Sci-
Wang and Xiaokang Ding from National Synchrotron Radiation Laboratory ence 359, 679–684.
for helping with the measurement of various spectra of light sources. We thank
Cooke, S.F., and Bear, M.F. (2013). How the mechanisms of long-term synap-
Dr. Yuen Wu and Xing Wang from School of Chemistry and Materials Science
tic potentiation and depression serve experience-dependent plasticity in pri-
(USTC) for helping with the measurement of the absorption spectrum of
pbUCNPs. We acknowledge support from the National Key Research and mary visual cortex. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130284.
Development Program of China (2016YFA0400900), the Strategic Priority Cooke, S.F., Komorowski, R.W., Kaplan, E.S., Gavornik, J.P., and Bear, M.F.
Research Program of the Chinese Academy of Science (XDA16020603, (2015). Erratum: Visual recognition memory, manifested as long-term habitua-
XDPB10, XDB02010000), the National Young Scientists 973 Program of China tion, requires synaptic plasticity in V1. Nat. Neurosci. 18, 926.
(2013CB967700), the National Natural Science Foundation of China
Cui, Z.Z., Feng, R.B., Jacobs, S., Duan, Y.H., Wang, H.M., Cao, X.H., and
(81790644, 61890953, 31322024, 81371066, 91432104, 31571073,
Tsien, J.Z. (2013). Increased NR2A: NR2B ratio compresses long-term depres-
81401025, 61727811, 91748212), the NIH (R01MH103133 to G.H.), a UMass
sion range and constrains long-term memory. Sci. Rep. 3, 1036.
OTCV award, a Worcester Foundation Mel Cutler Award to G.H., and the
Human Frontier Science Program (RGY-0090/2014). Dalke, C., Löster, J., Fuchs, H., Gailus-Durner, V., Soewarto, D., Favor, J.,
Neuhäuser-Klaus, A., Pretsch, W., Gekeler, F., Shinoda, K., et al. (2004). Elec-
troretinography as a screening method for mutations causing retinal dysfunc-
AUTHOR CONTRIBUTIONS
tion in mice. Invest. Ophthalmol. Vis. Sci. 45, 601–609.
Conceptualization, T.X. and G.H.; Methodology, Y.M., J.B., G.H., and T.X.; Desai, N. (2012). Challenges in development of nanoparticle-based therapeu-
Investigation, Y.M., J.B., Y. Zhang, Z.L., L.H., Y. Zhao, X.Z., C.W., G.H., and tics. AAPS J. 14, 282–295.
T.X.; Validation, J.B., G.H., and T.X.; Formal Analysis, Y.M. and J.B.; Writing – Do, M.T.H., and Yau, K.W. (2010). Intrinsically photosensitive retinal ganglion
Original Draft, J.B., Y.M., Y. Zhang, G.H., and T.X.; Writing – Review & Editing, cells. Physiol. Rev. 90, 1547–1581.
J.B., Y.M., G.H., and T.X.; Funding Acquisition, J.B., G.H., and T.X.; Supervi-
sion, J.B., G.H., and T.X. Do, M.T., Kang, S.H., Xue, T., Zhong, H., Liao, H.W., Bergles, D.E., and Yau,
K.W. (2009). Photon capture and signalling by melanopsin retinal ganglion
cells. Nature 457, 281–287.
DECLARATION OF INTERESTS
Dong, A., Ye, X., Chen, J., Kang, Y., Gordon, T., Kikkawa, J.M., and Murray,
T.X. and G.H. have a patent application related to this work. C.B. (2011). A generalized ligand-exchange strategy enabling sequential sur-
face functionalization of colloidal nanocrystals. J. Am. Chem. Soc. 133,
Received: June 6, 2018 998–1006.
Revised: November 9, 2018 Dubois, E. (2009). The Structure and Properties of Color Spaces and the Rep-
Accepted: January 24, 2019 resentation of Color Images (Morgan & Claypool Publishers).
Published: February 28, 2019
Fu, Y., Kefalov, V., Luo, D.G., Xue, T., and Yau, K.W. (2008). Quantal noise from
human red cone pigment. Nat. Neurosci. 11, 565–571.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit polyclonal anti-Iba1 Wako Pure Chemical Corporation Cat# 019-19741, RRID: AB_839504
Alexa Fluor 568 goat anti-rabbit IgG (H+L) Thermo Fisher Scientific Cat# A-11036, RRID: AB_143011
Chemicals, Peptides, and Recombinant Proteins
Y2O3 Sigma-Aldrich Cat# 205168
Yb2O3 Sigma-Aldrich Cat# 246999
Er2O3 Sigma-Aldrich Cat# 203238
NOBF4 Sigma-Aldrich Cat# 175064
Poly(acrylic acid) (PAA) Sigma-Aldrich Cat# 323667
1-ethyl-3-(3-dimethylaminopropyl) TCI America Cat# D1601
carbodiimide hydrochloride (EDC)
N-hydroxysuccinimide (NHS) TCI America Cat# B0249
Concanavalin A (ConA) Sigma-Aldrich Cat# C5275
Atropine Aladdin Cat# A109524
(R)-(-)-Phenylephrine Hydrochloride Aladdin Cat# G1316011
2,2,2-tribromoethyl alcohol Sigma-Aldrich Cat# T48402
2-Methyl-2-butanol Sigma-Aldrich Cat# 240486
Paraformaldehyde Sigma-Aldrich Cat# V900894
Optimal Cutting temperature (O.C.T) Sakura Cat# 4583
Compound
Vitamins Sigma-Aldrich Cat# M6895
Non-essential Amino Acid Solution Sigma-Aldrich Cat# M7145
Critical Commercial Assays
TUNEL (Terminal deoxynucleotidyl Vazyme Biotech Cat# A113
transferase dUTP nick end labeling)
Apoptosis detection kit
Experimental Models: Organisms/Strains
C57BL/6NCrl Beijing Vital River Laboratory Animal Cat# 213
Technology
Gnat1/ Calvert et al., 2000 N/A
Opn1LW-Cre Le et al., 2004 N/A
Ai9-lsl-tdTomato Jackson Laboratory Cat# 007909
Nrl-GFP Jackson Laboratory Cat# 021232
rd1/rd1 Pittler and Baehr, 1991 N/A
cl/cl (cone-DTA) Soucy et al., 1998 N/A
Software and Algorithms
Origin 8.0 OriginLab https://www.originlab.com/
Igor Pro 8.0 WaveMetrics https://www.wavemetrics.com/products/igorpro
MATLAB MathWorks https://www.mathworks.com/products/matlab.html
LAS X Leica https://www.leica-microsystems.com/products/
microscope-software/details/product/leica-las-x-ls/
LabVIEW National Instruments, USA http://www.ni.com/support/labview/
Anilab (China) behavior software Anilab Software & Instruments, China http://www.anilab.cn/product.asp
ImageJ National Institutes of Health (NIH), USA https://imagej.nih.gov/ij/download.html
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Other
65 RN 5uL SYR W/O NEEDLE Hamilton, Switzerland Cat# 7633-01
RN Needle (34/8 mm/3) Hamilton, Switzerland Cat# 207343
535 nm LED Starsealand, China Cat# XL001WP01WBGC/535
980 nm LED Starsealand, China Cat# XL001WP01IRC/980
Photometer Newport 1936-R
Spectrometer Avantes ULS2048
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tian Xue
(xuetian@ustc.edu.cn).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice
The experimental procedures on animals followed the guidelines of the Animal Care and Use committee of University of Science and
Technology of China. Mice were kept under Specific pathogen Free (SPF) housing facilities, with lighting period of 12h:12h (L:D), con-
stant temperature at 20-24 C and humidity around 40%–70%. The mouse lines in the Key Resources Table and their crossings have
been used in this study at the age of 2-3 months. Confirming non-injected mice and PBS-injected mice are identical in retinal histol-
ogy, we used non-injected mice as control mice in most other experiments. Both male and female mice were used in all experiments.
METHOD DETAILS
Synthesis of pbUCNPs
General chemicals
Y2O3 (99.9%), Yb2O3 (99.9%), Er2O3 (99.9%), CF3COONa (99.9%), CF3COOH, 1-octadecene, oleic acid, oleylamine, and other
organic solvents were purchased from Sigma-Aldrich and used directly without further purification. Lanthanide trifluoroactates,
Ln(CF3COO)3 were prepared according to literature method (Roberts, 1961).
Synthesis of b-NaYF4:20%Yb, 2%Er core
The b-NaYF4:20%Yb,2%Er core UCNPs were prepared by a modified two-step thermolysis method (Mai et al., 2006). In the first step,
CF3COONa (0.5 mmol) and Ln (CF3COO)3 ((Y+Yb+Er) 0.5 mmol in total, Y:Yb:Er = 78%:20%:2%) precursors were mixed with oleic
acid (5 mmol), oleyamine (5 mmol) and 1-octadecene (10 mmol) in a two-neck reaction flask. The slurry mixture was heated to 110 C
to form a transparent solution followed by 10 minutes of degassing. Then the flask was heated to 300 C with a rate of 15 C/min under
dry argon flow, and it maintained at 300 C for 30 minutes. The b-NaYF4: Ln intermediate UCNPs were gathered from the cooled re-
action solution by centrifugal washing with excessive ethanol (7500 RCF, 30 min). In the second step, the b-NaYF4: Ln intermediate
UCNPs were re-dispersed into oleic acid (10 mmol) and 1-octadecene (10 mmol) together with CF3COONa (0.5 mmol) in a new two-
neck flask. After degassing at 110 C for 10 minutes, this flask was heated to 325 C with a rate of 15 C/min under dry argon flow, and
remained at 325 C for 30 minutes. Then, b-NaYF4: Ln UCNPs were centrifugally separated from the cooled reaction media and pre-
served in hexane (10 mL) as stock solution.
Synthesis of b-NaYF4:20%Yb,2%Er@b-NaYF4 core/shell UCNPs
In this thermolysis reaction, as-synthesized-NaYF4:20%Yb, 2%Er UCNPs served as cores for the epitaxial growth of undoped-
NaYF4 shells. Typically, a stock solution of b-NaYF4: 20%Yb, 2%Er UCNPs (5 mL, ca. 1 mmol/L core UCNPs) was transferred into
a two-neck flask and hexane was sequentially removed by heating. CF3COONa (0.5 mmol) and Y(CF3COO)3 (0.5 mmol) were added
along with oleic acid (10 mmol) and 1-octadecene (10 mmol). After 10 minutes of degassing at 110 C, the flask was heated to 325 C
at a rate of 15 C /min under dry argon flow and was kept at 325 C for 30 minutes. The products were precipitated by adding 20 mL
ethanol to the cooled reaction flask. After centrifugal washing with hexane/ethanol (7500 RCF, 30 min), the core/shell UCNPs were
collected and re-dispersed in 10 mL of hexane.
Synthesis of pbUCNPs
As synthesized b-NaYF4:20%Yb,2%Er@b-NaYF4 UCNPs were first treated by surface ligand exchange using a modified literature
method (Dong et al., 2011). Generally, nitrosonium tetrafluoroborte/DMF solution (0.2 g NOBF4, 5 mL DMF) was added into 1 mL
UCNPs hexane stock solution, followed by 4 mL hexane and 3 hours of stirring at room temperature. Then oleic acid-free UCNPs
Cell 177, 1–13.e1–e6, April 4, 2019 e2

