WO2019093895A1

WO2019093895A1 - Label-free microscopy

Info

Publication number: WO2019093895A1
Application number: PCT/NL2018/050753
Authority: WO
Inventors: Andrea CANDELLI
Original assignee: Lumicks Technologies B.V.
Priority date: 2017-11-10
Filing date: 2018-11-12
Publication date: 2019-05-16
Also published as: NL2019891B1; DE112018005412T5

Abstract

A method of imaging at least part of a sample is provided which comprises: focusing at least part of a light beam in a sample plane in the sample, and in particular focusing the part of the light beam at or near the scatterer therein, thus providing unscattered light and scattered light; causing a displacement of at least part of the sample and at least part of the focus with respect to each other; and for plural relative positions of the sample and the focus: collecting the unscattered light and the scattered light with a detection system focused in at least part of the sample, and comprising a position dependent detector; and controlling the detection system to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light in the sample plane, in particular the image data representing at least part of an intensity pattern in the back focal plane of the detection system and/or in an optical conjugate plane of the back focal plane of the detection system. This step may also be described as: detecting with a position dependent detector at least part of the intensity pattern in the back focal plane and/or in an optical conjugate plane of the back focal plane of the detection system. A corresponding system is also provided.

Description

Label-free microscopy

TECHNICAL FIELD

The present disclosure relates to microscopy, in particular microscopy of biological specimens .

BACKGROUND

In microscopy there is a continuous strive towards imaging ever smaller details ever clearer and faster.

Current microscopy methods, in particular microscopy of biological and/or molecular interactions, heavily rely on (fluorescent) labelling strategies, which can be challenging and which potentially even interfere with the molecular interactions under scrutiny. Although (single molecule) fluorescence microscopy can be very powerful due to the high selectivity that can be achieved with labelling techniques there are a number of intrinsic drawbacks associated with it, such as the need for labelling steps, limited signal due to photobleaching , and limited photon flux which limits imaging speed. It is noted that several label free microscopy techniques have been developed previously. Standard bright field microscopy is based on differences in the transmitted light

intensity induced by a sample. The contrast is mostly based on absorption and or scattering of light induced by the sample. However, many specimens, especially biological specimens, do not absorb or scatter much light leading to poor contrast in many cases . Dark field microscopy uses a special illumination system which illuminates a sample with a hollow cone of light in such a way that the unscattered light cannot reach the detector. This leads to contrast based on the scattering properties of the sample. A different technique is Interferometric scattering microscopy "iSCAT" [Ortega-Arroyo et al . , Phys . Chem. Chem. Phys . , 14, 15625

(2012)] . This technique uses light reflected from a microscope coverglass as a reference field in order to detect scattered sample light

interferometrically with great sensitivity. It has been shown to have single-molecule sensitivity. This method however suffers from

irregularities in the coverglass which causes large background signals. Because of the scattering produced by the glass irregularities, for iSCAT imaging, it is required to first to obtain a "background" image of the surface sample prior to adding the biological specimen. In addition, this approach is limited to biological samples in close proximity to the glass surface, severely limiting its application to three-dimensional structures or suspended samples .

Hence, further improvements are desired.

It is noted that for characterisation of optical traps and detection of forces on trapped objects back-focal-plane interferometry- based techniques have been developed: Gittes and Schmidt [Gittes, F. and Schmidt, C. F., Optics Letters, 23, 7 (1998)] developed back-focal-plane interferometry to sensitively detect the displacement of microscopic beads trapped in optical tweezer systems in a nonimaging manner. This was expanded to a general three-dimensional case by Pralle et al . [Pralle, A., et al, Microscopy Research and Technique 44, 378-386 (1999)]. Jahnel et al. [Jahnel, M., et al, Optics Letters, 36, 1260-1262 (2011)] used back- focal-plane interferometry to map the force field of an optical trap. Liu et al . discloses back focal plane interferometry for 3D position tracking in optical tweezers [Liu, H., et al, 2012 Symposium on Photonics and

Optoelectronics, DOI : 10.1109/SOPO .2012.6270909 (2012)]. DeSantis and Cheng [DeSantis, M. C., & Cheng, W. , WIREs Nanomed Nanobiotechnol , 8, 717- 729. (2016)] describe how optical trapping combined with back-focal-plane interferometry can be used to detect individual virus particles and differentiate them from aggregates.

Further optical imaging techniques have also been disclosed, e.g. in WO 2008/092107, and by Roy et al. [Roy B., et al . ,

arXiv: 1201.2357vl [phys ics . optics ] , DOI: 10.1364/OE .20.008317 , (2012)], and by Wallin [Wallin A., Academic Dissertation, Univ. of Helsinki, ISBN: 978-952-1068-80-5 (2011)] .

SUMMARY

Herewith, a method and a system in accordance with the appended claims is provided.

An aspect comprises a method of imaging at least part of a sample, in particular a biological sample comprising a scatterer, e.g. a biological object. The method comprises:

focusing at least part of a light beam in a sample plane in the sample, and in particular focusing the part of the light beam at or near the scatterer therein, thus providing unscattered light and scattered light;

causing a displacement of at least part of the sample and at least part of the focus with respect to each other; and

for plural relative positions of the sample and the focus : collecting the unscattered light and the scattered light with a detection system focused in at least part of the sample, and comprising a position dependent detector;

controlling the detection system to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light in the sample plane, in particular the image data representing at least part of an intensity pattern in the back focal plane of the detection system and/or in an optical conjugate plane of the back focal plane of the detection system. This step may also be described as: detecting with a position dependent detector at least part of the intensity pattern in the back focal plane and/or in an optical conjugate plane of the back focal plane of the detection system.

The method further comprises: constructing an image of at least part of the sample, in particular at least part of the scatterer, on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample and the focus.

Thus, imaging and/or detection of a scatterer is provided based on back-focal-plane interferometry . The imaging is based on capturing image data corresponding to detecting the scattered (&

unscattered) light in the far field. The imaging may not affect the scatterer' s position and/or other properties. Each of the plural relative positions of the sample and the focus produces its own intensity pattern, resultant of interference between the non-scattered and the scattered light, in the back focal plane and/or in an optical conjugate plane of the back focal plane, representative of the interaction of the light beam focus with the portion of sample in which the light beam is focused.

In particular, the focus of the light beam and the focus of the collecting optical system may (be caused to) coincide, providing a confocal arrangement.

The light source may comprise a laser and the light beam may be a laser beam. The light beam may have any suitable beam shape but preferable is a Gaussian beam, which enables a particularly well-defined focus. Displacing the sample (part) and the focus (part) relative to each other in a controlled manner is also referred to as "scanning". The scanning may be 1-, 2- and/or 3-dimensional , scanning directions (i.e. directions of relative displacement) may be selected as desired and preferably be orthogonal to each other, preferably at least one of the scanning directions is perpendicular to the direction of propagation of the light beam; imaging may be based on 1-, 2- or 3 -dimens ional scanning, such as by scanning along a linear path (e.g. along a strand), scanning a plane (which may have any suitable shape and/or orientation in the sample) or scanning a volume, wherein the construction of the image and its possible interpretation may depend on properties of the detector, e.g. enabling position dependent detection in one or two spatial directions (see below) . Information on dynamics of the sample may be derived from consecutive scans of one or more sample portions, which may be depicted as a series of images, an image stack, a kymograph, a graph indicating the time-dependent changes and/or as a time varying image (a movie) . In another option, displacement of at least part of the sample with respect to the sample holder and to the focus may be caused by diffusion of at least part of the sample, which may have natural causes allowed over time, and/or which may be caused by causing flow of a sample fluid within the sample holder.

