WO2016065125A1

WO2016065125A1 - Method and apparatus for visualizing objects using oblique back-illumination

Info

Publication number: WO2016065125A1
Application number: PCT/US2015/056872
Authority: WO
Inventors: Timothy N. FORD; Alexander Q. ANDERSON; Guillermo J. Tearney
Original assignee: The General Hospital Corporation
Priority date: 2014-10-22
Filing date: 2015-10-22
Publication date: 2016-04-28

Abstract

[0041] Exemplary apparatus and method can be provided for obtaining image information for at least one portion of at least one biological structure. For example, at least one housing comprising at least one channel can be used to contain the biological structure(s) that includes a first flowing structure and a second non-flowing structure. At least one illumination arrangement can be used to provide at least one first electro-magnetic radiation to the channel(s). At least one detector arrangement can be used to detect at least one second electro-magnetic radiation which is an interaction between the first structure, the second structure, the channel(s) and the first electro-magnetic radiation(s). At least one computer arrangement can be used to generate the image information regarding the portion(s) based on the second electro-magnetic radiation(s) and further information regarding at least one characteristic of the channel(s).

Description

METHOD AND APPARATUS FOR VISUALIZING OBJECTS USING OBLIQUE BACK-ILLUMINATION

CROSS REFERENCE TO RELATED APPLICATION(S)

[0001] The present application relates to and claims priority from U.S. Provisional Patent Application Serial No. 62/067,071 filed October 22, 2014, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to exemplary embodiments of method, device and apparatus for an acquisition of image data using oblique back-illumination, e.g., in a flow chamber device.

BACKGROUND INFORMATION

[0003] Luminal surfaces of blood vessels, lymph channels, and other biological flow channels are environments rich in physical and biochemical effects that govern many vital physiological properties both healthy and aberrant. Many such processes occur between adherent endothelial cells comprising the luminal surface of vasculature and lymphatic ducts and whole blood and lymph which flows within these channels, including blood clotting cascades, endocrine signaling pathways, immune cell interactions, and more. These molecular and cellular mechanisms have important implications in medicine, for example in vascular diseases such as atherosclerosis, cancer medicine concerning metastasis, and organ transplant medicine concerning tolerance and rejection mechanisms.

[0004] A commonly-used and important reductive approach to studying these

mechanisms is to acquire image data from cells which have been removed from the body and grown in vitro in thin flow chambers approximating in geometric, physical, and chemical ways the cells' natural environment. Advancement in fields using the flow chamber approach is currently hindered by a lack of tools for visualizing adherent cells to study their behavior in the presence of flowing biological fluids such as blood. Many traditional and readily- available microscopy techniques cannot visualize cells through whole blood because of undesired optical properties of red blood cells, which are both highly light absorbing and strongly light scattering. As a consequence, the common strategy is to replace the native whole blood with optically transparent blood substitutes such as cell growth medium or very dilute blood. A major deficiency with the current state of the art is the reduction of physiological relevance of flow chamber experiments using whole blood substitutes as compared with the full biochemical complexity of the whole blood milieu.

[0005] Thus, there may be a need and benefit to provide methods, systems and apparatus for the acquisition of imaging data using oblique back-illumination, e.g., in within a flow chamber filled with flowing whole blood, which can overcome at least some of the above- described issues and/or deficiencies.

SUMMARY OF EXEMPLARY EMBODIMENTS

[0006] To address and/or overcome the above-described problems and/or deficiencies, exemplary embodiments of method and apparatus for recording image data from a flow chamber apparatus can be provided.

[0007] To that end, according to an exemplary embodiment of the present disclosure, it is possible to configure a standard microscope - comprising a sample holder stage arrangement, an optical imaging arrangement, an image detector and a computer to record image information - with one or more illumination arrangements that produce oblique back- illumination. Positioning a thin (for example, microfluidic) flow chamber housing with living or fixed cells adhered therein on the microscope sample holder stage element, it is possible to flow optically-scattering biological fluids such as whole blood or lymph, through the channel and observe through image information interactions between the adhered cells and flowing cells or other particles. The illumination arrangements are configured to couple light into the channel filled with optically scattering fluid and is aided by optical reflections at the channel walls. With many multiple scattering paths traversed between the illumination channel launch point and the back-illumination exit point into the detection optical system, and multiple optical reflections in the channel acting as an optical waveguide, an oblique illumination field is developed and trans-illuminates the cells in the field of view of the optical imaging system.