were precipitated by adding 5 mL isopropanol and purified by centrifugal wash with DMF. UCNPs solids were re-dispersed in poly
(acrylic acid)/DMF (10 mg/mL, 5 mL) solution to coated UCNPs surface with PAA. After overnight stirring, PAA coated UCNPs
(paaUCNPs) were purified by centrifugal and wash with DI-water. Then ConA proteins were conjugated to paaUCNPs surface by
tranditional EDC/NHS coupling. Generally, 10 mg paaUCNPs in 1 mL DI-water were treated with 1 mL EDC/NHS water solution
(1 g/L). After stirring at room temperature for 1 hour, 30 mL ConA solution was introduced (5 g/L) and the mixture was further stirred
overnight. The pbUCNPs were purified by washing with deionized water, centrifugation and dispersed in water for further use.
Sub-retinal injection
For sub-retinal injection, pupils were dilated with atropine (100 mg/mL, Sigma-Aldrich), and animals were anesthetized by Avertin
(450 mg/kg, Sigma-Aldrich). A 33 Gauge needle was inserted through the cornea to release the intra-ocular pressure. Nanoparticles
dissolved in 2 mL sterile PBS to reach 25 mg/ml was injected into the sub-retinal space through a beveled, 34-gauge hypodermic
needle (Hamilton, Switzerland). During and after the injection the animal was kept on a warming blanket and eyes were kept wet
to avoid cataract.
Distribution and spectrum analysis

To observe the distribution of nanoparticles in the sub-retinal space, fresh retinas were isolated from eyes following execution. Ret-
inas were then fixed in 4% paraformaldehyde (PFA), frozen and cut into 20 mm slices with Leica CM3050 S Cryostat (Leica, Germany)
(Figure 1I). All retinal slices were scanned by Leica two-photon microscope (SP8, Leica, Germany). The excitation sprectrum was
obtained by exciting the nanoparticles with two-photon laser beam (wavelength from 700-1040 nm, Dl = 5 nm), and emitted light
intensity was measured at 535 nm. The emission spectrum was obtained by exciting nanoparticles with a 980 nm laser beam and
collecting the emitted light with a photomultiplier behind a light grating slit with 5 nm wavelength step from 435 nm to 790 nm
(SP8, Leica, Germany). Data were acquired with Leica Imaging software and analyzed with Origin 8.0 (Origin Lab Corp). High reso-
lution fluorescence images of rods and cones were confocal images with 63 3 oil immersion objective from 6-mm retina slices.
Retinal histology
To analyze whether nanoparticles are potentially toxic to retina, we injected nanoparticles in different concentrations and then per-
formed hematoxylin-eosin (HE) staining on fixed retinal slices (Burstyn-Cohen et al., 2012). Cell bodies of photoreceptors were
located in the outer nucleus layer (ONL) and we counted the number of cell layers as a parameter to evaluate the damage. The number
of cell layers were counted at 5 different locations of injection sites and repeated at 5 randomly selected different slices of each retina,
then averaged.
TUNEL Apoptosis detection

We detect photoreceptors apoptosis using TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) Apoptosis detec-
tion kit (Vazyme Biotech, Ltd-A113). Before TUNEL staining, retinal slices were washed twice by PBS solution and then incubated in
1% Triton X-100 (Sangon-A110694) solution for 2-3 hours. Afterward retinal slices were equilibrated in equilibration buffer (provided
in the kit) for 10-30 min and then incubated in TdT incubation buffer for 60 min. Finally, retinal slices were incubated in DAPI-PBS
solution for 5 min and then washed 3 times by PBS solution. All retinal slices were scanned by Leica two-photon microscope to detect
TUNEL signals. The number of positive cells were counted at 6 different locations of injection site from each retina and then averaged.
Microglia staining
To detect immune reactions in nanoparticles injected retinas, we implemented Iba1 (ionized calcium binding adaptor molecule 1, one
marker protein of microglia) staining assay. Retinal slices were washed twice by PBS solution and then blocked in 1% Triton X-100
(Sangon-A110694) and 5% goat serum solution (blocking solution) for 2-3 hours. Then slices were incubated in blocking solution with
rabbit anti-Iba1 antibody (Wako-019-19741, 1:1000) at 4 C overnight. Afterward, slices were washed 3 times by PBS solution,and
then incubated in Alexa Fluor 568 goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific-1832035, 1:800) at
room temperature for 2-3 hours. Finally, retinal slices were incubated in DAPI-PBS solution for 5 min and then washed 3 times by
PBS solution. All retinal slices were scanned by Leica two-photon microscope to analyze retinas immune activities. The number
of positive cells were counted at 6 different locations of injection site from each retina and then averaged.
Single cell electrophysiology

Before rod suction pipette recordings, pbUCNP-injected or non-injected mice were dark-adapted overnight. Animals were anesthe-
tized with Tribromoethanol (Avertin, 450 mg/kg, Sigma-Aldrich) and eyes were enucleated after euthanasia. Retina was carefully iso-
lated from the eye and flat-mounted onto the recording chamber. Recordings were carried out on an Olympus upright infrared-DIC
microscope. The extracellular bath solution was bicarbonate-buffered Ames medium (in mM): 120 NaCl, 22.6 NaHCO3, 3.1 KCl,
0.5 KH2PO4, 1.5 CaCl2, 1.2 MgSO4, 6 Glucose, equilibrated with 5% CO2/95% O2 and heated to 35 C (Warner Instruments Corp,
TC-3448). The perfusion speed was 5 ml/min through the 3 mL recording chamber. The outer segment of a rod was gently sucked
into a 1.6 mm diameter glass pipette filled with modified Ames solution (in mM): 135 NaCl, 10 mM HEPES, 3.1 KCl, 0.5 KH2PO4,
1.5 CaCl2, 1.2 MgSO4, 6 Glucose, pH adjusted to 7.4 by NaOH. Stimulation light was applied through the imaging objective.
e3 Cell 177, 1–13.e1–e6, April 4, 2019