A scatterer, e.g. a scattering particle located in a focal plane of a light beam, in particular a Gaussian beam, of which the incoming field is defined as Ei, will produce a scattered field Es . It is noted that direct detection of the scattered field Es is possible (e.g. in darkfield microscopy) but this is limited to relatively large structures because of a size dependence of the scattering cross-section r scaling with r⁶. However, present method relies on the notion that when the scattered field Es is allowed to interfere with the unscattered field Eu, the interference term scales with a r³-size dependence instead. This facilitates detection of smaller details.

The scatterer should have an index of refraction that differs from the surrounding medium in the sample for at least a portion of wavelengths of the light of the light beam. The image in particular shows one or more of position, shape and size of the scatterer in the sample. The sample may comprise plural scatterers and/or scatterers comprising different structures and/or differently scattering structures.

Suitable biological objects for acting as a scatterer in a biological sample as discussed herein may comprise or be a cellular body or a substructure thereof, such as any one of cells, proteins, small molecules interacting with proteins, viruses, DNA and RNA molecules, chromosomes, organelles, filaments, a sub-cellular structures, but also tissues, antibody stained tissues, protein-small molecule complexes etc. The detection system, e.g. based on a condenser (or an objective), is positioned after the sample as seen in the direction of propagation of the light beam, to collect at least a portion of the scattered light. The intensity pattern is created by interference in the back focal plane of the optical system, the pattern depending on the relative position between the scatterer, e.g. a refractive portion of a particle, and the focus. Capturing image data, e.g. by detection of this intensity pattern by the position sensitive detector in the back focal plane, or an optical conjugate plane thereof, allows sensitive detection of the scatterer. The image may be constructed by converting the image data associated with each relative position of the sample and the focus into one or more pixel values of the constructed image. By varying the relative positions of at least part of the sample and the focus, e.g.

scanning the positions of the sample and the focus relative to each other, an image can be obtained that is dependent on the scattering properties of the scatterer, in particular on diffraction and refraction (but also absorption) properties of the scatterer and/or any structures thereof and/or therein. As a consequence, the scatterer need not be labelled or otherwise affected.

The light intensity pattern in the back focal plane or the optical conjugate plane thereof, and consequently the intensity

distribution on the position sensitive detector, is proportional to interaction of the light and the scatterer, in particular proportional to the amount of deflection of the light beam induced by the sample. Note that, unless specified otherwise, all references to directions and/or relative position like "lateral" are to be understood as referencing to the direction of propagation of the light beam; generally the direction.

The image data may comprise one or more of the intensity distribution on the position sensitive detector or a fraction thereof, a total intensity on the position sensitive detector, colour information, variation date, time stamps, etc. The image data used for construction of the image may also comprise spatial and/or time averages of detection signals and/or statistical information regarding detection signals, e.g. root-mean-square deflection noise (RMS deflection noise) . The position sensitive detector may comprise e.g. a camera, a diode array, a quadrant photodiode (QPD) , a position sensitive diode (PSD) , or any combination thereof. A detector that is position sensitive in two dimensions, e.g. a camera, a PSD or a QPD enables independent and/or simultaneous detection of the interaction of the scatterer and the light beam projected onto these two dimensions, i.e. it enables quantification of the interaction in X and Y directions independently and allows the total, or absolute, signal to be calculated as S = sqrt(Sx² + Sy²) . Quantification of the signal might be used to obtain certain properties of the sample e.g. scatterer' s size, permittivity of the solvent medium, relative refractive index of the scatterer compared to the medium and incident wavelength of the light. A relatively simple detector such as a QPD or PSD may be faster than a camera, enabling detection of rapid changes in the sample. Such a detector may also have a larger dynamic range enabling measurement of small signals on a large background.

A scatterer in the sample plane, particularly a scatterer slightly above or below the focal plane of the light beam, might cause a symmetric change to the interference pattern in the back-focal plane (i.e. the chief ray of the scattered beam might not be deflected in the lateral direction but the marginal rays of the light beam might be deflected to cause a divergence or convergence of the scattered light beam) . Detection of this deflection may also be implemented for providing information for the imaging step. As an example, the total intensity of the transmitted light within a certain region in the back focal plane of the optical system may be monitored, e.g. using an aperture to restrict the range of acceptance angles of the detection system.

The image may be constructed as an array of pixels. Each pixel or groups of pixels may correspond to one relative position of the sample and the focus. Also or alternatively, each pixel or groups of pixels may correspond to a plurality of relative positions of the sample and the focus. This facilitates scaling the image.

The method may further comprise recording the image data, e.g. detected intensity patterns, as a function of the relative positions of the sample and the focus and constructing an image of the at least part of the sample on the basis of the recorded image data. The image data may be stored in a transient or permanent memory and/or be transmitted through the internet to a remote controller or computer. Detection and image construction may therefore be done separately.

The resolution may depend on the relative sizes of the focus and scatterer or, respectively any structure (s) of the scatterer to be studied. This is particularly interesting for biological samples which may comprise scattering particles varying in one or more of sizes, shapes and internal structures . Preferably, the light beam focus is smaller than the scatterer; this may facilitate resolving details of the scatterer smaller than the scatterer itself (i.e. sub-scatterers ) . In each case, dependent also on the detector used, the image data may be used for constructing the image per direction independently and/or in combination, e.g. the image being based on signals Sx, Sy, Sz (signals in X-, Y- and Z-directions independently) and/or as an absolute value Sabs (Sabs = sqrt{Sx² + Sy² + Sz²}), wherein "S" indicates a signal strength which may correspond to or may be proportional to the amount of beam deflection. Note that a two- dimensional position dependent detector may be formed by a combination of two one-dimensional position dependent detectors oriented (to detect) at an angle to each other, in particular perpendicular to each other.

However, more complex detectors, including cameras, may also be used, wherein averaging over detector data (e.g. averaging over parts of the camera image) may be used to define part of the image data.

The image may be constructed from a combination of image data associated with one or more individual directions, e.g. corresponding to amounts of scattering in one or more directions (e.g.: Sx, Sy) and/or to an absolute value thereof (e.g.: Sabs) . Suitably, the image size in one or more individual directions may cover at least about 5 times a Full Width at Half Maximum (FWHM) of the light beam focus size in that direction; note that in a Gaussian beam focus the focus will generally be symmetric in directions perpendicular to the beam propagation direction of the beam. E.g. the image size may represent more than about 5 times, preferably at least 10 times, or even 20 times the FWHM of the beam in two perpendicular directions; the larger the image size, the more structure and/or

morphology information may be obtained. An image size of a few micrometer e.g. about 3 micrometers per direction may suffice for imaging a

chromosome or a bacterium, an image size of several tens of micrometers may enable imaging an entire cell or even a multicellular object.

At least part of the image may be rendered in a brightness scale and/or an essentially single-colour-scale, e.g. a grey scale wherein degrees of brightness may correspond to amounts of beam deflection in one or more directions and/or to an absolute value thereof (e.g. Sx, Sy, Sabs as discussed above) . Instead of a grey scale any other sequential colormap may be used, where sequential means that the perceived lightness value increases or decreases monotonically through the colormap, e.g. a colour temperature scale (also known as thermal red scale) ranging from purplish red via bright red, orange and yellow to white. In such scale, images will exhibit the well-known shadow effect which gives standard DIC (Nomarsky phase microscopy) images their three-dimensional appearance. The position dependent detector may be a two-dimensional position dependent detector, in particular a detector capable of detecting two perpendicular directions simultaneously, conveniently called X- and Y- directions. Then, both X and Y beam deflection data may be simultaneously available as (part of) the image data. In such case different linear combinations of the X and Y beam deflection data may be used for construction of the image, in particular for generating pixel values, and rendering the image with a shadow effect oriented in any chosen direction. This may increase a (perceived) resolution or contrast of the image. This is an advantage over known techniques, e.g. in DIC microscopy one needs to physically rotate the

Wollaston prism which determines the shearing direction in order to choose the orientation of the shadow effect, i.e. one needs to physically manipulate (the beam line of) the optical setup itself.