[0008] The image information can be that of phase gradient contrast microscopy, where image intensity signal is related to differences in optical path length traversed through the biological objects of interest near the focal plane, which may be present due to variations in optical refractive index and/or object thickness near the focal plane. More specifically, recorded image intensity can be related to both the gradient of the optical path lengths along the illumination obliquity axis, and light absorption near the focal plane. In some exemplary embodiments, two or more images are detected with different illumination configurations providing different illumination obliquity characteristics. In these embodiments, further image information may be then derived from the first image information and knowledge of the illumination obliquity characteristics. These further image information may include separation of phase gradient contrast information from absorption contrast information. In exemplary embodiments of the present disclosures, the light detected contains information regarding the interactions between the adhered cells and the flowing biological fluid, but also the channel boundaries and reflections therefrom.

[0009] The image data features high spatial resolution to visualize individual cells and sub-cellular components. The image method described can also be referred to as oblique back-illumination microscopy (OBM). As will be become apparent from this disclosure, the described method and apparatus offer improvements in current flow chamber microscopy apparatuses in that physiologically relevant volumes of turbid media such as whole blood may be flowed while visualizing unstained samples. Some embodiments of the present disclosure allow for simultaneous visualization using OBM and other standard microscopy methods that rely on exogenous staining with contrast agents such as fluorescent molecules, phosphorescent molecules, or others.

[0010] To that end, exemplary apparatus and method can be provided for obtaining image information for at least one portion of at least one biological structure. For example, at least one housing comprising at least one channel can be used to contain the biological structure(s) that includes a first flowing structure and a second non-flowing structure. At least one illumination arrangement can be used to provide at least one first electro-magnetic radiation to the channel(s). At least one detector arrangement can be used to detect at least one second electro-magnetic radiation which is an interaction between the first structure, the second structure, the channel(s) and the first electro-magnetic radiation(s). At least one computer arrangement can be used to generate the image information regarding the portion(s) based on the second electro-magnetic radiation(s) and further information regarding at least one characteristic of the channel(s).

[0011] According to an exemplary embodiment of the present disclosure, a wavelength of each of the first and second electro-magnetic radiations can be substantially the same. The first structure can include blood, leukocytes, serum and/or platelets. The second structure can include cells which are provided substantially stationary at at least one wall of the channel(s). The cells can be substantially fixed to the wall(s) of the channel(s). At least one area of the cells can include a monolayer of cells and/or endothelial cells.

[0012] In a further exemplary embodiment of the present disclosure, the housing(s) can have at least two walls, where at least one of the walls can have an index of refraction that is substantially different from an index of refraction of the first structure. At least one of the walls can contain a coating that increases a reflectivity of an electromagnetic radiation that impacts upon the wall. For example, the illumination arrangement can be configured to provide the first electro-magnetic radiation(s) at at least two different locations. The different locations can be illuminated using different illumination sources. The different illumination sources can have different wavelength bands distinguishable by optical filters. The different locations can be illuminated using the same illumination source and an optical arrangement to determine the illumination location. The detection arrangement can be configured to detect the different radiations (i) separately from one another, (ii) using different detectors, (iii) on different portions of the same detector, and/or (iv) during different non-overlapping spans of time. The detection arrangement can be configured to distinguish between the different radiations by (i) passing the different radiations through optical color filters and/or (ii) temporally switching between active illumination sources with every image sensor detection event. The time delay between successive image sensor detection can be as short as possible by use of a frame transfer camera or double-shutter camera.

[0013] The computer arrangement can be configured to generate further image information from the image information generated regarding the detection of the different second electro-magnetic radiations. For example, a direction of one of the different radiations can be (i) different from a direction of another one of the different radiations, (ii) along a direction of extension of the channel, and/or (iii) orthogonal to a direction of extension of the channel. The light sources can include at least four light sources which generate at least four different radiations. A direction of two of the different radiations can be orthogonal to a direction of another two of the different radiations.