535-nm light was from a filter block in front of a white light LED. Infrared light was generated by 980-nm laser. Flash light intensities in
Figures 3B–3E, S4A, S4B, S4D, S4E, S4G, and S4H: 535-nm - 0.64, 2.57, 12.55, 49.38, 162.5, 1102.21; 980-nm - 1.50 3 105, 6.31 3
105, 1.85 3 106, 5.81 3 106, 2.26 3 107, 1.17 3 108; unit - photons$mm-2.
To measure the action spectrum of rods, light flashes in a series of wavelengths were delivered to elicit flash photoresponses. All
the recorded rods were given a strong light test pulse to check if a normal saturating photocurrent can be elicited, before the family of
light pulses with different wavelengths was given. Flash light intensities were selected to generate photocurrents within the linear
range. Light pulses were from LEDs or lasers. The sensitivity was calculated as photoresponse / (flash intensity 3 area) and then
normalized to the peak sensitivity.
To quantify light adaptation, we applied background light and allowed at least 2-min adaptation for the recorded rod (Karnas et al.,
2013; Schmidt and Kofuji, 2010). Then on top of the background light, a light flash was applied to elicit a transient photocurrent. Flash
sensitivity (SF) was calculated by dividing the peak amplitude of the transient photocurrent with the intensity of the flash light. For a
series of applied background light with different intensities, flash sensitivities were measured and normalized to the flash sensitivity in
the dark (SD). The background light intensities are: 535-nm - dark, 12.9, 49.20, 113.24, 266, 542.79, 2700.06, 5965.32; 980-nm - dark,
4.08 3 106, 1.03 3 107, 2.02 3 107, 4.60 3 107, 7.44 3 107, 1.94 3 108, 4.36 3 108; unit - photons$mm-2$s-1). The intensity-response
curves except under dark background were measured under three different conditions: visible light flashes on top of a visible or NIR
light background (535-nm - 776 photons$mm-2$s-1 or 980-nm - 1.3 3 108 photons$mm-2$s-1) and NIR light flashes on top of a visible
light background (535-nm - 776 photons$mm-2$s-1).
Data were lowpass filtered at 50 Hz and sampled at 25 kHz by Axon 700B Amplifier and Digital 1440A interface. Data were analyzed
with custom routines in Origin 8.0 and presented as mean ± SD. Single cell electrophysiology was carried out 5-6 weeks after
pbUCNPs injection. Data for noise analysis of rods were filtered with low-pass Bessel filter (cutoff frequency - 5Hz) (Baylor et al.,
1980; Fu et al., 2008). Power spectra density was calculated with function ‘periodogram()’ in MATLAB (Mathworks, USA).
Electroretinography
Mice were anesthetized by Avertin (450 mg/kg, Sigma-Aldrich) after their pupils were dilated with atropine (100 mg/mL, Sigma-Al-
drich). During the experiment, the anesthetized animal was kept on a warming blanket and eyes were kept wet to avoid cataract
(Dalke et al., 2004). Mice were placed into a Faraday cage and a glass recording electrode with a tip diameter of 10 mm was put tightly
against the center of the cornea. A ground electrode was inserted into subcutaneous space of the tail and a reference electrode was
inserted into subcutaneous space of the head. A 535-nm LED light (8.26 3 103 photons$mm-2) and a 980-nm laser beam with a spot
diameter of 1.8 mm (9.83 3 108 photons$mm-2) was placed in front of the pupil for stimulation. Data acquisition was carried out by a
differential amplifier (AM-SYSTEM INC) and Digital 1440A (Axon CNS). Data were analyzed with custom routines in Origin 8.0 (Origin
Lab Corp). ERG was carried out 4 weeks after pbUCNPs injection.
Pupillary light reflex

Head-fixed mice were used for pupillometry of long-duration measurements (Xue et al., 2011). C57BL/6 wild-type mice were anes-
thetized with intraperitoneal injection of Avertin. A patch of skin overlying the skull was excised, and four bone screws were threaded
into the skull, with care taken to prevent any damage to the brain. These screws were covered with dental cement, and served as the
foundation for a stainless-steel post. Under anesthesia, eyes were kept wet with eye gel (5% Sodium carboxymethyl cellulose in PBS,
Sigma-Aldrich) to avoid cataract. Mice were kept in 12/12 hours light/dark cycles. All PLR experiments were performed during the
day: from 2 hours after light-on to 2 hours before light-off with > 1 h dark adaptation. To measure the PLR of the pbUCNP-injected
eye, we built a pupillometer with a miniature, infrared CCD camera and 850-nm LED light for video recording via a Ganzfeld sphere. A
laser beam with a spot diameter of 1.8 mm was placed 1 mm away in front of the injected eye. Light intensity of 980-nm light in Fig-
ure 4B: paaUCNP-injected - 9.49 3 107, 2.36 3 108, 3.80 3 108, 6.67 3 108, 1.42 3 109, 2.97 3 109, 4.44 3 109, 6.06 3 109, 1.13 3
1010, 1.97 3 1010, 2.56 3 1010; pbUCNP-injected - 1.33 3 106, 3.15 3 106, 1.01 3 107, 2.44 3 107, 4.18 3 107, 6.54 3 107, 1.21 3 108,
1.72 3 108, 2.80 3 108; unit - photons$mm-2$s-1. Videos for contralateral eyes were recorded at a frame rate of 5 Hz. A data-acqui-
sition board (NI USB-6211, National Instruments) and custom written software were used for triggering recordings and light stimu-
lations. Videos were recorded and analyzed with XCAP-Ltd V3.x. The normalized pupil area was calculated by normalizing the area
measured at maximum constriction during the 40 s light stimulation to that before light stimulation. We measured PLR of injected
mice 2-3 weeks after injection.
Light-dark box
Mice were placed in a 59 cm 3 28.5 cm 3 28.5 cm custom-made light and dark double box (Bourin and Hascoët, 2003). On the
three sides of the light box, 20 980-nm LEDs (1 Watt) and twenty 535-nm LEDs (1 Watt) were evenly placed for light stimulation.
Intensity of 980 nm light at the center of the light box was 8.1 3 107 photons$mm-2$s-1, and intensity of 535-nm light was 9.1 3
102 photons$mm-2$s-1. Animals were introduced to the box and allowed for 5-min adaptation. A series of light stimulation in the order
of 5 min in dark, 5 min in 980-nm light, and 5 mins in 535-nm light was programed. All these experiments were carried out in the dark
environment and videos were acquired by an infrared camera and custom-made software. Experiments were performed 4-5 weeks
after injection.
Cell 177, 1–13.e1–e6, April 4, 2019 e4

Light induced fear conditioning

Fear conditioning experiments were carried out in a 21.5 cm 3 21.5 cm 3 24 cm custom-made box (Cui et al., 2013) (Figure 4E). On
the four sides of the box, 20 980-nm LEDs (1 Watt) and 20 535-nm LEDs (1 Watt) were evenly placed for light stimulation. The training
protocol consisted of 5-min adaptation, 20 s 535-nm light as conditional stimulus (CS) followed with a 2 s electrical foot shock as
unconditional stimulus (US). Paired CS and US was repeated 5 times with 2-min interval. One day after the training, a test protocol,
which was the same as the training protocol but no unconditional stimulus (US), was used to test freezing time of mice and in which
2 times 20 s 535-nm light and 2 times 980-nm light randomly as conditional stimulus (CS). Anilab (China) behavior software was used
to control 980-nm or 535-nm LEDs light stimulation and the electrical shock. Intensity of 980-nm light at the center of the box was
1.07 3 108 photons$mm-2$s-1, and intensity of 535-nm light was 1.47 3 103 photons$mm-2$s-1. Freezing time ratios of Pre-CS (40 s
before conditional stimulus) and CS (40 s, total time of the 2 times 980-nm or 535-nm conditional stimulus) were analyzed to compare
the effect of light induced freezing. Experiments were conducted 4-5 weeks after injection.
Visually evoked potential

1-2 weeks after pbUCNPs injection, VEP was carried out as described in literatures (Cooke and Bear, 2013; Cooke et al., 2015). Mice
were anesthetized by pentobarbital sodium (LUPI-P8410) at the dose of 1 g/kg body weight and then fixed on the stereotaxic appa-
ratus. The skull on either right or left visual cortex was grinded off and removed carefully avoiding any damage to the visual cortex. An
electrical recording glass pipette with a tip diameter of 15 mm was inserted into right/left visual cortex and targeted to the following
coordinates (relative to bregma): 1-(2.15, 2.8, 0.4), 2-(2.75, 2.8, 0.4), 3-(2.0, 3.28, 0.4), 4-(2.75, 3.28, 0.4), 5-(2.25, 4.24,
0.4), 6-(3.0, 4.24, 0.4) mm. A ground electrode was inserted into subcutaneous space of the tail and a reference electrode was
inserted into subcutaneous space of the head. The contralateral eye of the exposed visual cortex was illuminated by 980-nm light
(7.07 3 108 photons$mm-2$s-1) or 535-nm light (3.37 3 103 photons$mm-2$s-1) during recording. Signal was amplified by a differential
amplifier (AM-SYSTEM INC) and digitized by Digital 1440A (Axon CNS).
Y shaped water maze