The output of a quadrant photo diode ("QPD") and/or a position sensitive diode ("PSD") may not only be a difference signal in

perpendicular directions, but also a sum signal proportional to the total intensity detected on the diode. The former may relate to the lateral change in propagation/deflection of the light. The latter may correspond to the total intensity of the transmitted beam or reflected beam

(dependent on whether the method is performed in-line or reflectively, see below) . In such case, simultaneously with the deflection based contrast also an intensity based contrast image may be constructed. The method therefore also gives access to absorption/extinction parameters of the sample. In addition, the simultaneous measurement of both the deflection and the intensity of the transmitted beam allows to correct for artefacts such as caused by sudden laser emission intensity variations, e.g. by normalizing the deflection signal by the total intensity signal.

The method may be performed in an in-line arrangement. For that, the method may comprise arranging the light source on one side of the sample and the detection system on a second side of the sample, in particular the first and second sides being opposite each other, such that at least part of the light beam traverses the sample from the first side to the second side before reaching the detection system and the detector. Such method may further comprise focusing at least part of a light beam in the sample from the first side and collecting the unscattered light and the scattered light on the second side of the sample and controlling the detection system to capture image data representing at least part of the intensity pattern resulting from the collected light, as above. Thus, the method is based on forward-scattered light, or rather on interference of forward-scattered light with unscattered transmitted light.

Also or alternatively, the method may be performed in a reflection arrangement. For that, the method may comprise arranging the light source and the detection system on one side of the sample, and arranging a reflector for at least part of the light beam, such that at least part of the light beam traverses at least part of the sample from a first side and returns to the first side before reaching the detection system and the detector. Then, the method may further comprise focusing at least part of a light beam in the sample, from the first side and collecting the unscattered light and the scattered light on the first side of the sample and controlling the detection system to capture image data representing at least part of the intensity pattern resulting from the collected light, as above. Thus, the method is based on backward-scattered light, or rather interference of backward-scattered light with unscattered reflected light. The reflected light may be reflected from at least one of a portion of the sample, a portion of a sample holder and/or a separate reflector .

Also or alternatively, the method may be performed in a ci rcumventional arrangement, wherein the unscattered light is light not having traversed and/or otherwise interacted with the sample at all.

Spatial filtering of the scattered and/or the unscattered light in the optical system and before the detector, may be employed, such that at least part of the scattered and/or of the unscattered light passes through a spatial filter prior to reaching the detector.

For accurate beam deflection and absorption measurements it is advantageous to collect as much as possible of the scattered light cone for which one may use a condenser with a numerical aperture larger than the refractive index of the sample medium. This particularly applies to forward-scattered light in an in-line arrangement. In particular for detection of symmetric changes to the back focal plane interference pattern it may be advantageous to restrict the acceptance angle of the detected light cone. Therefore an iris may be arranged in the back focal plane of the condenser, which may be adjustable. An iris may also be otherwise employed for spatial filtering. Also, or alternatively, a dual- detection system may be provided and used, wherein a portion of the light cone, preferably substantially the full light cone, is collected by the detection system, e.g. a condenser, after which one part of the collected light is detected by the position sensitive detector, e.g. for lateral deflection determination and/or for absorption measurement and/or for capturing image data representative thereof, to construct the image, while a second detector may be provided and used for measuring another portion of the beam selected by a spatial filter for detecting changes to the collimation of the scattered beam and/or for capturing image data

representative thereof.

The method may comprise trapping at least one object in the sample, in particular optically trapping, wherein the object comprises the scatterer or the scatterer interacts with at least one of the objects, e.g. being attached to an object. The interaction may comprise one or more of being attached to the object, moving with respect to the object, reacting with the object in a chemically and/or a biological and/or a physical sense, etc. Thus, at least one of the position and orientation of the scatterer may be controlled and/or adjusted in the sample. This may facilitate studying the scatterer. The object may be a microsphere and the scatterer may be a biological object, e.g. a cellular body, a filament, a macromolecule etc. Optical trapping may obviate (presence of) attachment structures for holding the scatterer, which might otherwise affect the scatterer, and/or it may support the object free from (i.e. not in contact with) a solid substrate. E.g., this might avoid unwanted contributions of the sample holder to the signal and it might decouple the sample from unwanted motions (vibrations or drift) of the sample holder. Thus, image resolution and stability may be improved.

The method may comprise trapping, in particular optically trapping, plural objects attached to each other by at least one connecting element, wherein at least one of the objects and/or the connecting element comprises the scatterer, and/or wherein the scatterer interacts with at least one of the objects and/or the connecting element(s), e.g. being attached to an object or to the connecting element (s) . In particular the objects may be microspheres and the connecting element (s) may comprise a filament, a microtubule, a DNA-strand, etc.

In case of optical trapping of at least one object in the sample with one or more optical trapping beams, the light beam may differ from at least one of the optical trapping beams in at least one intensity, wavelength and polarization. Thus, interaction between the trapping beam(s) and the (detection) light beam may be prevented and/or the different beams may be separately controlled by wavelength-specific and/or polarization-specific optics. The method is flexible and may comprise modifying one or more of: the focus size of the light beam, the intensity of the light beam and/or the wavelength of the light beam, as well as - in the case of trapping - modifying one or more of: the focus size of a trapping light beam, the intensity of a trapping light beam and/or the wavelength of a trapping light beam. The modification may be done within one image and/or between different images and it may be controlled by a controller. The modification allows detection of different image details and/or image data capturing with different scanning settings. In a particular embodiment, the light beam may serve as a trapping beam. The described modification facilitates switching between both functions, e.g. by dithering power and/or wavelength.

The method may comprise that at least one of the light beam is polarized, in particular linearly polarized. Also, (the part of) the intensity pattern of the scattered and unscattered light represented in the image data may be detected through at least one polarization dependent optical element, such as a polarizer, a Polarizing Beam Splitter Cube ("PBSC") , a Wollaston prism, etc. comprised in the collecting optical system. The light may be split in different fractions according to multiple polarizations and each split fraction may be detected separately on a position dependent detector and image data representing one or several of the fractions may provide information on polarization dependent characteristics of the sample, e.g. polarization altering characteristics of (at least part of) the sample. One or more of the polarization of the light beam and the at least one polarization dependent optical element may be adjustable with respect to polarisation directions, which may be controller operable; the light beam may be sent through a polarisation changing element such as a quarter wave and/or a half wave plate.

The method may comprise providing at least part of the sample with an optically effective label, possibly comprising optically

activating or de-activating the label. Although they are not required with the presently provided techniques, labeling and associated techniques may be exploited: e.g. it might be advantageous to scan for example biological samples such as cells, tissues, biomolecules which have been (partly) fluorescently labelled and to simultaneously detect fluorescence thereof. The fluorescence may be excited or de-excited (e.g. quenched, bleached, etc.) by the scanning beam. In particular in the latter case the

fluorescence might label specific structures of interest in the sample (the principal stain) while the scattering contrast may act as the counterstain for providig a composite image with more context than the primary stain alone.