[0014] These and other objects, features and advantages of the exemplary embodiments of the present disclosure will become apparent upon reading the following detailed description of the exemplary embodiments of the present disclosure, when taken in conjunction with the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the present disclosure, in which: [0016] Figure 1 is a side-view diagram of a flow chamber apparatus, flowing and non- flowing biological objects, an oblique back-illumination configuration and a light detection configuration according to an exemplary embodiment of the present disclosure;

[0017] Figure 2 is a top-view diagram of a flow chamber and a configuration of two illumination sources with opposing illumination directions according to an exemplary embodiment of the present disclosure;

[0018] Figure 3 is a side-view diagram of an embodiment showing a flow chamber apparatus, flowing and non-flowing biological objects, an oblique trans-illumination configuration and a light detection configuration;

[0019] Figure 4 is a side-view diagram of e the oblique back-illumination arrangement that is configured and aligned with the detection optical arrangement using an objective-lens optical fiber-adapter according to an exemplary embodiment of the present disclosure;

[0020] Figure 5 is a cross sectional-view of an objective-lens and objective-lens optical fiber-adapter with two illumination sources providing two different illuminations with equal amounts of obliquity but opposing directions of obliquity according to an exemplary embodiment of the present disclosure;

[0021] Figure 6 is a perspective view of the under-side of an objective-lens optical fiber- adapter showing the objective-lens cavity according to an exemplary embodiment of the present disclosure;

[0022] Figure 7 is side-view of multiple optical reflections at the channel boundary layers driven by index-of-refraction mismatches between the biological fluid channel and the surrounding flow chamber material according to an exemplary embodiment of the present disclosure;

[0023] Figure 8 is a side-view of a compact oblique back-illumination arrangement comprised of discrete LEDs adhered to the microscope sample stage holder to allow the objective turret to rotate freely and easily switch among a set of objective lenses according to an exemplary embodiment of the present disclosure;

[0024] Figure 9 is a top-view diagram of a flow chamber channel and four oblique back- illumination sources that provide illumination along and orthogonal to the direction of biological fluid flow according to an exemplary embodiment of the present disclosure;

[0025] Figure 10 is a figure showing approximate image contrast of a moving objective as a function of its speed along the illumination obliquity axis; and

[0026] Figure 11 is a side-view diagram of an embodiment showing an oblique back- illumination channel and a trans-illumination channel for dual-modality imaging.

[0027] Throughout the figures, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components or portions of the illustrated embodiments. Moreover, while the subject invention will now be described in detail with reference to the figures, it is done so in connection with the illustrative

embodiments. It is intended that changes and modifications can be made to the described exemplary embodiments without departing from the true scope and spirit of the subject disclosure, as defined by the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0028] In one exemplary embodiment of the present disclosure, biological objects of interest can be adhered to the walls of a flow chamber using standard cell culture protocols. Figure 1 shows a side view of an exemplary apparatus, and a flow chamber apparatus is provided that can include a housing substrate 101 with one or multiple channels 102 through which turbid media 103, such as blood or lymph, is flowed. In some exemplary embodiments, the solid substrate 101 can be optically transparent media such as glass or a polymer such as poly (methyl methacrylate) (PMMA). In some exemplary embodiments, the bottom layer of the flow chamber may be constructed from a standard microscope cover slip with optical index approximately 1.5 and thickness approximately 0.17 mm, to be compatible with standard cover-slip corrected microscope objectives. The turbid media 103, such as whole blood, can then be flowed through the chamber in a direction 104 that has the effect of providing a shear force on the adhered cells 105 lining the chamber. A sub-set of these adherent cells 106 can be referred to as the cells under observation. A goal of the apparatus and method described in the present disclosure is to provide image data of the cells under observation 106 using OBM.

[0029] In an exemplary embodiment of the present disclosure, a light source 107 is configured which provides illumination to one portion of the sample. In one embodiment, the illumination source is a light-emitting diode (LED). In another exemplary embodiment, the light source is a thermal source such as a xenon or mercury-xenon arc lamp. In some exemplary embodiments, the wavelength of the illumination source is in the visible range between 400 nm and 700 nm. In an exemplary embodiment, the wavelength of the

illumination source can be approximately in the red region between 600 nm and 700 nm. In certain exemplary embodiments, the wavelength of the illumination source can be in the near- infrared region between 700 nm and 1000 nm. In some exemplary embodiments, the wavelength of the illumination source is in the region between 1000 nm and 2000 nm. In some embodiments, the spectral bandwidth of the illumination source is 50 - 200 nm. In a preferred embodiment, the spectral bandwidth of the illumination source is 10 - 50 nm.