Y-shaped water maze experiments were performed in either scotopic (dark) or photopic (light) condition (Prusky et al., 2000). During
adaptation periods, mice were released in the water close to stimulating light boxes for discovering the hidden platform. During adap-
tation period, the released place became further from the light boxes with time and ultimately mice were released at the release chute.
The adaptation lasted for 2 days with one section (12 trials per section) per day. After adaptation mice were trained to find a hidden
platform associated to the task stimuli. Trainings usually lasted for 7 days: one section per day and 12 trials per section. The platform
and the associated stimuli were placed right or left randomly across trials in each section, such as LRLRLLRRLRRL. Upon completion
of the trainings, the test sections were conducted in two separated days. In Task #1 animals were trained with visible light horizontal
and vertical light gratings (0.8 circles per degree, c/d, 7.26 3 103 photons$mm-2$s-1 at the release chute) and then tested to discrim-
inate visible (7.26 3 103 photons$mm-2$s-1 at the release chute) and NIR (8.01 3 107 photons$mm-2$s-1 at the release chute) horizon-
tal/vertical light gratings in the dark background. For the visual acuity test in the dark background, 0.054 c/d, 0.08 c/d, 0.107 c/d,
0.134 c/d, 0.161 c/d, 0.214 c/d, 0.268 c/d, 0.322 c/d, 0.35 c/d, 0.38 c/d, 0.435 c/d gratings were tested for 980-nm light stimuli
(8.01 3 107 photons$mm-2$s-1 at the release chute) and 535-nm light stimuli (7.26 3 103 photons$mm-2$s-1 at the release chute).
In Task #2 animals were trained and tested to discriminate NIR light gratings made of LEDs arrays under the background visible
room light (background light intensity equivalent to 535-nm light was 4.31 3 103 photons$mm-2$s-1). Two lines of 535-nm LEDs
and two lines of 980-nm LEDS were placed perpendicular to each other. Each line was made of 18 LEDs. The left pattern was
90 degrees rotated compared to the right pattern. The training lasted for 9 days with one section per day and 12 trials per section
with 980-nm LEDs on only. In Task #2, the intensity of 980-nm light pattern measured at the release chute was 8.01 3 107 pho-
tons$mm-2$s-1 and 535-nm light pattern was 7.26 3 103 photons$mm-2$s-1. In Task #3 animals were trained with visible light triangle
and circle patterns and tested to discriminate visible and NIR light triangle/circle patterns under the dark background. In Task #4
animals were tested to discriminate visible light triangle/circle patterns under the visible light background (7.26 3 103 pho-
tons$mm-2$s-1) or NIR light background (8.01 3 107 photons$mm-2$s-1). In Task #3 and #4, the intensity of 980-nm light pattern
measured at the release chute was 8.01 3 107 photons$mm-2$s-1 and 535-nm light pattern was 7.26 3 103 photons$mm-2$s-1. In
Task #5 animals were tested to discriminate patterns in the mixture of visible and NIR light patterns. The hidden platform was
associated with the triangle pattern and four different stimuli were presented randomly (see Figure 6I). In Task #5, the intensity of
980-nm light pattern measured at the release chute was 2.32 3 108 photons$mm-2$s-1 and 535-nm light pattern was 1.57 3 103 pho-
tons$mm-2$s-1. All videos were acquired by infrared camera and analyzed by Corel Screen Cap X6 and Origin 8.0. Y shaped water
maze experiments were implemented 3-8 weeks after pbUCNP-injection. The spectrum of 980-nm LEDs was measured using spec-
trometer (Avantes USB2.0) to confirm there is no detectable visible light emission.
QUANTIFICATION AND STATISTICAL ANALYSIS
Imaging quantifications
Fluorescence analysis was based on careful match of the confocal imaging parameters. The intensities of pixels or the number of
cells were then quantified in ImageJ (NIH).
e5 Cell 177, 1–13.e1–e6, April 4, 2019

Statistics
Statistical analysis was performed using Microsoft Excel or Matllab softwares. Unpaired two-tailed Student’s test was used to deter-
mine statistical significance. The ‘‘n’’ numbers for each experiment are provided in the text and figure legends. Data are all presented
as mean ± SD. For immunocytochemistry and toxicological detection experiments were repeated on at least 3 animals.
Fitting procedures
Fittings were conducted in MATLAB (Mathworks, USA) with an unconstrained nonlinear optimization routine. The upconversion rela-
tionship was fitted by a linear function in the log-log scale. The dose response curve of PLR - the normalized pupil area versus light
intensity - was fitted by the Hill function. The action spectrum of rods was fitted to absorption-spectrum template (Fu et al., 2008;
Govardovskii et al., 2000). The relative flash sensitivity of background light adapted rods was fitted to Weber-Fechner equation (Bay-
lor et al., 1980; Morshedian et al., 2018; Fu et al., 2008).
Cell 177, 1–13.e1–e6, April 4, 2019 e6

Supplemental Figures
Figure S1. Properties of UCNPs and Distributions in Subretinal Space, Related to Figure 1
(A) Fourier Transform-Infrared (FT-IR) spectra of UCNPs before (black) and after (red) ConA surface modifications. After ConA conjugation, new peaks at 1681 and
1456 cm-1 emerged, which are attributed to amide bond formation.
(B) Absorption spectrum of UCNPs before (black) and after (red) ConA conjugation.
A new absorption peak at 285 nm is assigned to ConA. NIR range absorption is shown in inset, and the absorption peak at 980 nm is attributed to the absorption
for upconversion.
(C) Dynamic light scattering (DLS) spectrum of pbUCNPs (0.2 mg/mL) upon the introduction of different concentrations of b-cyclodextrins (0, 50, 100 and 200 nM)
showing aggregation of pbUCNPs.
(D) Dynamic light scattering spectrum of paaUCNPs (0.2 mg/mL) upon the introduction of different concentrations of b-cyclodextrins (0, 50, 100 and 200 nM).
(E) Illustration of subretinal injection of pbUCNPs.
(F) Distribution of pbUCNPs (green) in the subretinal space after a single injection. Scale bar: 250 mm.
(G) Quantified green light intensity distribution along the spread of pbUCNPs in the subretinal space (n = 4). Data are mean ± SD. X axis is the distance from the
injection site and y axis is the total green pbUCNPs emission intensity from a 50 3 50 mm2 measuring window on a retina slice along the distribution of pbUCNPs
from the injection site.
Figure S2. Toxicity and Biocompatibility Evaluation of Retina Slices in a Time Series up to 10 Weeks after Injection, Related to Figure 2
(A) Microglia marker Iba1 staining of retina slices. Retinal injection of 6 mM H2O2 was used as the positive control. Red: Iba1; Green: pbUCNPs emission upon
excitation by NIR light; Blue: DAPI (4’, 6-diamidino-2-phenylindole) signal indicating cell nucleuses. Scale bar: 50 mm.
(B) TUNEL staining of retina slices. Retinal injection of 6 mM H2O2 was used as the positive control. Red: TUNEL staining; Green: pbUCNPs emission upon
excitation by NIR light; Blue: DAPI. Scale bar: 50 mm.
(C) and (D) Excitation (C) and emission (D) spectra of pbUCNPs in solution (black), fixed retina (gray) and fresh retina (violet).
Figure S3. Action Spectra of Rods and ERG of Gnat1/ Mice, Related to Figure 3
(A) Left: Action spectra of rods from non-injected (gray) and pbUCNP-injected (red) retina. Fit of action spectrum of rods from non-injected retina to absorption-
spectrum template (Fu et al., 2008; Govardovskii et al., 2000) is shown in dashed blue line. Open squares are theoretical predicted values from the fit under
detection threshold. The predicted sensitivity of non-injected rods to the NIR light (980 nm) is extremely small. With the help of pbUCNPs, rods gained 23 orders of
magnitude higher sensitivity to 980-nm NIR light. Right: Same action spectra were plotted with excitation spectrum of pbUCNPs (cyan) normalized to the action
spectrum value at 980 nm of rods from pbUCNP-injected mice. All data are mean ± SD.
(B) ERG recordings of Gnat1/ mice. Light intensities are 7.26 3 103 photons$mm-2 for 535-nm light and 8.01 3 107 photons$mm-2 for 980-nm light.
Figure S4. Adaptation and Noise Properties of Rods from pbUCNP-Injected Retina, Related to Figure 3
(A) Visible light flash responses of rod photoreceptors under dark and visible light background. Background and flash light stimulations are illustrated on top of the
recording traces (A, D and G).
(legend continued on next page)

(B) Intensity-response curves of rods in (A) under dark background (black trace) and the specified background with 535-nm light intensity of 736 photons$mm-2$s-1
(gray trace) (n = 3). All data are mean ± SD.
(C) Normalized flash sensitivity is plotted as a function of background light intensity and fitted with Weber-Fechner equation SF/SD = 1/[1+IB/I0] (Baylor et al.,
1980; Morshedian et al., 2018; Fu et al., 2008). The visible background light intensity that decreased the visible flash light sensitivity by half, I0(V/V), is 137 pho-
tons$mm-2$s-1, similar to values in previous studies (Karnas et al., 2013; Schmidt and Kofuji, 2010) (n = 3). All data are mean ± SD.
(D) NIR light flash responses of rod photoreceptors under dark and visible light background.
(E) Intensity-response curve of rods in (D) under dark background and the specified background 535-nm light intensity of 736 photons$mm-2$s-1 (n = 3).
(F) Normalized flash sensitivity is plotted as a function of background light intensity and fitted with Weber-Fechner equation (n = 3). The visible background light
intensity that decreased the NIR flash light sensitivity by half, I0(N/V), is 313 photons$mm-2$s-1.
(G) Visible light flash responses of rod photoreceptors under dark and NIR light background.
(H) Intensity-response curve of rods in (G) under dark background and the specific background light intensity of 1.3 3 108 photons$mm-2$s-1 (n = 3)
(I) Normalized flash sensitivity is plotted as a function of background light intensity and fitted with Weber-Fechner equation (n = 3). The NIR background light
intensity that decreased the visible light flash sensitivity by half, I0(V/N), is 2.76 3 107 photons$mm-2$s-1. All data are mean ± SD.
(J) Adaptation of visible (green) and NIR (red) light flash sensitivity under the visible light background (from C and F) were re-plotted. The discrepancy came from
the nonlinearity of upconversion by pbUCNPs (as quantified in Figure S5B). NIR light flash adaptation (red) was converted to equivalent visible light flash
adaptation (violet) according to the pbUCNP upconversion relation fit from Figure S5B, which matched very well with the visible light flash adaptation
curve (green).
(K) Representative traces of dark current and steady-light current from rods of non-injected and pbUCNP-injected mice.
(L) Differential power spectrum density between dark and light current was the difference between the power spectrum density in darkness and in saturated
steady light (Baylor et al., 1980; Fu et al., 2008). There is no significant difference in the rod dark current noise spectrum with respect to non-injected versus
pbUCNP-injected animals, indicating that pbUCNPs did not interfere with the membrane biophysical properties and the phototransduction cascade of rods. All
data are mean ± SD.
Figure S5. Demonstration of the Nonlinearity of pbUCNP Photon Upconversion and PLR Measured from Gnat1/; cl/cl mice, Related to
Figure 4
(A) To calculate the upconversion efficiency of pbUCNPs, pbUCNPs were spreaded evenly on the surface of a cover glass: 2.4 cm 3 2.4 cm. 980 nm light was
applied from the top of the cover glass covering a 1.5 cm 3 1.5 cm area and the intensity of emission light from the other side of the cover glass was measured
behind a combined filters (bandpass filter at 510 - 560 nm and short-pass filter cutoff at 805 nm). The numerical aperture of the sensor in the light intensity meter is
0.75, which collected a proximately 17% of the isotropically emitted photons from pbUCNPs.
(B) The upconversion relationship between NIR and visible light intensities. From a linear fit in the log-log scale, we extracted the power relation between up-
converted 535-nm emissions and 980-nm excitation light to be 1.6.
(C) Light induced pupillary light reflex (PLR) dose response curves and their Hill function fitting (markers and solid lines) for visible (black) and NIR (red) light. The
Hill coefficients for visible and NIR light PLR dose response are 0.78 and 1.10, respectively. To calculate the theoretical NIR light PLR dose response curve, NIR
light intensities were applied to the upconversion relationship to generate upconverted corresponding visible light intensities, and then these intensities were
(legend continued on next page)