In accordance with the method a system is provided herewith. The system comprises: a sample holder to hold a biological sample, a light source providing a light beam, and, operably arranged along an optical path of at least part of the light beam: a source optical system, which may comprise one or more optical elements, in particular a focusing lens and/or an objective, and which is arranged to focus at least part of the light beam in a sample held in the sample holder; a detection system comprising a position dependent detector, e.g. one or more of a split photodiode, a quadrant photodiode, a photodiode array, a camera, a position-sensitive photodiode. The detection optical system, e.g.

comprising one or more optical elements, in particular a condenser lens, provides a back focal plane and is arranged to collect at least part of the light beam comprising both light not scattered by the sample, i.e. unscattered light, and light scattered by at least one scatterer in the sample, i.e. scattered light, and to provide from them an intensity pattern in the back focal plane .

The detection system is arranged to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light in the sample plane, in particular the image data representing at least part of an intensity pattern in the back focal plane of the detection system and/or in an optical conjugate plane of the back focal plane of the detection system. E.g. The position dependent detector is arranged to detect at least part of the intensity pattern in the back focal plane and/or in an optical conjugate plane of the back focal plane.

At least part of at least one of the sample holder, the light source and the source optical system is adjustable to controllably displace the focus of the light beam and at least part of the sample relative to each other, e.g. being connected to a position controller which the system may comprise. The system further comprises a controller connected with the position dependent detector and programmed to construct an image of at least part of the sample, in particular at least part of the scatterer, on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample and the focus. The relative positions may result from a 1-, 2- or 3- dimensional scan of at least part of the sample. The image may be 1-, 2- or 3- dimensional and it may be rendered in a sequential colour scale. The source optical system and the detection system preferably are configured in a confocal arrangement.

The system may comprise a spatial filtering system, e.g.

comprising a pinhole and/or an iris, which may be adjustable with respect to position and/or aperture, for spatial filtering detection light between the collecting optics (condenser, objective, ...) of the detection system and the position dependent detector. The spatial filtering system may be connected with a controller. The spatial filtering system may further comprise relay optics.

The system may comprise a trapping arrangement to trap and/or hold one or more objects in the sample. In particular, an optical trapping arrangement may be provided. A multiple trapping arrangement to trap and/or hold one or more objects in the sample in multiple traps may be preferred. An optical trapping arrangement may comprise one or more lights sources, e.g. lasers, focusing optics and detection optics arranged to provide one or more optical trapping beams in the sample.

Further, in accordance with the principles disclosed herein, an optical detection module is provided to be placed in an optical train of a sample or beam scanning microscope comprising a sample holder to hold a biological sample, a light source providing a light beam, and, operably arranged along an optical path of at least part of the light beam, a source optical system arranged to focus at least part of the light beam in a sample held in the sample holder, and wherein at least part of at least one of the sample holder, the light source and the source optical system is adjustable to controllably displace the focus of the light beam and at least part of the sample relative to each other, e.g. being connected to a position controller. The detection module comprises:

a detection optical system comprising a position dependent detector, e.g. one or more of a split photodiode, a quadrant photodiode, a photodiode array, a camera, a position-sensitive photodiode;

wherein the detection system provides a back focal plane and is arranged to collect at least part of the light beam comprising both light not scattered by the sample, i.e. unscattered light, and light scattered by at least one scatterer in the sample, i.e. scattered light, and to provide from them an intensity pattern in the back focal plane;

wherein the detection system is arranged to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light in the sample plane, in particular the image data representing at least part of an intensity pattern in the back focal plane of the detection system and/or in an optical conjugate plane of the back focal plane of the detection system;

the detection system further comprising a controller connected with the position dependent detector and programmed to construct an image of at least part of the sample, in particular at least part of the scatterer, on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample and the focus .

Another aspect is a method of imaging at least part of a sample, in particular a biological sample comprising a scatterer, the method comprising:

controlling a source optical system to focus at least part of a light beam in the sample and in particular at or near the scatterer therein, thus providing unscattered light and scattered light, which form an intensity pattern in a back focal plane of the source optical system;

controlling at least one of the source optical system and a sample positioning system to position the focus and the sample at a plurality of different positions with respect to each other,

for plural relative positions of the sample and the focus, controlling a detection system to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light in the sample plane, in particular the image data representing at least part of an intensity pattern in the back focal plane of a detection system and/or in an optical conjugate plane of the back focal plane; and

constructing an image of at least part of the sample on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample and the focus.

Further, a computer-implemented method for imaging at least part of a sample, in particular a biological sample comprising a

scatterer, is provided, the method comprising:

controlling a source optical system to focus at least part of a light beam in the sample and in particular at or near the scatterer therein, thus providing unscattered light and scattered light;

controlling at least one of the source optical system and a sample holder to displace at least part of the sample and at least part of the focus with respect to each other for achieving plural relative positions of the sample and the focus; at each relative position of said plural relative positions, controlling a detection system to detect at least part of an intensity pattern, e.g. an interference pattern, caused by the unscattered light and the scattered light combining;

constructing an image of the at least part of the sample on the basis of the detected interference intensity patterns respectively associated with the plural relative positions.

One distinct aspect of this disclosure relates to a controller comprising a processor that is configured to execute one or more of the steps of the computer-implemented methods as described herein.

One distinct aspect of this disclosure relates to a computer program comprising instructions to cause a controller as described herein to carry out one or more of the steps of the computer-implemented methods as described herein.

One distinct aspect of this disclosure relates to a computer- readable medium comprising a computer program as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-described aspects will hereafter be more explained with further details and benefits with reference to the drawings showing a number of embodiments by way of example.

Fig. 1A illustrates an optical system 2 according to an embodiment;

Fig. IB illustrates a method for imaging at least part of a sample according to one embodiment;

Fig. 2 is an embodiment of a system for label free imaging using back-focal-plane interferometry; Fig. 2A shows a typical deflection signal of the system of Fig. 2 as a light beam is scanned over a small object in the sample;

Fig. 3 is an embodiment of another in-line arrangement using sample scanning;

Fig. 4 is a detail of an embodiment of imaging with a dual beam optical tweezer system;

Fig. 5 is an embodiment of a reflection arrangement, including optional confocal fluorescence detection;

Fig. 6 shows an exemplary embodiment for de-scanned detection in transmission geometry; Fig. 6A shows a typical deflection signal of the system of Fig. 6 as a light beam is scanned over a small object in the samp1e ;

Fig. 7-10 are exemplary images formed in accordance with the principles presently disclosed;

Fig. 11 shows a typical intensity pattern as used in accordance with the principles presently disclosed.

DETAILED DESCRIPTION OF EMBODIMENTS

It is noted that the drawings are schematic, not necessarily to scale and that details that are not required for understanding the present invention may have been omitted. The terms "upward", "downward", "below", "above", and the like relate to the embodiments as oriented in the drawings, unless otherwise specified. Further, elements that are at least substantially identical or that perform an at least substantially identical function are denoted by the same numeral, where helpful individualised with alphabetic suffixes.

Further, unless clearly otherwise indicated, terms like

"detachable" and "removably connected" are intended to mean that

respective parts may be disconnected essentially without destruction of either part, e.g. excluding structures in which the parts are integral (e.g. welded or molded as one piece) , but including structures in which parts are attached by or as mated connectors, fasteners, releasable self- fastening features, etc.