[0030] The illumination can be communicated onto a region of the sample by means of illumination optics 108. In some embodiments the illumination optics 108 are free-space lenses. In other embodiments the illumination optics 108 are light guides such as a multimode optical fiber, optical fiber bundle, liquid waveguide, or a planar waveguide. The illumination optical arrangement 108 can define an illumination direction 109, and illuminate an area separated by the cells under observation by a source-detector separation 111. In some exemplary embodiments, a detection optics arrangement 112 can be provided and configured to project an image of the cells under observation 106 onto a light detector arrangement 113. For example, the light detector arrangement is comprised of a multi-pixel camera sensor such as a charge-coupled device (CCD) or an array complementary metal oxide semiconductor (CMOS) detector. Further, the image information can be digitized and stored with a computer arrangement 114.

[0031] In some exemplary embodiments of the present disclosure, only one light source can used. As shown in Figure 2, the housing substrate 201 contains a flow chamber channel 202 through which turbid media 203 is flowed in direction 204. Cells under observation 205 can be illuminated by a first illumination source 206 through a first illumination optical arrangement 207 in a first direction 208. The detection optical arrangement 213 is used to provide image information of the cells under the first illumination. Subsequently, the cells under observation 205 can be illuminated by a second illumination source 209 through a second illumination optical arrangement 210 in a second and opposing illumination direction 211. The detection optical arrangement 213 is used to provide further image information of the cells under the second illumination.

[0032] For example, the illumination optical arrangements 207 and 210 can be configured to illuminate areas of the flow chamber separated by the cells under observation 205 by the same source-detector separation 212 and incidence angle. In one exemplary embodiment, the illumination directions 208 and 211 can be substantially opposite to each other, and both parallel with the direction of channel elongation 202, and approximately coincident with the channel centerline. For example, the computer arrangement is used to provide further image information from the first and second image information based on an OBM image-processing algorithm.

[0033] In some exemplary embodiments of the present disclosure, one or more light sources are positioned above the sample instead of below. As shown in Figure 3, the housing 301 can contain a flow chamber channel 302 through which a turbid medium 303 flows in a direction 304. Adherent cells 305 can be affixed to the chamber walls, a subset of those 306 are referred to as the cells under observation. An illumination source 307 can be provided and configured to be located above the substrate. The illumination can be communicated by an illumination optical configuration 308 to illumination the sample in an illumination direction 309 having incident angle 310 and source-detector separation 311. A detection configuration 312 can be provided to generate image information of the cells under observation 306 onto a detector 313 and digitized and stored with a computer arrangement 314.

[0034] In some exemplary embodiments of the present disclosure, an illumination housing configuration can be provided to hold the illumination optical arrangement such that the illumination direction and source-detector separation can be controlled. As shown in Figure 4, a flow chamber housing 401 contains a flow channel 402 through which a turbid media 403 flows in direction 404. Adherent cells are affixed to the walls of the chamber 402, a sub-set of which are referred to as the cells under observation 406. An illumination source 407 can be provided with an illumination optical arrangement 408 which can define an illumination direction 409 and source-detector separation. According to one exemplary embodiment, an illumination housing configuration 410 can be configured to attach securely and reversibly to the detection optical arrangement 411. The illumination housing

configuration fixes the illumination direction 409 and a source-detector separation to optimal values dictated by the optical properties of the turbid media 403 at the specific wavelength of the illumination source 407.

[0035] Figure 5 shows an exemplary illumination housing configuration in cross section, a first illumination source 501 can be coupled to a first illumination optical configuration 502 that resides inside the housing configuration 507 in a first closed channel 508 such that first illumination direction 503 is controlled to an optimal value. In an exemplary embodiment of the present disclosure, a second illumination source 504 can be coupled to a second illumination optical configuration 505 that also resides inside the housing configuration 507 through a second closed channel 509 opposite of first closed channel 508. A third closed channel 510 can be provided to allow optical free space to exist between the cells under observation and the detection optical arrangement 511. The solid and opaque walls of the illumination housing configuration can serve to block direct illumination of the detection optical configuration with light from either illumination source. Instead, light from either illumination source generally first travels through the turbid media and through the cells under observation in the manner desired for optimized oblique back-illumination imaging. Figure 6 shows the exemplary illumination housing configuration 601 in perspective view, illustrating the detection channel through which the detection optical arrangement sits.