applied to visible light PLR dose response Hill function. The theoretical NIR light PLR dose response curve (red dashed line) matched very well to the experimental
measurement. Therefore, the non-linearity shown in the light induced behavior is attributed to the non-linearity of the upconversion process.
(D) Spectrum of 980-nm LED light in log scale. Inset: expanded view of this spectrum in linear scale.
(E) Expanded view of (D) at above 900 nm in log scale.
(F) PLR from Gnat1/; cl/cl mice, which have intrinsically photosensitive retinal ganglion cells (ipRGCs) as the only functional photoreceptors. All data are
mean ± SD.
Figure S6. Simultaneous NIR and Visible Light Pattern Vision, Related to Figure 6
(A) Diagram of the light grating stimuli made from LED arrays and transparent acrylic (TPA) boards.
(B) Correct rates made by each individual animal in discriminating NIR light gratings.
(C) Correct rates made by each individual animal in discriminating visible light gratings.
(D) The visual imaging resolution for each individual animal for visible (dark) and NIR (gray) light gratings.
(E) Correct rates made by each individual animal in discriminating NIR light triangle and circle under dark background.
(F) Correct rates made by each individual animal in discriminating visible light triangle and circle under dark background.
(G) Correct rates made by each individual animal in discriminating visible light triangle and circle under visible light background.
(H) Correct rates made by each individual animal in discriminating visible light triangle and circle under NIR light background.
(I) Correct rates made by each individual animal in discriminating NIR and visible light triangle and circle simultaneously.
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CHAPTER XIV
LETIZIA THE FIRST
“Mrs. Fuller has been so excited ever since the letter came to say
that you would pay a visit to Lebanon House,” the nurse stopped to
tell Nancy at the head of the stairs, before showing the way along
the landing to the old lady’s room.
“Are we going to see more aunts, muvver?” Letizia anxiously
inquired.
“No, darling, you’re going to see dear father’s grannie whom he
loved very much; and he would like you to love her very much too.”
“Well, I will love her,” Letizia promised.
“You must remember that your name is Letizia and that her name
is Letizia. You were christened Letizia because father loved his
grannie. And remember she is very old and that’s why you’ll find her
in bed.”
“Will she be like Red Riding Hood’s grannie?” Letizia asked.
“Perhaps she will be a little.”
“But a naughty wolf won’t come and eat her?” Letizia pressed on a
note of faint apprehension.
“Oh, no,” her mother assured her. “There are no wolves in this
house.”
Was it a trick of the gathering dusk, or did the bright-eyed young
woman raise her eyebrows and smile to herself at this confident
reply?
Nancy had never been so much surprised in her life as she was by
the aspect of old Mrs. Fuller’s room. The old lady, wrapped in a bed-
jacket of orange and yellow brocade and supported by quantities of
bright vermilion cushions, was sitting up in a gilded four-post bed,
the curtains and valance of damasked maroon silk and the canopy
sustained by four rouged Venetian amorini with golden wings. Over
the mantelpiece was a copy of Giorgione’s “Pastorale.” Mirrors in
frames of blown glass decorated with wreaths of pink glass rosebuds
and blue glass forget-me-nots hung here and there on the white
walls, the lighted candles in which gave the windowpanes such a
bloom in the March dusk as is breathed upon ripe damsons.
Bookcases on either side of the fireplace were filled with the sulphur
backs of numerous French novels. On a mahogany table at the foot
of the bed stood a green cornucopia of brilliantly tinted wax fruits that
was being regarded with slant-eyed indifference by two antelopes of
gilded wood, seated on either side.
Of course, Nancy had known that old Mrs. Fuller was different
from the rest of the family; but this flaunting rococo bedroom made a
sharper impression of that immense difference than could all the
letters to her grandson. It was strange, too, that Bram should never
have commented on this amazing room, set as it was in the heart of
the house against which his boyhood had so bitterly revolted. In her
astonishment at her surroundings she did not for the moment take in
the aspect of the old lady herself; and then suddenly she saw the
dark eyes of Bram staring at her from the middle of those vermilion
cushions, the bright eyes of Bram flashing from a death’s head
wrapped in parchment. She put her hand to her heart, and stopped
short on her way across the room to salute the old lady.
“What’s the matter?” snapped a high incisive voice.
“Oh, you’re so like Bram,” cried Nancy, tears gushing like an
uncontrollable spring from her inmost being, like blood from a
wound, and yet without any awareness of grief, so that her voice was
calm, her kiss of salutation not tremulous.
“Might I lift my little girl on Mrs. Fuller’s bed, nurse?” she asked.
“Don’t call her nurse,” the old lady rapped out. “This ain’t a
hospital. It’s only that sanctimonious ghoul Caleb who calls her
nurse. She’s my companion, Miss Emily Young. And why should the
wretched child be lifted up to see an old bogey like myself?”
“I think she’d like to kiss you, if she may,” said Nancy.
“Yes, I would like to kiss you,” said Letizia.
The old woman’s eyes melted to an enchanting tenderness, and,
oh, how often Nancy had seen Bram’s eyes melt so for her.
“Lift her up, Emily, lift her up,” said Mrs. Fuller.
Miss Young put Letizia beside her and the old woman encircled
the child with her left arm. The other hung motionless beside her.
“I’m not going to maul you about. I expect your aunts have
slobbered over you enough downstairs. Just give me one kiss, if you
want to. But if you don’t want to, now that you’re so close to my
skinny old face, why, say so, and I shan’t mind.”
But Letizia put both arms round her great-grandmother’s neck and
kissed her fervidly.
“And now sit down and tell me how you like Lebanon House,” she
commanded.
“Is this Lebbon House?”
The old woman nodded.
“I like it here, but I don’t like it where those aunts are. Have you
seen those aunts, grannie?”
“I made them.”
“Why did you make them, grannie? I don’t fink they was very
nicely made, do you? I don’t fink their dresses was sewed on very
nicely, do you?”
“You’re an observant young woman, that’s what you are.”
“What is azervant?”
“Why, you have eyes in your head and see with them.”
“I see those gold stags,” said Letizia, pointing to the antelopes.
“Ah-ha, you see them, do you?”
“Did Santy Claus give them to you? He gived me a lamb and a
monkey and lots and lots of fings, so I aspeck he did.”
“I expect he did too. But they’re antelopes, not stags.”
“Auntylopes?” Letizia repeated dubiously. “Will Santy Claus put
gold aunts in your stocking at Christmas?”
“Mon dieu, I hope not,” the old lady exclaimed. “So you like
antelopes better than aunts?”
“Yes, I do. And I like puss-cats better. And I like all fings better
than I like aunts.”
“Well, then I’ll tell you something. When Santa Claus brought me
those antelopes, he said I was to give them to you.”
Letizia clapped her hands.
“Fancy! I fought he did, grannie.”
“So, if you’ll take them into the next room with Miss Young, she’ll
wrap them up for you while I’m talking to your mother.”
“How kind of you to give them to her,” said Nancy, from whose
eyes the silent tears had at last ceased to flow.
“Letizia darling, say ‘thank you’ to your kind grannie.”
“Senza complimenti, senza complimenti,” the old woman muttered,
“The pleasure in her eyes was all the thanks I wanted.”
“I aspeck they won’t feel very hungry wivout the apples and the
pears,” Letizia suggested anxiously.
“Of course they won’t, darling,” her mother interrupted quickly.
“You’d better wrap up some of the fruit as well, Emily,” said the old
lady with a chuckle.
“No, please ...” Nancy began.
“Hoity-toity, I suppose I can do what I like with my own fruit?” said
the old lady sharply. “Draw the curtains before you go, Emily.”
When Letizia had retired with Miss Young, and the gilded
antelopes and a generous handful of the wax fruit, the old lady bade
Nancy draw up one of the great Venetian chairs. When her
grandson’s wife was seated beside the bed, she asked her why she
had come to Brigham.
Nancy gave her an account of her struggles for an engagement
and told her about Bram’s death and that unuttered wish.
“He may have worried about your future,” said the old lady. “But it
was never his wish that Letizia should be brought up here. Never! I
know what that wish was.”
“You do?”
“He was wishing that he had become a Catholic. He used to write
to me about it, and I’m afraid I was discouraging. It didn’t seem to me
that there was any point in interrupting his career as a clown by
turning religious somersaults as well. I’m sorry that it worried his
peace at the last, but by now he is either at rest in an eternal
dreamless enviable sleep or he has discovered that there really is a
God and that He is neither a homicidal lunatic, nor a justice of the
peace, nor even a disagreeable and moody old gentleman. I used to
long to believe in Hell for the pleasure of one day seeing my late
husband on the next gridiron to my own; but now I merely hope that,
if there is another world, it will be large enough for me to avoid
meeting him, and that, if he has wings, an all-merciful God will clip
them and put him to play his harp where I shan’t ever hear the tune.
But mostly I pray that I shall sleep, sleep, sleep for evermore. And so
young Caleb objected to bring up my namesake? By the way, I’m
glad you’ve not shrouded her in black.”
“I knew Bram wouldn’t like it,” Nancy explained.
“I loved that boy,” said the old lady gently. “You made him happy.
And I can do nothing more useful than present his daughter with a
pair of gilded antelopes.” Her sharp voice died away to a sigh of
profound and tragic regret.
Nancy sat silent waiting for the old lady to continue.
“Of course, I could have written and warned you not to ask young
Caleb for anything,” she suddenly began again in her high incisive
voice. “But I wanted to see you. I wanted to see Letizia the second. I
must die soon. So I didn’t attempt to stop your coming. And, as a
matter of fact, you’ve arrived before I could have written to you. No,
don’t hand your child over to young Caleb, girl. Just on sixty-six
years ago my mother handed me over to old Caleb. I suppose she
thought that she was doing the best thing for me. Or it may have
been a kind of jealousy of my young life, who knows? Anyway she
has been dead too long to bother about the reason for what she did.
And at least I owe her French and Italian, so that with books I have
been able to lead a life of my own. Letizia would hear no French or
Italian in this house except from me. And even if I could count on a
few more years of existence, what could I teach that child? Nothing,
but my own cynicism, and that would be worse than nothing. No, you
mustn’t hand her over to young Caleb. That would be in a way as
wrong as what my mother did. Your duty is to educate her. Yes, you
must educate her, girl, you must be sure that she is taught well. She
seems to have personality. Educate her. She must not be stifled by
young Caleb and those two poor crones I brought into this world. It
would be a tragedy. I had another daughter, and I was not strong
enough in those days to secure her happiness. Perhaps I was still
hoping for my own. Perhaps in trying to shake myself free from my
husband I did not fight hard enough for her. She ran away. She went
utterly to the bad. She died of drink in a Paris asylum. Caterina
Fuller! You may read of her in raffish memoirs of the Second Empire
as one of the famous cocottes of the period. If my mother had not
married me to Caleb, I daresay I should have gone to the bad
myself. Or what the world calls bad. But how much worse my own
respectable degradation! It was only after Caterina’s death that I
ceased to lament my prison. It was as if the sentient, active part of
me died with her. Thence onward I lived within myself. I amused
myself by collecting bit by bit over many years the gewgaws by
which you see me surrounded. They represent years of sharp
practice in housekeeping. The only thing for which I may thank God
sincerely is that I wasn’t married to young Caleb. I should never have
succeeded in cheating him out of a penny on the household bills. I
should never have managed to buy a solitary novel, had he been my