Figure 1A illustrates an optical system 2 according to an embodiment. The system 2 comprises a sample holder 6 to hold a sample 70, e.g. a biological sample, comprising a scatterer (not shown) . System 2 further comprises a source optical system 4 that is configured to focus at least part of a light beam 12 in the sample 70 and in particular at or near the scatterer therein, thus providing unscattered light 16 and scattered light 14. Optionally, the source optical system 4 comprises a light source for providing the light beam 12, such as a laser. As indicated by the arrows x, y, z, at least part of the sample 70 and the focus of light beam 12 can be displaced with respect to each other.

Herewith plural relative positions of the sample 70 and the focus can be achieved. In one example, the source optical system 4 is configured to move the focus with respect to the sample 70. Additionally or

alternatively, the sample holder 6 may be configured to move the focus with respect to the sample. The unscattered light 16 and the scattered light 14 combine and cause an intensity pattern, e.g. an interference pattern. The optical system 2 further comprises a detection system 8 to detect at least part of this intensity pattern. The detection system 8 comprises a position dependent detector, e.g. one or more of a split photodiode, a quadrant photodiode, a photodiode array, a camera, a position-sensitive photodiode. The detection system 8 may provide a back- focal plane and may be arranged to collect at least part of the scattered light 14 and unscattered light 16 in this back focal plane and/or in an optical conjugate plane of the back-focal plane. System 2 further

comprises a controller 120 that is configured to control the source optical system 4 and the detection system 8 and optionally the sample holder 6 to perform their respective functions as described herein.

Figure IB illustrates a method for imaging at least part of a sample according to one embodiment. This method may be implemented to control at least one of the source optical system 4, the sample holder 6 and the detection system 8.

In step S2, the embodiment comprises controlling a source optical system 4 to focus at least part of a light beam 12 in the sample 70 and in particular at or near the scatterer therein, thus providing unscattered light 16 and scattered light 14. Controlling the source optical system 4 to focus at least part of light beam 12 in the sample may consist of controlling a light source, such as a laser, to generate a light beam 12, which light beam 12 passes through passive elements, such as lenses prisms, mirrors, filters et cetera.

In step S4, the embodiment comprises controlling at least one of the source optical system 4 and a sample holder 6 to cause displacement of at least part of the sample 70 and at least part of the focus with respect to each other for achieving a relative position of the sample 70 and the focus. In order to achieve this position, the source optical system may be controlled, which may comprise controlling an orientation of a mirror in the source optical system 4 for directing the light beam 12. Alternatively or additionally, the plural positions may be achieved by controlling the sample holder 70, which may comprise controlling an orientation and/or position of the sample holder 70.

Step S6 comprises controlling the detection system 8 to detect at least part of an intensity pattern, e.g. an interference pattern, caused by the unscattered light 16 and the scattered light 14 combining. In an example, the detection system 8 comprises a position-dependent light detector, such as an imaging system, that comprises a plurality of pixels. Each pixel may be configured to output a light intensity value that is indicative of the light intensity incident on the pixel, and/or indicative of other image data such as RMS deflection noise. Thus, a plurality of pixels may output light intensity values that are indicative of a light intensity pattern. A pixel for example outputs a light intensity value in the form of a voltage signal. The pixels may continuously output

respective light intensity values, that may vary with time. The position- dependent detector may further comprise an image data capture module, that may be embodied as a software module in a computer. The image data capture module may continuously receive the light intensity values from the pixels of the position-dependent detector. It should be appreciated that controlling the detection system 8 to detect at least part of the intensity pattern may consist of transmitting an instruction to the image data capture module to store the light intensity values that it is currently receiving from the respective pixels. Herewith, the image data capture module captures the light intensity values and may thus capture the intensity pattern as image data.

Steps S4 and S6 are repeated at least once, so that at least two intensity patterns are at least partially detected for two respective relative positions of the sample and the focus. However, steps S4 and S6 may be repeated numerous times.

Step S8 comprises constructing an image of the at least part of the sample on the basis of the detected image data, e.g. the detected intensity patterns, respectively associated with the plural relative positions. After steps S4 and S6 have been repeated a number of times for a plurality of relative positions, a plurality of intensity patterns have been detected by the detection system 8, wherein each intensity pattern is associated with a relative position of the sample and focus. Step S8 may comprise, for each detected intensity pattern, determining an image pixel value, for example a greyscale value, for an image pixel in the to be constructed image. Step S8 may further comprise constructing the image based on the determined image pixel values and their associated relative pos itions .

Figure 2 also shows an exemplary embodiment of a system arranged for performing at least one embodiment of the method disclosed herein, the system having a source optical system 4. Fig. 2 shows a light source 10 projecting a light beam 20, which might be a laser beam, onto a scanning device 30, here being controlled by an optional controller in the form of a central processing unit (CPU) . The scanning device can for example be a tip/tilt mirror or an acousto/electronic optical deflection sys tern .

The beam 20 is relayed using telescope lenses 40, 50 to the back-focal-plane of a microscope objective 60.

The microscope objective 60 focusses the light onto a sample held in a sample holder 70. The sample, which might be a biological sample, can be scanned by the focused beam by means of the scanning device. A condenser lens 80 or similar optical system is used together with a relay optical system (e.g. a single lens 90) of a detection system 8 to project the light beam that has passed through the sample onto a position sensitive detector 100 which can for example be a quadrant photodiode (QPD) , a position sensitive diode (PSD) or a camera of the detection system 8, positioned in a conjugate of the back-focal plane of the condenser. The signals from the position sensitive detector are optionally amplified and combined in an electronic circuit 110 and are sent to the CPU 120 or other processing unit. An optional beam splitter 130 can be used to send part of the light beam that has passed through the sample to a spatial filter 140, positioned in a conjugate of the back- focal plane of the condenser, that can be used to select only a part of the beam. A detector 150, e.g. a photodiode, can be used to detect e.g. changes in collimation of the scattered which correlate to axial

displacements of the scatterer. Based on the detector signals and the current position of the scanner an image can be constructed by the CPU.

The light source 10 can be a laser and the light beam 20 can be a laser beam. However, other light sources and light beams may be provided. The sample can be a biological sample, comprising scatterers such as a cell or sub-cellular structure, a filament (e.g. actin, microtubule), a protein on the surface of a substrate (e.g. a microscope cover glass) or a structure suspended in an optical trap, e.g. a dual optical tweezer setup. The sample can furthermore comprise any scatterer with topological features or variation in refractive index.

The image contrast of the image to be constructed is predominantly based on deflection of the light beam caused by interaction with (the scatterer in) the sample. Any object in the sample, in

particular a sample plane in which the light beam focus is located, having a refractive index (polarizability) that differs from a refractive index of a medium surrounding the object will cause a deflection of (part of) the beam, hence the name "scatterer". This deflection can be measured, e.g. by monitoring the difference signal (Vx and/or Vy) of a quadrant photodiode, wherein the measurement results provide image data. It has been shown that this deflection can be detected directly, e.g. in

darkfield microscopy, but this has the disadvantage that the scattered intensity scales with r⁶ (a radius of the scattering feature raised to the 6th power) which makes it hard to detect small objects such as e.g. single proteins or small filaments, or structures thereof and changes in size and or mass of the biological object under study due to proteins, small molecules or other entities, binding to the initial biological structure of interest. This binding can be either dynamic or static in nature and can be detected as a variation the deflection signal.

Taking advantage of interference between the deflected / scattered and the unscattered part of the light beam it has now been found that one can directly detect the field amplitude instead of the power amplitude of the scattered light. This reduces the scaling factor to r³, making it much easier to image or detect small structures and/or objects such as single proteins or protein complexes.