[0036] Figure 7 shows a cross-sectional view of an exemplary embodiment of the flow channel housing which illustrates the different indices of refraction of the layers. The flow chamber housing substrate 701 can include an optically transparent lower medium with index of refraction n\ and optically transparent upper medium with index of refraction n^. The flow channel 702 can be filled with a biological fluid 703, such as whole blood with index of refraction ri2. Light from an illumination arrangement 707 can be coupled into the channel with starting direction 709. Some portion of the illumination power 710 can be optically scattered throughout the volume of biological medium 703, while the remainder portion (e.g., the ballistic light) can strike the channel wall 702. Some portion of this ballistic light can be reflected at the wall due to the index of refraction mismatch, and can be directed back into the channel where it reflects further 721. The collective action of many multiples of optical scattering events with many optical reflections at the boundaries redirect light towards the cells under observation and into the detection optical system 712 to be detected at detector 713 and digitized with computer arrangement 714. The interaction of the illumination with the optical properties of the biological fluid and channel boundary conditions can generate an oblique field trans-illuminating the cells under observation.

[0037] In a further exemplary embodiment of the present disclosure, as illustrated in Figure 8, a flow chamber apparatus can be provided comprising a housing substrate 801 with one or multiple channels 802 through which biological fluid 803 is flowed in direction 804. Adhered cells 805 lining the chamber include a subset 806 under observation. For example, the illumination can be provided by discrete LED elements 807 positioned at one or more locations on the underside of the flow chamber housing. The LEDs may further contain illumination-shaping optics 808 which direct the illumination direction 809 into the flow channel fluid. The LEDs can be set into a housing that sets the separation distance 811 symmetrically about the detection optical axis. Flexible wires 815 provide electrical power to the LEDs and are connected to and controlled by an illumination computer arrangement 816. Obliquely trans-illuminated light is collected by optical imaging system 812, detected by detector 813 and recorded with computer arrangement 814. For example, the LEDs 807 and illumination-shaping optical arrangements 808 can be embedded in the same housing 801 as the flow chamber housing for optimal alignment and optical throughput into the flow chamber. In other exemplary embodiments, the LEDs 807 and illumination-shaping optical arrangements 808 can be held together in a housing separate from both the flow-chamber housing 801 and the imaging optical system 812 to facilitate quick positioning of the flow chamber housing independent from the illumination and detection systems, and for quick selection of objective lenses from a standard object lens turret.

[0038] In yet another exemplary embodiment of the present disclosure, as illustrated in Figure 9, a flow chamber apparatus can be provided comprising a housing substrate 901 with one or multiple channels 902 through which biological fluid 903 is flowed in direction 904. Cells under observation 905 can be illuminated with light from source 906 coupled into the channel 902 through optical arrangement 907 such that it is directed towards the cells under observation in direction 908. For example, the entire illumination apparatus 920 can be presented in multiple locations, including the opposing direction along the channel 921, and the orthogonal direction 922 and 923. In this exemplary four-illumination embodiment, image information taken from four images each taken under a different illumination source can be combined with a priori information regarding the illumination geometry and channel characteristics to produce further image information. For example, the two illumination directions 920 and 921 along the channel direction 904 can be used to identify and selectively image stationary and slow-moving biological objects from fast-moving objects and particles. In addition, e.g., two illumination directions 922 and 923 orthogonal to the channel direction 904 can be used to image all objects in the field regardless of their velocity.

[0039] In further exemplary embodiments of the present disclosure, the exposure time of the detection system is selected according to a preferred image information content. Figure 10 shows a plot showing the approximate relationship between the speed of a moving object and the apparent contrast of the object as seen by oblique back-illumination imaging. It is an exemplary feature of oblique back-illumination that objects traveling across the field of view orthogonal to the optical axis and parallel to the illumination obliquity axis, and with sufficient speed that they travel more than one object diameter during the time of the sensor exposure substantially disappear from the image. This can be in contrast to objects that travel orthogonal to the optical axis and orthogonal to the illumination obliquity axis, which appear in moderate contrast, or to objects that do not move with sufficient speed, or at all, during the time of the sensor exposure, which appear with very high contrast. It is possible to set the exposure time of the image sensor, and thus the characteristic object speed separating visible from substantially invisible objects. For example, with favorable orientation of the oblique back-illumination axes (parallel to the direction of fluid flow) and favorable selection of the image sensor exposure time (exposure time greater than approximately 0.5 x object diameter / object speed), background signal from fast-moving objects can be substantially reduced revealing subtle information from stationary and slow-moving objects.

