WO2011022824A1

WO2011022824A1 - Rapid multi-component optical protein patterning by photobleaching

Info

Publication number: WO2011022824A1
Application number: PCT/CA2010/001315
Authority: WO
Inventors: Santiago Costantino; Jonathan BÉLISLE
Original assignee: Hôpital Maisonneuve-Rosemont
Priority date: 2009-08-24
Filing date: 2010-08-24
Publication date: 2011-03-03

Abstract

A method is provided for optical protein patterning by photobleaching using a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes. The method comprises positioning a substrate surface with a protein-coupled fluorescent dye at the focal plane of the microscope objective, providing at least one image within the focal plane of the camera port, and illuminating the image so as to photobleach the fluorescent dye. The image is projected on the substrate surface via the microscope objective thereby binding the protein-coupled fluorescent dye on the substrate surface according to the pattern of the image projected thereon. A device is provided for optical protein patterning by photobleaching also adapted for use with a microscope. The device comprises at least one spatial filter and at least one light source. The spatial filter includes an image that is projected at the focal plane of the camera port. The light source illuminates the spatial filter thereby projecting the image at the focal plane of the objective.

Description

TITLE

RAPID MULTI-COMPONENT OPTICAL PROTEIN PATTERNING BY

PHOTOBLEACHING

FIELD

[0001] The present disclosure broadly relates to optical protein patterning. More specifically, but not exclusively, the present disclosure relates to a method for adsorbing proteins with control over their individual concentrations and spatial distributions.

BACKGROUND

[0002] Spatial distributions of proteins are crucial for development, growth and normal life of organisms. Protein patterning may allow reproducing complex cellular environments in miniature version. Protein patterning may be seen as protein immobilization within a specific localization in a two or three dimensional space. Immobilization of proteins may be performed for example, by light with the advantage of being compatible with biological functions.

[0003] Laser-Assisted Protein Adsorption by Photobleaching (LAPAP) was recently introduced in an attempt to overcome most of the limitations that typically constrained the applications of protein patterning [I]. Briefly, in LAPAP, biotin-4-fluorescein (B4F) molecules are photobleached by a blue laser in order to adsorb them to a glass surface. The photobleaching of fluorescein creates free radicals that react and bind the B4F molecules to the surface when they are close enough [2]. The laser is focused at the glass surface and scanned across the substrate while varying the illumination intensity and scanning velocity to produce a predetermined pattern. In a subsequent step, streptavidin is incubated for attachment to biotin and to finally link biotinylated proteins or antibodies. Simplicity and robustness made LAPAP the alternative to established techniques to obtain protein gradients and even arbitrary spatial distributions of molecules [3].

[0004] LAPAP-generated protein gradients have proven to be more stable and reproducible as compared to micropipette puffed generated gradients [4]. Unlike the patterns generated by microcontact printing, the protein concentrations (i.e. gradients) can be varied over three orders of magnitude using LAPAP [5]. A very simple setup and even the ability to be implemented on commercial confocal microscopes, make LAPAP advantageous over microfluidic implementations [6]. Finally, the use of low power visible lasers and commercially available reagents is simpler than U. V. illumination [7].

[0005] The present disclosure refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY

[0006] As broadly claimed, the present disclosure relates to optical protein patterning.

[0007] In an embodiment, the present disclosure relates to a method for optical protein patterning using a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the method comprising:

[0008] positioning a substrate surface with a sample protein at the focal plane of the microscope objective;

[0009] providing at least one image within the focal plane of the camera port; and [0010] illuminating the image so as to project the image on the substrate surface via the microscope objective thereby binding the protein sample on the substrate surface according to the pattern of the image projected thereon.

[0011] In an embodiment, the method further comprises treating the substrate surface with a blocking solution prior to adding the sample protein thereon.

[0012] In an embodiment, the method further comprises revealing the protein pattern formed on the substrate surface by adding fluorophores that bind to the protein patterned on the substrate surface. In an embodiment, the fluorophores comprise fluorescently conjugated antibodies. In an embodiment, the protein pattern on the substrate surface is imaged by fluorescence microscopy.

[0013] In an embodiment, the method further comprises modulating the concentration of protein that binds to the substrate surface. In an embodiment, the image comprises pixels and modulating the concentration of protein that binds to the substrate surface is provided by modulating the pixel value within the image. In an embodiment, modulating the concentration of protein that binds to the substrate surface is provided by modulating the time period of illumination. In an embodiment, modulating the concentration of protein that binds to the substrate surface is provided by modulating the intensity of illumination.

[0014] In an embodiment, the method further comprises binding fluorophores to the substrate surface prior to adding the sample protein thereon. In an embodiment, the fluorophores comprise fluorescently conjugated antibodies. In an embodiment, the fluorophores are bound to the substrate surface by standard illumination. In an embodiment, the fluorophores are bound to the substrate surface by widefield illumination.