The deflection provides a varying interference resulting in a difference signal of the QPD; as the beam is scanned over a scatterer the light detected on the QPD will first deflect to one side and then to the other. This is illustrated in Fig. 2A for a single and smooth scattering structure, e.g. a microsphere or a protein smaller than the beam focus.

Scanning an object whose optical properties such as position, size and/or mass change over time will result in a varying amount of deflected light and hence of varying signal.

Scanning an object with different scattering structures will result in a more complex signal shape. Scanning the beam in two dimensions allows the build-up of a 2D image of the scatterer.

Due to aberrations and imperfections in the optical path a scanning beam approach as illustrated in this exemplary embodiment might lead to a non-zero and/or structured background image, on top of which it is hard to detect small signals of a scattering object in the sample. This may be resolved by careful subtraction of a background image. Such a background subtraction can for example be achieved by scanning the image multiple times where at least a portion of the sample, e.g. the scatterer, is moved by a known amount between the consecutive images e.g. using a sample stage or by moving optical traps . Subtracting such consecutive images may lead to a background free image, possibly with two displaced copies of the sample. If the sample is larger than the displacement, postprocessing might be useful to recover a single-copy image. Another method for background subtraction is to take advantage of any dynamics that might be present in the sample: for samples which are changing over time it is possible to achieve high quality subtraction of a (static) background by subtracting an average over many images from one or more individual images .

By repeatedly scanning 2-dimensional images of the sample while the sample and focus are displaced with respect to each other along the direction of beam propagation (the z-direction) so that the light beam is focused in different layers of the sample is it is possible to construct a 3 -dimens ional image.

It is advantageous to use a condenser with a numerical aperture ("NA") higher than the index of refraction of the sample medium in order to allow optimal capturing of the scattered light.

Multi-beam scanning can be done to improve imaging speed and/or accuracy. For this, it is preferred to collect and detect the deflection of multiple beams simultaneously. At least some of the multiple beams may differ in one or more optical characteristic such as

polarization state (e.g. different linear polarization directions), wavelength, wavelength modulation, intensity modulation, etc. which is detectable by the detection system by suitable (combinations of)

techniques such as polarisation separation, wavelength selective filtering and/or absorption, demodulation techniques, etc.

According to an alternative exemplary embodiment it is possible to scan the sample stage instead of (a focus of) the light beam. This might have the advantage that in this case there is less background signal caused by aberrations in the optical system. Figure 3 shows a scheme of such an embodiment. However, scanning both at least part of the sample and at least part of the light beam is also possible. Fig. 3 shows a light source 10 of a source optical system 4 projecting a beam of light 20 into the back-focal-plane of a microscope objective 60. The objective 60 focusses the light into a sample 70 which is mounted onto a sample holder. The sample holder can be scanned in one or more directions (here: three mutually perpendicular directions X, Y and Z) , preferably two directions that are parallel to a sample plane. The scanning can be controlled by a controller, e.g. via signals provided by a central processing unit (CPU) 120 shown here. From the sample, when the light beam is at least partly scattered, both the unscattered light beam 20 and the scattered light beam 25 are collected by the condenser 80. Via a relay lens 90 of the detection system 8 the back focal plane of the condenser 80 is imaged onto a position sensitive detector 100. Signals from the position sensitive detector 100 are amplified and combined, as indicated at reference number 110, and sent to the CPU 120. The CPU 120 uses information on the position of the sample holder 70 and the signals from the detector 100 as image data to construct an image of at least part of the sample on the basis of the image data and the relative positions of the sample holder and the focus .

Such system may be less susceptible to spurious background signals caused by aberrations and optical imperfections. Optionally one could also add a pinhole or other spatial filter 85 in the focal point of the optical relay system to the position sensitive detector. Spatial filtering may reject unwanted background light scattered from different portions of the sample, e.g. different focal planes. Further,

reconstruction of three dimensional datasets of scattering contrast may be facilitated and/or enhanced by scanning the sample stage in a direction along the direction of light beam propagation (here: the Z-direction) in addition to one or more lateral directions (here: X- and Y- directions) .

Part of another embodiment is illustrated in Figure 4. An object, e.g. a DNA molecule 230 with bound proteins 240, is tethered between two beads 210 held in a sample medium (220) in trapping beams 200 of a dual optical trap, known per se. A light beam 250 is scanned along the DNA molecule 230. The molecule 230 and the proteins 240 each scatter the light beam 250 to some extent, dependent on their optical properties relative to the surrounding sample medium. At least part of the scattered light and unscattered light are collected and detected as generally indicated before. Thus, one or more images of (part of) the DNA molecule 230 and/or the proteins 240 may be constructed based on the image data representative of the back-focal-plane interferometry signal of the light beam. In such case effective background subtraction can be done e.g. by (slightly) moving the optical traps between consecutive images and subtracting the images .

As an ezample, the light beam 250 may serve as an additional trapping beam, and/or one of the trapping beams 200 may be used as an imaging beam. The switching between both functions (imaging and trapping) of such beam may be done by dithering power and/or wavelength of the respective beam. Also or alternatively, focus positions may be rapidly switched. Several of such methods may be combined. E.g., a trapping beam focus may be suddenly moved from a trapping position to another, non- trapping, position so rapidly that the trapped object cannot follow the movement and is effectively released from the trap. At the new, non- trapping, position, image data can be taken at a single relative position (single pixel image data-image) or at different relative positions of the sample and the focus (multi-pixel image data by stepwise scanning or continuous scanning) . Thereafter, the light beam may quickly return to a position at or near the trapping position for re-trapping the previously trapped object to continue to trap and/or manipulate the object. The effective trapping force may then scale with a duty cycle defined by the ratio of trapping duration and imaging duration per repetition. This may be repeated for the same or different parts of the sample. The image data thus captured for different relative positions of the sample and the focus can be used for constructing the image of at least part of the sample.

This allows imaging of at least part of the sample, which may comprise one or more of the trapped object (s), another scatterer and an optional connecting element, without the need for (a system providing) an extra light beam in addition to the trapping beam.

Using the trapping beam as the light beam for imaging may also be facilitated by rapidly lowering the power of the trapping beam to facilitate release of the trapped object before scanning the image.

Similarly the power of the light beam may be rapidly increased after the light beam returns to the previously trapped object in order to continue to trap and/or manipulate the object. The switching of powers and/or focus positions of one or more beams may be done very rapidly, for example using acousto- or electro-optic modulators which allow interleaving of trapping and image scanning functions in a time shared manner. Thus, the same beam can be used for trapping/manipulation and for imaging in a sequential and/or an interleaved fashion in any order.