[0040] In yet another exemplary embodiment of the present disclosure, as illustrated in Figure 11, a flow chamber apparatus can be provided comprising a housing substrate 1101 with one or multiple channels 1102 through which biological fluid 1103 can be flowed in direction 1104. Cells adhered to the walls of the channel 1105 can include a subset under observation 1106. The oblique back-illumination can be provided by an illumination source 1107 through an optical arrangement 1108 in a particular direction 1109. Light obliquely trans-illuminating the cells under observation can be collected by optical imaging system 1112, detected with detector 1113 and/or recorded with computer arrangement 1114. In an exemplary configurations, a second illumination source 1120 can be provided to directly trans-illuminate the cells under observation with illumination optical arrangement 1121. For example, this illumination source 1120 can provide white light imaging from a lamp or broadband white light source. Further, the illumination source 1120 can provide light suitable for phase contrast imaging including Zernike phase contrast and differential interference contrast (DIC) imaging. In addition, the illumination source 1120 can provide an excitation light suitable for fluorescence or phosphorescence imaging. Additionally, the illumination source 1120 and oblique back-illumination source 1107 can be used separately in a time- multiplexed way and/or simultaneously, and a filter optical arrangement 1122 can be used to distinguish between sources. For example, the filter optical arrangement 1122 can be or include a dichroic beamsplitter or prism to direct the image information from illumination source 1120 to a different sensor than the image information from illumination source 1107. Further, the filter optical arrangement 1122 can be or include a dichroic beam splitter or prism to direct the image information from illumination source 1120 to a different part of the same sensor than the image information from illumination source 1107. The foregoing merely illustrates the principles of the invention. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. Indeed, the arrangements, systems and methods according to the exemplary embodiments of the present invention can be used with any OCT system, OFDI system, SD- OCT system or other imaging systems, and for example with those described in International Patent Application PCT/US2004/029148, filed September 8, 2004, U.S. Patent Application No. 11/266,779, filed November 2, 2005, and U.S. Patent Application No. 10/501,276, filed July 9, 2004, the disclosures of which are incorporated by reference herein in their entireties. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements and methods which, although not explicitly shown or described herein, embody the principles of the invention and are thus within the spirit and scope of the present invention. In addition, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly being incorporated herein in its entirety. All publications referenced herein above are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for obtaining image information for at least one portion of at least one biological structure, comprising:

at least one housing, comprising at least one channel which is configured to contain the at least one biological structure that includes a first flowing structure and a second non- flowing structure;

at least one illumination arrangement which is configured to provide at least one first electro-magnetic radiation to the at least one channel;

at least one detector arrangement which is configured to detect at least one second electro-magnetic radiation which is an interaction between the first structure, the second structure, the at least one channel and the at least one first electro-magnetic radiation; and at least one computer arrangement which is configured to generate the image information regarding the at least one portion based on the detected at least one second electro-magnetic radiation and further information regarding at least one characteristic of the at least one channel.

2. The apparatus according to claim 1, wherein a wavelength of each of the first and second electro-magnetic radiations are substantially the same.

3. The apparatus according to claim 1, wherein the first structure includes blood.

4. The apparatus according to claim 1, wherein the first structure includes leukocytes.

5. The apparatus according to claim 1, wherein the first structure includes serum.

6. The apparatus according to claim 1, wherein the first structure includes platelets.

7. The apparatus according to claim 1, wherein the second structure includes cells which are provided substantially stationary at at least one wall of the at least one channel.

8. The apparatus according to claim 7, wherein the cells are substantially fixed to the at least one wall of the at least one channel.

9. The apparatus according to claim 8, wherein at least one area of the cells includes a monolayer of cells.

10. The apparatus according to claim 8, wherein at least one area of the cells includes endothelial cells.

11. The apparatus according to claim 1 , wherein the at least one housing has at least two walls, wherein at least one of the walls has an index of refraction that is substantially different from an index of refraction of the first structure.