accountant. I should have remained for ever what I was when I
married, raw, noisy, impudent, scatterbrained, until I died as a bird
dies, beating its wings against the cage. Educate Letizia, educate
her. I wish I had a little money. I have no means of getting any now. I
had some, but I spent it on myself, every penny of it. Don’t despair
because you’ve not had an engagement since Christmas. It’s only
early March. Mon dieu, I haven’t even a ring that you could pawn.
But I don’t worry about you. I’m convinced you will be all right. Easy
to say, yes. But I say it with belief, and that isn’t so easy. I shall live
on for a few weeks yet, and I know that I shall have good news from
you before I die.”
All the while the old lady had been talking, her face had been
losing its expression of cynicism, and by the time she had finished it
was glowing with the enthusiasm of a girl. It was as if she had beheld
reincarnate in little Letizia her own youth and as if now with the
wisdom of eighty-three years she were redirecting her own future
from the beginning. Presently, after a short silence, she told Nancy to
search in the bottom drawer of a painted cabinet for a parcel
wrapped up in brown paper, and bring it to her. With this she fumbled
for a while with her left hand and at last held up a tunic made
apparently of thick sackcloth and some fragments of stuff that looked
like a handful of cobwebs.
“The silk has faded and perished,” she murmured. “This was once
a pair of blue silk tights. I wore them when I made my descent down
that long rope from the firework platform. It was a very successful
descent, but my life has perished like this costume—all that part of it
which was not fireproof like this asbestos tunic: Take this miserable
heap of material and never let your daughter make such a descent,
however brightly you might plan the fireworks should burn, however
loudly you might hope that the mob would applaud the daring of her
performance, however rich and splendid you might think the costume
chosen for her. Yes, this wretched bundle of what seemed once such
finery represents my life. Wrap it up again and take it out of my sight
for ever, but do you, girl, gaze at it sometimes and remember what
the old woman who once wore it told you a few weeks before she
died.”
There was a tap at the door, and the elderly parlourmaid came in
to say that the fly for which Mr. Fuller had telephoned was waiting at
the door.
“Do you mean to say that Mr. Fuller hasn’t ordered the brougham
to take Mrs. Fuller to the station?” the old lady demanded angrily.
“I think that the horse was tired, ma’am,” said the elderly maid,
retreating as quickly as she could.
“I wish I had my legs. I wish I had both arms,” the old lady
exclaimed, snatching at the small handbell that stood on the table at
the left of the bed, and ringing it impatiently.
Miss Young brought Letizia back.
“Emily, will you drive down with my visitors to the station? I shan’t
need anything for the next hour.”
It was useless for Nancy to protest that she did not want to give all
this trouble. The old lady insisted. And really Nancy was very grateful
for Miss Young’s company, because it would have been dreary on
this cold March night to fade out of Brigham with such a humiliating
lack of importance.
“Good-bye, little Letizia,” said her great-grandmother.
“Good-bye, grannie. I’ve told my auntylopes about my lamb and
about my dog and about all my fings, and they wagged their tails and
would like to meet them very much they saided.”
On the way to the station Miss Young talked about nothing else
except Mrs. Fuller’s wonderful charm and personality.
“Really, I can hardly express what she’s done for me. I first came
to her when she was no longer able to read to herself. I happened to
know a little French, and since I’ve been with her I’ve learnt Italian.
She has been so kind and patient, teaching me. I used to come in
every afternoon at first, but for the last two years I’ve stayed with her
all the time. I’m afraid Mr. Fuller resents my presence. He always
tries to make out that I’m her nurse, which annoys the old lady
dreadfully. She’s been so kind to my little brother too. He comes in
two or three times a week, and sometimes he brings a friend. She
declares she likes the company of schoolboys better than any. She
has talked to me a lot about your husband, Mrs. Fuller. I thought that
she would die herself when she heard he had been killed like that.
And the terrible thing was that she heard the news from Mr. Fuller,
whom, you know, she doesn’t really like at all. He very seldom
comes up to her room, but I happened to be out getting her
something she wanted in Brigham, and I came in just as he had told
her and she was sitting up in bed, shaking her left fist at him, and
cursing him for being alive himself to tell her the news. She was
calling him a miser and a hypocrite and a liar, and I really don’t know
what she didn’t call him. She is a most extraordinary woman. There
doesn’t seem to be anything she does not know. And yet she has
often told me that she taught herself everything. It’s wonderful, isn’t
it? And her room! Of course, it’s very unusual, but, do you know, I
like it tremendously now. It seems to me to be a live room. Every
other room I go into now seems to me quite dead.”
And that was what Nancy was thinking when the dismal train
steamed out of Brigham to take Letizia and herself back to London,
that melancholy March night.
CHAPTER XV
THE TUNNEL
The only other occupant of the railway-carriage was a nun who sat
in the farther corner reading her breviary or some pious book. Letizia
soon fell fast asleep, her head pillowed on her mother’s lap, while
Nancy, watching the flaring chimneys in the darkness without, was
thinking of that old lady who had flared like them in the murk of
Lebanon House. After two hours of monotonous progress Letizia
woke up.
“Muvver,” she said, “I fink I’ve got a funny feeling in my tummy.”
“I expect you’re hungry, pet. You didn’t eat a very good tea.”
“It was such a crumby cake; and when I blowed some of the
crumbs out of my mouf, one of those aunts made a noise like you
make to a gee-gee, and I said, ‘Yes, but I’m not a gee-gee,’ and then
the plate what I was eating went out of the room on a tray.”
“Well, I’ve got a sponge cake for you here.”
Letizia worked her way laboriously through half the cake, and then
gave it up with a sigh.
“Oh, dear, everyfing seems to be all crumbs to-day.”
“Try some of the lemonade. Be careful, darling, not to choke,
because it’s very bubbly.”
Letizia made a wry face over the lemonade.
“It tastes like pins, muvver.”
The nun who overheard this criticism put down her book and said,
with a pleasant smile, that she had a flask of milk which would be
much better for a little girl than lemonade. She had, too, a small
collapsible tumbler, from which it would be easier to drink than from
a bottle.
“Is that a glass, muvver?” Letizia exclaimed. “I was finking it was a
neckalace.”
“Thank Sister very nicely.”
“Is that a sister?” Letizia asked incredulously.
The various relationships to which she had been introduced this
day were too much for Letizia, and this new one seemed to her even
more extraordinary than the collapsible metal tumbler. Nancy
explained to the nun that they had been making a family visit to
hitherto unknown relations in Brigham, to which the nun responded
by saying that she, too, had been making a kind of family visit
inasmuch as she had been staying in Lancashire at the mother
house of the Sisters of the Holy Infancy.
“Right out on the moors. Such a lovely position, though of course
it’s just a little bleak at this time of year.”
She had laid aside her pious book and was evidently glad to talk
for a while to combat the depression that nocturnal journeys
inevitably cast upon travellers in those days before corridors were at
all usual in trains. In those days a railway compartment seemed such
an inadequate shelter from the night that roared past in torrents of
darkness on either side of it. The footwarmers, glad though one was
of them, only made the chilly frost that suffused the upper portion of
the carriage more blighting to the spirit. The dim gaslit stations
through which the train passed, the clangour of the tunnels, the
vertical handle of the door which at any moment, it seemed, might
become horizontal and let it swing open for the night to rush through
and sweep one away into the black annihilation from which the train
was panting to escape, the saga of prohibitions inscribed above the
windows and beneath the rack which gradually assumed a
portentous and quasi-Mosaic significance—all these menacing,
ineluctable impressions were abolished by the introduction of the
corridor with its assurance of life’s continuity.
Nancy told the nun that she was a Catholic, and they talked for a
time on conventional lines about the difficulty of keeping up with
one’s religious duties on tour.
“But I do hope that you will go on trying, my dear,” said the nun.
The young actress felt a little hypocritical in allowing her
companion to presume that until this date she had never
relinquished the struggle. Yet she was not anxious to extend the
conversation into any intimacy of discussion, nor did she want the
nun to feel bound by her profession to remonstrate with her for past
neglect. So instead of saying anything either about the past or the
future, she smiled an assent.
“You mustn’t let me be too inquisitive a travelling companion,” said
the nun, “but I notice that you’re in deep mourning. Have you lost
some one who was very dear to you?”
“My husband.”
The nun leaned over and with an exquisite tenderness laid her
white and delicate hand on Nancy’s knee.
“And you have only this little bright thing left?” she murmured.
Letizia had been regarding the nun’s action with wide-eyed
solemnity. Presently she stood up on the seat and putting her arms
round her mother’s neck, whispered in her ear:
“I fink the lady tied up with a handkie is nice.”
“You have conquered Letizia’s heart,” said Nancy, smiling through
the tears in her eyes.
“I’m very proud to hear it. I should guess that she wasn’t always an
easy conquest.”
“Indeed, no!”
“Letizia?” the nun repeated. “What a nice name to own! Gladness!”
“You know Italian? My husband’s grandmother was Italian. I often
wish that I could speak Italian and teach my small daughter.”
“What is Italian, muvver?” Letizia asked.
“Italian, Letizia,” said the nun, “is the way all the people talk in the
dearest and most beautiful country in the world. Such blue seas, my
dear, such skies of velvet, such oranges and lemons growing on the
trees, such flowers everywhere, such radiant dancing airs, such
warmth and sweetness and light. I lived in Italy long ago, when I was
young.”
Nancy looked up in amazement as the nun stopped speaking, for
her voice sounded fresh and crystalline as a girl’s, her cheeks were
flushed with youth, her eyes were deep and warm and lucent as if
the Southern moon swam face to face with her in the cold March
night roaring past the smoky windows of the carriage. Yet when
Nancy looked again she saw the fine lines in the porcelain-frail face,
and the puckered eyelids, and middle-age in those grave blue eyes.
In Italy, then, was written the history of her youth, and in Italy the
history of her love, for only remembered love could thus have
transformed her for a fleeting instant to what she once was. At that
moment the train entered a tunnel and went clanging on through
such a din of titanic anvils that it was impossible to talk, for which
Nancy was grateful because she did not want Letizia to shatter the
nun’s rapture by asking questions that would show she had not
understood a great deal about Italy or Italian. Presently the noise of
the anvils ceased, and the train began to slow down until at last it
came to a stop in a profound silence which pulsed upon the inner ear
as insistently as a second or two back had clanged those anvils. The
talk of people in the next compartment began to trickle through the
partition, and one knew that such talk was trickling all the length of
the train, and that, though one could not hear the words through all
the length of the train, people were saying to one another that the
signals must be against them. One felt, too, a genuine gratitude to
those active and vigilant signals which were warning the train not to
rush on through that din of anvils to its doom.
And then abruptly the lights went out in every single compartment.
The blackness was absolute. People put up windows and looked out