According to yet another embodiment, indicated in Figure 5, instead of detection of scattered light and unscattered light in the forward direction (i.e. in transmission through the sample) back-scattered light may be used. In this case the light beam 20 generated by the light source 10 travels through a beam splitter 300 and travels to the sample 70 via an optional beam scanner 30, an optional relay system, which here is indicated as two lenses 40, 50, and an objective 60 which focuses the light beam 20 into the sample 70. The sample 70 is mounted on a sample holder, here comprising a coverslip 340 that is at least partly

transparent to the wavelength of the light beam 20. The sample 70 and/or the sample holder 340 holding the sample 70 may at least partly be movable, preferably controllably movable as discussed above, e.g. controlled by a controller. At or near the sample 70 at least part of the light beam 20 is reflected. E.g., a substrate-sample transition in the coverslip 340 may reflect part of the light from the light beam prior to a remaining part of the light beam having interacted with the actual sample . The reflected light can be used as the reference field for interferometric detection. Both the light back-scattered from the sample and the reflected reference light are collected with the objective 60, then serving as detection optical system and providing the back focal plane. Note that, in this embodiment, the illumination light, the sample and the detection light are, automatically, in a confocal arrangement and the source optical system 4 and the detection system 8 share a significant number of optical elements (300, 320, 30, 40, 50, 60) . The detection light travels back through the objective 60 and the relay system 50, 40, and via the scanning mirror 30 to the beam splitter 300 where at least a part of the light is reflected and sent to a position sensitive detector 100 via a further optional relay 90 and an optional pinhole 85 or iris for spatial

filtering. For light efficiency one might choose to use linearly polarized light form the light source, e.g. using a polarizing beam splitter 300 which transmits p-polarized light and a quarter wave plate 320 as shown. If the quarter wave plate 320 is rotated such that the illumination light travelling to the sample 70 has a circular polarization, the back- reflected detection light, after passing for a second time through the quarter wave plate 320, will have a linear polarization rotated 90 degrees with respect to the incoming light and therefore has s-polarization . This will be efficiently reflected by the polarizing beam splitter 300 ensuring optimal light efficiency directed towards the position dependent detector 100.

If one would like to simultaneously observe any fluorescence light of the sample 70, e.g. being excited in the sample 70 by the illumination/scanning beam, this can be easily achieved by adding a dichroic beam splitter 310 which e.g. transmits the scanning excitation beam but reflects the fluorescence emission. This is shown in Fig. 4 but such optical fluorescence detection system may be added to any embodiment. In Fig. 4, the emission travels to a sensitive point detector 330 or any other suitable detector or camera, via another optional relay 90 and optional spatial filter 85. Any detection signals from the point detector 330 may be combined with data from the position sensitive detector 100 as part of image data for constructing the image. In another embodiment, not shown, polarization sensitive detection can be implemented. For this, a polarizing beam splitter may be located before the detector 100, to split the detection beam(s) according to polarization. In this way the two orthogonally polarized components of the detection light (scattered and unscattered light) each give rise to their own detection signals which may be treated separately or in any suitable combination as image data for constructing the image. If the scattering by the sample is polarization dependent this leads to slight differences in the detection signals from the individual beams which can be analysed for example in terms of birefringence. The signals may be detected with a quadrant position sensitive detector or with two position sensitive detectors each associated with one of the polarization

directions, such detectors then possibly being unidirectionally sensitive. In case of two detectors, for optimization of the signal on both detectors the polarization of the illumination/scanning beam may be tuned, e.g. to

45 degrees, with the aid of a half wave plate. Similarly, the polarization of the illumination/scanning beam could be modulated in conjunction with polarization insensitive detection in order to characterize polarization- dependent scattering (e.g. implementing time-multiplexed polarization dependent detection) .

In another embodiment, shown in Fig. 6, the beam scanning implementation and the forward scattered detection can be further

implemented using a de-scanning tip-tilt mirror 160 after the light has travelled through the sample and has been collected by the detection system and passed through a pair of optical relay lenses 90. The de- scanning tip-tilt mirror ensures that the scanning beam is transformed into a stationary beam. It is now possible to use a spatial filtering assembly consisting of a pair of lenses 170 and a pinhole or spatial filter 140 before the beam is detected by the position sensitive detector 110 (see Fig. 6A for an indicative signal) . This spatial filtering can be employed to reject background light and improve e.g. the z-sectioning of the scattering signal.

Other optical techniques like wavelength variation and/or wavelength dependent detection and/or detection of angle-dependent scattering may be exploited as well.

Figs. 7-10 show exemplary images, which were obtained with a stage-scanning implementation in a microscopy system otherwise generally in accordance with Fig. 2. The illumination light beam 20 was set to a very low power level, parked at the center of the field of view in a confocal setup and the microscope sample stage was raster scanned while acquiring image data representative of deflection data. From the image data the images of Figs. 7-10 were constructed.

Figures 7 and 8 are images of a human cheek epithelial cell, constructed from a one-directional deflection signal detected by scanning a cheek epithelial cell through a static light beam by stage scanning. Fig. 7 is constructed from deflection in one direction (X), whereas Fig. 8 is constructed from deflection in a perpendicular direction (Y) . The scale of both images is 80 x 60 micrometer.

Figure 9 is an absolute signal image of the cell of Figs. 7-8.

The image is obtained by combining the X and Y-direction data of Figs . 7 and 8 according to Sabs = sqrt(Sx² + Sy²) and using a different brightness colour scale relative to Figs. 7-8.

Figure 10 is an image of a single chromosome.

The images of Figs. 7-10 illustrate that (as described above) , when the light beam focus is smaller than the scatterer so that the size of the scatterer is significantly larger than the light beam focus, the reconstructed images may resolve details of the scatterer. Thus, images according to the present concepts provide a map of the relative-position- specific interaction of (a scatterer in) the sample part and the focussed light beam, which shows at least some of the shape, structure and/or morphology of the sample part, so that a (very) clear impression of a morphology of at least part of the scatterer may be obtained. In fact, images that may be obtained according to the present concepts may be of similar quality as, or even better quality than, images obtained by differential interference contrast microscopy, which is nowadays a standard technique. E.g. a comparably higher contrast may be achieved in the image. Also a better depth sectioning and/or background rejection may be achieved, in particular if a pinhole and/or spatial filter is

implemented as indicated in Fig. 3.

Fig. 11 shows a typical intensity pattern of a back focal plane. The pattern is offset from the centre, as indicated by the cross hairs dividing the picture. Intensity differences in X- and Y-direction (Xdiff, Ydiff) and total intensity (Sum) may be calculated as shown.

The disclosure is not restricted to the above described embodiments which can be varied in a number of ways within the scope of the claims. For instance, the interference pattern beam line may be combined with another imaging beam line with which a focal plane or an optical conjugate thereof may be imaged. Such beam lines may partly overlap, e.g. sharing the condenser and being separated by a partial beam splitter to two different optical detectors, e.g. a quadrant photodiode for the interference pattern beam line and a camera for the imaging beam line and/or having different wave lengths and being separable using a dichromatic mirror and/or a filter.

Elements and aspects discussed for or in relation with a particular embodiment may be suitably combined with elements and aspects of other embodiments, unless explicitly stated otherwise.

Claims

1. A method of imaging at least part of a sample (70) , in particular a biological sample comprising a scatterer, e.g. a biological object, the method comprising:

focusing at least part of a light beam (12, 20, 250) in a sample plane in the sample (70), and in particular focusing the part of the light beam at or near the scatterer therein, thus providing

unscattered light (16; 20) and scattered light (14; 25) ;

causing a displacement of at least part of the sample (70) and at least part of the focus with respect to each other; and

for plural relative positions of the sample (70) and the focus: collecting the unscattered light (16) and the scattered light (14) with a detection system (8) focused in at least part of the sample, and comprising a position dependent detector (100) ;

controlling the detection system (8) to capture image data, the image data representing at least part of the intensity pattern related to an outgoing angular distribution of the scattered and unscattered light (14, 16; 20, 25) in the sample plane, the image data representing at least part of an intensity pattern in a back focal plane of the detection system (8) and/or in an optical conjugate plane of the back focal plane of the detection system (8) , the method further comprising constructing an image of at least part of the sample (70), in particular at least part of the scatterer, on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample (70) and the focus .