12. The apparatus according to claim 11, wherein at least one of the walls contains a coating that increases a reflectivity of an electromagnetic radiation that impacts upon the wall.

13. The apparatus according to claim 1, wherein the illumination arrangement is configured to provide the at least one first electro-magnetic radiation at at least two different locations

14. The apparatus according to claim 13, wherein the at least two different locations are illuminated using different illumination sources.

15. The apparatus according to claim 14, wherein the at least two different illumination sources have different wavelength bands distinguishable by optical filters.

16. The apparatus according to claim 13, wherein the at least two different locations are illuminated using the same illumination source and an optical arrangement to determine the illumination location.

17. The apparatus according to claim 13, wherein the detection arrangement is configured to detect the different radiations separately from one another.

18. The apparatus according to claim 13, wherein the detection arrangement detects the different radiations using different detectors.

19. The apparatus according to claim 13, wherein the detection arrangement detects the different radiation on different portions of the same detector.

20. The apparatus according to claim 13, wherein the detection arrangement detects the different radiations during different non-overlapping spans of time.

21. The apparatus according to claim 13, wherein the detection arrangement is configured to distinguish between the different radiations by passing the different radiations through optical color filters.

22. The apparatus according to claim 20, wherein the detection arrangement is configured to distinguish between the different radiations by temporally switching between active illumination sources with every image sensor detection event.

23. The apparatus according to claim 22, wherein the time delay between successive image sensor detection is reduce by use of a frame transfer camera or double-shutter camera.

24. The apparatus according to claim 17, wherein the computer arrangement is configured to generate further image information from the image information generated regarding the detection of the different second electro-magnetic radiations.

25. The apparatus according to claim 17, wherein a direction of one of the different radiations is different from a direction of another one of the different radiations.

26. The apparatus according to claim 17, wherein the direction of the different radiations is along a direction of extension of the at least one channel.

27. The apparatus according to claim 17, wherein the direction of the different radiations is orthogonal to a direction of extension of the at least one channel.

28. The apparatus according to claim 13, wherein the light sources include at least four light sources which generate at least four different radiations.

29. The apparatus according to claim 28, wherein a direction of two of the different radiations is orthogonal to a direction of another two of the different radiations.

30. A method for obtaining image information for at least one portion of at least one biological structure, comprising:

providing the at least one biological structure that includes a first flowing structure and a second non-flowing structure in at least one channel of at least one;

providing at least one first electro-magnetic radiation to the at least one channel; detecting at least one second electro-magnetic radiation which is an interaction between the first structure, the second structure, the at least one channel and the at least one first electro-magnetic radiation; and

generating the image information regarding the at least one portion based on the detected at least one second electro-magnetic radiation and further information regarding at least one characteristic of the at least one channel.

31. The method according to claim 30, wherein a wavelength of each of the first and second electro-magnetic radiations are substantially the same.

32. The apparatus according to claim 30, wherein the first structure includes at least one of blood, leukocytes, serum or platelets.

33. The apparatus according to claim 30, wherein the second structure includes cells which are provided substantially stationary at at least one wall of the at least one channel.

34. The apparatus according to claim 33, wherein cells are substantially fixed to the at least one wall of the at least one channel.

35. The apparatus according to claim 34, wherein at least one area of the cells includes at least one of a monolayer of cells or endothelial cells.

36. The apparatus according to claim 30, wherein the at least one housing has at least two walls, wherein at least one of the walls has an index of refraction that is substantially different from an index of refraction of the first structure.

37. The apparatus according to claim 36, wherein at least one of the walls contains a coating that increases a reflectivity of an electromagnetic radiation that impacts upon the wall.

38. The apparatus according to claim 30, wherein the illumination arrangement is configured to provide the at least one first electro-magnetic radiation at at least two different locations

39. The apparatus according to claim 38, wherein the at least two different locations are illuminated using different illumination sources.

40. The apparatus according to claim 39, wherein the at least two different illumination sources have different wavelength bands distinguishable by optical filters.

41. The apparatus according to claim 38, wherein the at least two different locations are illuminated using the same illumination source and an optical arrangement to determine the illumination location.

42. The apparatus according to claim 38, wherein the detection arrangement is configured to detect the different radiations separately from one another.