into the viewless tunnel, until the vapours drove them back within.
Now down the line were heard hoarse shouts and echoes, and the
bobbing light of the guard’s lamp illuminated the sweating roof of the
tunnel as he passed along to interview the engine driver. In a few
minutes he came back, calling out, “Don’t be frightened, ladies and
gentlemen, there’s no danger.” Heads peered out once more into the
mephitic blackness, and the word went along that there had been a
breakdown on the line ahead and that their lighting had by an
unfortunate coincidence broken down as well. Everybody hoped that
the signals behind were as vigilant as those in front and that the red
lamps were burning bright to show that there was danger on the line.
“I aspeck the poor train wanted a rest, muvver,” said Letizia. “I
aspeck it was sleepy because it was out so late.”
“I know somebody else who’s sleepy.”
“P’r’aps a little bit,” Letizia admitted.
“Dear me, she must be tired,” her mother said across the darkness
to the nun. “Well, then, put your head on my lap, old lady, and go
right off to sleep as soon as ever you can.”
For some time the two grown-ups in the compartment sat in
silence while the little girl went to sleep. It was the nun who spoke
first.
“I wonder whether it will disturb her if we talk quietly? But this utter
blackness and silence is really rather dispiriting.”
“Oh, no, Sister, we shan’t disturb her. She’s sound asleep by now.”
“Does she always travel with you when you’re on tour?” the nun
asked.
“Until now she has. You see, my husband only died at Christmas
and we were always together with her. I am a little worried about the
future, because I can’t afford to travel with a nurse and landladies
vary and of course she has to be left in charge of somebody.”
“Yes, I can understand that it must be a great anxiety to you.”
Nancy thought how beautiful the nun’s voice sounded in this
darkness. While the train was moving, she had not realised its
quality, but in the stillness now it stole upon her ears as magically as
running water or as wind in pine-tree tops or as any tranquil and
pervasive sound of nature. In her mind’s eye she was picturing the
nun’s face as it had appeared when she was speaking of Italy, and
she was filled with a desire to confide in her.
“That is really the reason I’ve been to Brigham,” Nancy said. “I
thought that I ought to give my husband’s relations the opportunity of
looking after Letizia. Not because I want to shirk the responsibility,”
she added quickly. “Indeed I would hate to lose her, but I did feel that
she ought to have the chance of being brought up quietly. My own
mother died when I was very young, and my father who is on the
stage allowed me to act a great deal as a child, so that really I didn’t
go to school till I was over twelve, and it wasn’t a very good school,
because I was living in Dublin with an aunt who hadn’t much money.
Indeed I never really learnt anything, and when I was sixteen I went
back to the stage for good. I’m only twenty-four now. I look much
older, I think.”
“I shouldn’t have said that you were more than that,” the nun
replied. “But how terribly sad for you, my dear, to have lost your
husband so young. Many years ago before I became a nun I was
engaged to be married to a young Italian, and he died. That was in
Italy, and that is why I still always think of Italy as the loveliest
country and of Italian as the most beautiful language. But you were
telling me about your relations in Brigham.”
Nancy gave an account of her visit, and particularly of the
interview with Letizia’s great-grandmother.
“I think the old lady was quite right. I cannot imagine that bright
little sleeping creature was intended to be brought up in such
surroundings. Besides, I don’t think it is right to expose a Catholic
child to Protestant influences. Far better that you should keep her
with you.”
“Yes, but suppose I cannot get an engagement? As a matter of
fact, I have only a pound or two left, and the prospect is terrifying
me. I feel that I ought to have gone on acting at Greenwich. But to
act on the very stage on which my husband had died in my arms! I
couldn’t. I simply couldn’t have done it.”
“My dear, nobody would ever dream of thinking that you could. It’s
cruel enough that you should have to act on any stage at such a
time. However, I feel sure that you will soon get an engagement.
Almighty God tries us in so many ways—ways that we often cannot
understand, so that sometimes we are tempted to question His love.
Be sure that He has some mysterious purpose in thus trying you
even more hardly. Nobody is worth anything who cannot rise above
suffering to greatness of heart and mind and soul. Do not think to
yourself that a foolish old nun is just trying to soothe you with the
commonplaces of religious consolation. To be sure, they are
commonplaces that she is uttering, but subtleties avail nothing until
the truth of the great commonplaces has been revealed to the
human soul. Our holy religion is built up on the great commonplaces.
That is why it is so infinitely superior to the subtleties of proud and
eccentric individuals as encouraged by Protestantism. What a long
time we are waiting in this darkness! Yet we know that however long
we have to wait we shall sometime or other get out of this tunnel.”
“Yes, but if we wait much longer,” said Nancy, “I will have another
problem to face when we get to London, for I will never dare arrive
back at my present landlady’s too late.”
“I can solve that problem for you, at any rate,” said the nun. “I shall
be met at Euston by a vehicle, and I know that our guest-room is free
to-night. So don’t let your night’s lodging worry you.”
After this they sat silent in the darkness for a long time. The
presence of the nun filled Nancy with a sense of warm security and
peace of mind. Gradually it seemed to her that this wait in the tunnel
was a perfect expression of the dark pause in her life, which,
beginning with the death of Bram, had ended in her visit to Brigham.
A conviction was born in her brain, a conviction which with every
minute of this immersion in absolute blackness became stronger,
that somehow the presence of the nun was a comforting fact, the
importance of which was not to be measured by her importance
within the little space of the railway-carriage, but that the existence of
this nun was going to influence the whole of her life, which must
soon begin again when the train emerged from the tunnel. The
curtain would rise once more upon the pantomime, and, whatever
the vicissitudes that she as the heroine of it might have to endure,
there would always be a Fairy Queen waiting in the wings to enter
and shake her silver wand against the powers of Evil. It was very
childish and sentimental to be sitting here in the dark dreaming like
this, Nancy kept telling herself; but then once more the mystery of
the tunnel would enfold her as one is enfolded by those strange half-
sleepy clarities of the imagination that flash through the midway of
the night when one lies in bed and hopes that the sense of
illumination that is granted between a sleep and a sleep will return
with daylight to illuminate the active life of the morning. Her thoughts
about the nun reassumed their first portentousness; the comparison
of her own life to a pantomime appeared once more with the
superlative reality of a symbol that might enshrine the whole
meaning of life. Then suddenly the lights went up, and after a few
more minutes the train was on its way again.
Nancy was glad indeed on arriving at Euston toward two o’clock of
a frore and foggy night to drive away with Sister Catherine in the
queer conventual vehicle like a covered-in wagonette with four small
grilled windows. To have argued with Miss Fewkes about her right to
enter the tall thin house in Blackboy Passage at whatever hour she
chose would have been the climax to the Brigham experience.
The Sisters of the Holy Infancy were a small community which was
founded by one of several co-heiresses to a thirteenth-century
barony by writ, dormant for many centuries. Instead of spending her
money on establishing her right to an ancient title Miss Tiphaine de
Cauntelo Edwardson preferred to endow this small community and
be known as Mother Mary Ethelreda. The headquarters of the
community were at Beaumanoir where Sister Catherine, the right-
hand of the now aged foundress, had been visiting her. This was a
Lancashire property which had formerly been held by Miss
Edwardson’s ancestors and repurchased by her when she decided
to enter the religious life. In London the house of the community was
situated in St. John’s Wood where the Sisters were occupied in the
management of an extremely good school. There was a third house
in Eastbourne which was used chiefly as a home for impoverished
maiden ladies.
Sister Catherine was head-mistress of St. Joseph’s School, and it
was there that she took Nancy and Letizia from Euston. The
porteress was overjoyed to see her, having been working herself up
for the last two hours into a panic over the thought of a railway
accident. The white guest-room was very welcome to Nancy after
the fatigue of this long day, so long a day that she could not believe
that it had only been fifteen hours ago that she set out from Euston
to Brigham. She seemed to have lived many lives in the course of it
—Bram’s life as a boy with his brother, old Mrs. Fuller’s eighty years
of existence, Sister Catherine’s bright youth in Italy, and most
wearingly of all, Letizia’s future even to ultimate old age and death.
And when she did fall asleep she was travelling, travelling all the
time through endless unremembered dreams.
In the morning Letizia greatly diverted some of the nuns by her
observations on the image of the Holy Child over the altar, which
was a copy of the famous image of Prague.
“Muvver, who is that little black boy with a crown on His head?”
“That is the baby Jesus, darling.”
“Why is He dressed like that? Is He going out to have tea with one
of His little friends?”
Nancy really did not know how to explain why He was dressed like
that, but hazarded that it was because He was the King of Heaven.
“What has He got in His hand, muvver? What toy has He got?”
“That’s a sceptre, and a thing that kings hold in their hands.”
“Are you quite sure that He is the baby Jesus, muvver?” Letizia
pressed.
“Quite sure, darling.”
“Well, I don’t fink he is. I fink he’s just a little friend of the baby
Jesus, who He likes very much and lets him come into His house
and play with His toys, but I don’t fink that little black boy is the baby
Jesus. No, no, no, no, no!” she decided, with vigorous and repeated
shakes of the head.
Nancy was sorry when they had to leave St. Joseph’s School and
return to Blackboy Passage.
“I fink here’s where the little friend lives,” Letizia announced.
“Oh, darling, you really mustn’t be so terribly ingenious. You quite
frighten me. And what am I going to do about you next week when
the dear Kinos will be gone?”
CHAPTER XVI
BLACKBOY PASSAGE
As Nancy had anticipated, Miss Fewkes was more than doubtful
about her ability to keep an eye on Letizia while her mother was
haunting the offices of theatrical agents.
“I’m not really at all used to children,” she sniffed angrily.
“Supposing if she was to take it into her silly little head to go and
jump out of the window? There’s no knowing what some children
won’t do next. Then of course you’d blame me. I’ve always been
very nervous of children. I could have been married half-a-dozen
times if I hadn’t have dreaded the idea of having children of my own,
knowing how nervous they’d be sure to make me.”
“I wondered if perhaps Louisa might be glad to keep an eye on
her, that is, of course, if you’d let me give her a little present. It
probably won’t be for more than a week.” It certainly wouldn’t, Nancy
thought, at the rate her money was going, for she could not imagine
herself owing a halfpenny to Miss Fewkes. And even that little
present to Louisa, the maid-of-all-work, would necessitate a first visit
to the nearest pawnbroker.
“Louisa has quite enough to do to keep her busy without looking
after the children of my lodgers,” the landlady snapped.
Poor Louisa certainly had, Nancy admitted to herself guiltily, at the
mental vision of the overworked maid toiling up and downstairs all
day at Miss Fewkes’s behest.
“I don’t see why you don’t take her out with you,” said the landlady
acidly.
“Oh, Miss Fewkes, surely you know something of theatrical
agents!” Nancy exclaimed. “How could I possibly drag Letizia round
with me? No, I’ll just leave her in my room. She’ll be perfectly good,