2. The method according to claim 1, wherein the position dependent detector (100) is a two-dimensional position dependent detector; and/or

wherein the method further comprises rendering at least part of the image in a brightness scale and/or an essentially single-colour- scale; and/or

wherein the method further comprises constructing the image from a combination of image data associated with one or more individual directions, e.g. corresponding to amounts of scattering in one or more directions (Sx, Sy) and/or to an absolute value thereof (Sabs) ; and/or wherein method further comprises constructing a contrast image based on an intensity of the light beam after interaction thereof with the sample (70) .

3. The method according to any preceding claim, comprising arranging a light source (10) for providing the light beam (12; 20; 250) on one side of the sample (70) and the detection system (8) on a second side of the sample (70), in particular the first and second sides being opposite each other, such that at least part of the light beam (12; 20; 250) traverses the sample (70) from the first side to the second side before reaching the detection system (8) and the detector (100) .

4. The method according to any preceding claim, comprising arranging a light source (10) for providing the light beam (12; 20; 250) and the detection system (8) on one side of the sample, and arranging a reflector for at least part of the light beam such that at least part of the light beam (12; 20; 250) traverses at least part of the sample (70) from a first side and returns to the first side before reaching the detection system (8) and the detector (100) .

5. The method according to any preceding claim, comprising spatial filtering of the scattered and/or the unscattered light in the detection system (8) before the detector (100), such that at least part of the scattered and/or of the unscattered light (14, 16; 20, 25) passes through a spatial filter (35) prior to reaching the detector (100) .

6. The method according to any preceding claim, comprising trapping at least one object (210, 230) in the sample (70), in particular optically trapping, wherein the object (210, 230) comprises the scatterer or the scatterer interacts with at least one of the objects (210, 230), e.g. being attached to an object (210, 230) .

7. The method according to claim 6, comprising trapping, in particular optically trapping, plural objects (210) attached to each other by at least one connecting element (230), in particular the objects (210) being microspheres and the connecting element(s) (230) comprising a microtubule, a DNA-strand, etc.,

wherein at least one of the objects (210) and/or the connecting element (230) comprises the scatterer, and/or wherein the scatterer interacts with at least one of the objects (210) and/or the connecting element(s) (230), e.g. being attached to an object (210) and/or to the connecting element (s) (230) .

8. The method according to claim 6 or 7, comprising optical trapping of at least one object (210, 230) in the sample with one or more optical trapping beams (200), wherein the light beam (250) differs from at least one of the optical trapping beams (200) in at least one intensity, wavelength and polarization.

9. The method according to any preceding claim, wherein the method comprises modifying one or more of: the focus size of the light beam (12, 20, 250), the intensity of the light beam (12, 20, 250) and/or the wavelength of the light beam; and/or wherein the method comprises providing at least part of the sample (70) with an optically effective label, possibly comprising optically activating and/or de-activating the label .

10. A system (2) for the method of any preceding claim, wherein the system (2) comprises:

a sample holder (6, 340) to hold a biological sample (70), a light source (10) providing a light beam (12, 20,250) , and, operably arranged along an optical path of at least part of the light beam (12, 20, 250) :

a source optical system (4) configured to focus at least part of the light beam (12, 20, 250) in a sample (70) held in the sample holder (6, 340),

a detection optical system (8) comprising a position dependent detector (100), e.g. one or more of a split photodiode, a quadrant photodiode, a photodiode array, a camera, a position- sensitive photodiode;

wherein the detection system (8) provides a back focal plane and is arranged to collect at least part of the light beam comprising both light not scattered by the sample, i.e. unscattered light, and light scattered by at least one scatterer in the sample, i.e. scattered light, and to provide from them an intensity pattern in the back focal plane;

wherein the detection system (8) is arranged to capture image data, the image data representing at least part of the intensity pattern related to an outgoing angular distribution of the scattered and unscattered light in the sample plane, the image data representing at least part of an intensity pattern in a back focal plane of the detection system (8) and/or in an optical conjugate plane of the back focal plane of the detection system (8);

wherein at least part of at least one of the sample holder (6,

340), the light source (10) and the source optical system (4) is

adjustable to controllably displace the focus of the light beam (12, 20, 250) and at least part of the sample (70) relative to each other, e.g. being connected to a position controller (120);

the system further comprising a controller (120) connected with the position dependent detector (100) and programmed to construct an image of at least part of the sample (70) , in particular at least part of the scatterer, on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample (70) and the focus.

11. The system (2) according to claim 10, comprising a spatial filtering system (35) for spatial filtering detection light between collecting optics (60) of the detection system (8) and the position dependent detector (100) .

12. The system (2) according to claim 10 or 11, comprising a trapping arrangement to trap and/or hold one or more objects in the sample (70), in particular an optical trapping arrangement, preferably a multiple trapping arrangement to trap and/or hold one or more objects (210, 230) in the sample in multiple traps .

13. A detection module for placement in an optical train of a sample or beam scanning microscope comprising a sample holder (6, 340) to hold a biological sample (70) , a light source (10) providing a light beam (12, 20, 250) , and, operably arranged along an optical path of at least part of the light beam (12, 20, 250), a source optical system (4) arranged to focus at least part of the light beam (12, 20, 250) in a sample held in the sample holder (6, 340) , and wherein at least part of at least one of the sample holder (6, 340), the light source (10) and the source optical system (4) is adjustable to controllably displace the focus of the light beam and at least part of the sample (70) relative to each other, e.g. being connected to a position controller (120);

wherein the detection module comprises : a detection optical system (8) comprising a position dependent detector (100), e.g. one or more of a split photodiode, a quadrant photodiode, a photodiode array, a camera, a position-sensitive photodiode;

wherein the detection system (8) provides a back focal plane and is arranged to collect at least part of the light beam (12, 20, 250) comprising both light not scattered by the sample, i.e. unscattered light, (16, 20) and light scattered by at least one scatterer in the sample, scattered light, (14, 25) and to provide from them an intensity pattern in the back focal plane;

wherein the detection system (8) is arranged to capture image data, the image data representing at least part of the intensity pattern related to an outgoing angular distribution of the scattered and

unscattered light (14, 16; 20, 25) in the sample plane, the image data representing at least part of an intensity pattern in the back focal plane and/or in an optical conjugate plane of the back focal plane;

the detection system (8) further comprising a controller (120) connected with the position dependent detector (100) and programmed to construct an image of at least part of the sample (70) on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample (70) and the focus.

14. A method of imaging at least part of a sample (70) , in particular a biological sample comprising a scatterer, the method

comprising :

controlling a source optical system (4) to focus at least part of a light beam (12, 20, 250) in the sample (70) and in particular at or near the scatterer therein, thus providing unscattered light (16, 20) and scattered light (14, 25), which form an intensity pattern in a back focal plane of a detection optical system (8);

controlling at least one of the source optical system (4) and a sample positioning system to position the focus and the sample (70) at a plurality of different positions with respect to each other,

for plural relative positions of the sample (70) and the focus, controlling the detection system (8) to capture image data, the image data representing at least part of the intensity pattern related to the outgoing angular distribution of the scattered and unscattered light (14, 16; 20, 25) in the sample plane, the image data representing at least part of an intensity pattern in the back focal plane of the detection system and/or in an optical conjugate plane of the back focal plane; and constructing an image of at least part of the sample (70) on the basis of the image data associated with the plural relative positions as a function of the relative positions of the sample (70) and the focus.

15. A computer program comprising instructions to cause the system (2) according to any of claims 10-12 to execute the steps of the method according to any of the claims 1-9.

16. A computer-readable medium having stored thereon the computer program according to claim 15.