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Mammalian Near-Infrared Image Vision

through Injectable and Self-Powered

Mammalian Near-Infrared Image Vision through

d The nanoparticles are safe and enable NIR light sensation

d This NIR pattern vision is compatible with native daylight

d This method offers options for mammalian vision repair and

Ma et al., 2019, Cell 177, 1–13

Mammalian Near-Infrared Image Vision

*Correspondence: baojin@ustc.edu.cn (J.B.), gang.han@umassmed.edu (G.H.), xuetian@ustc.edu.cn (T.X.)

Cell 177, 1–13, April 4, 2019 ª 2019 Elsevier Inc. 1

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2 Cell 177, 1–13, April 4, 2019

Figure 1. Characterzations of pbUCNPs

Cell 177, 1–13, April 4, 2019 3

Figure 2. Biocompatibility of pbUCNPs

4 Cell 177, 1–13, April 4, 2019

Cell 177, 1–13, April 4, 2019 5

(legend on next page)

6 Cell 177, 1–13, April 4, 2019

Figure 4. NIR Light Sensation of pbUCNP-

Figure 3. NIR Light-Mediated Photoreceptor Activation through pbUCNPs

Cell 177, 1–13, April 4, 2019 7

8 Cell 177, 1–13, April 4, 2019

(legend on next page)

Cell 177, 1–13, April 4, 2019 9

Figure 6. NIR Light Pattern Vision of pbUCNP-Injected Mice

10 Cell 177, 1–13, April 4, 2019

Cell 177, 1–13, April 4, 2019 11

12 Cell 177, 1–13, April 4, 2019

Cell 177, 1–13, April 4, 2019 13

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

e1 Cell 177, 1–13.e1–e6, April 4, 2019

CONTACT FOR REAGENT AND RESOURCE SHARING

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell 177, 1–13.e1–e6, April 4, 2019 e2

Distribution and spectrum analysis

TUNEL Apoptosis detection

Single cell electrophysiology

e3 Cell 177, 1–13.e1–e6, April 4, 2019

Pupillary light reflex

Cell 177, 1–13.e1–e6, April 4, 2019 e4

Light induced fear conditioning

Visually evoked potential

Y shaped water maze

QUANTIFICATION AND STATISTICAL ANALYSIS

e5 Cell 177, 1–13.e1–e6, April 4, 2019

Cell 177, 1–13.e1–e6, April 4, 2019 e6

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