[0015] In an embodiment, at least two protein samples are added on the substrate surface. In an embodiment, multiple wavelengths are used for illuminating the image. In an embodiment, the method further comprises controlling the binding of each protein sample by the intensity of a corresponding wavelength. In an embodiment, the protein samples comprise distinct fluorophores. In an embodiment, the distinct fluorophores comprise respective fluorescently conjugated antibodies. In an embodiment, the distinct fluorophores have distinct absorption spectra maxima.

[0016] In an embodiment, the method further comprises adding at least one other protein sample on the substrate surface following patterning of the initial protein sample during the illumination step. In an embodiment, the method further comprises at least another illumination step so as to bind the other protein sample on the substrate surface according to the pattern of the image projected thereon. In an embodiment, another image is illuminated for patterning of the other protein sample. In an embodiment, the wavelength used to illuminate the image for the other protein sample is distinct from the wavelength used to illuminate the image for the initial protein sample.

[0017] In an embodiment, a plurality of images are provided within the focal plane of the camera port. In an embodiment, each image of the plurality is illuminated by a respective light source.

[0018] In an embodiment, illumination is provided by a plurality of light sources.

[0019] In an embodiment, the present disclosure relates to a substrate surface having a protein pattern obtained by the method of the present disclosure. In an embodiment, the present disclosure relates to a biosensor obtained by the method of the present disclosure.

[0020] In an embodiment, the present disclosure relates to a device for optical protein patterning adapted for use with a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the device comprising:

[0021] at least one spatial filter comprising an image for being projected at the focal plane of the camera port; and

[0022] at least one a light source for illuminating the spatial filter,

[0023] wherein the illumination of the spatial filter when positioned at the focal point of the camera port provides for projecting the image at the focal plane of the objective.

[0024] In an embodiment, the present disclosure relates to an assembly for optical protein patterning comprising:

[0025] a microscope having an objective and at least one camera port, the objective and the camera port defining respective focal planes; and

[0026] a device comprising at least one spatial filter comprising an image for being projected at the focal plane of the camera port and at least one a light source for illuminating the spatial filter,

[0027] wherein the illumination of the spatial filter when positioned at the camera port focal point provides for projecting the image at the focal plane of the objective.

[0028] In an embodiment, the present disclosure relates to a kit for optical protein patterning adapted for use with a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the kit comprises:

[0029] a device comprising at least one spatial filter comprising an iimmaaggee for being projected at the focal plane of the camera port and at least one a light source for il lllnummiinnflattiinngσ t thhee s spnaattiiaall f fiilltteerr.

[0030] wherein the illumination of the spatial filter when positioned at the focal point of the camera port provides for projecting the image at the focal plane of the objective.

[0031] In an embodiment, the device further comprises a magnifying lense providing for projecting the image at the focal plane of the cameral port. In an embodiment the magnifying glass is interposed between the spatial filter and the camera port.

[0032] In an embodiment, the spatial filter is positioned at the focal plane of the camera port.

[0033] In an embodiment, the device further comprises at least one beam expander interposed between the spatial filter and the light source. In an embodiment, the device further comprises at least one collimator interposed between the spatial filter and the light source. In an embodiment, the device further comprises at least one polarizer interposed between the spatial filter and the light source. In an embodiment, the device further comprises at least one polarizer interposed between the spatial filter and the camera port. In an embodiment, the device further comprises at least one dichroic mirror interposed between the spatial filter and the light source. [0034] In an embodiment, the device further comprises a plurality of light sources for illuminating the same spatial filter. In an embodiment, the device further comprises at least one beam expander interposed between the spatial filter and the plurality of light sources. In an embodiment, the device further comprises at least one collimator interposed between the spatial filter and the plurality of light sources. In an embodiment, the device further comprises least one polarizer interposed between the spatial filter and the plurality of light sources. In an embodiment, the device further comprises at least one dichroic mirror interposed between the spatial filter and the plurality of light sources. In an embodiment, the device further comprises a plurality of dichroic mirrors interposed between the spatial filter and the plurality of light sources, each dichroic mirror of the plurality of dichroic mirrors being illuminated by a respective light source of the plurality of light sources, each dichroic mirror deflecting light to illuminate the spatial filter.

[0035] In an embodiment, the device further comprises a plurality of spatial filters and a plurality of light sources. In an embodiment, each light source of the plurality of light sources illuminates a respective spatial filter of the plurality of spatial filters. In an embodiment, the device further comprises at least one beam expander interposed between the plurality of spatial filters and the plurality of light sources. In an embodiment, the device further comprises at least one collimator interposed between the plurality of spatial filters and the plurality of light sources. In an embodiment, the device further comprises at least one polarizer interposed between the plurality of spatial filters and the plurality of light sources. In an embodiment, the device further comprises at least one polarizer interposed between the plurality of spatial filters and the camera port. In an embodiment, the device further comprises at least one dichroic mirror interposed between the spatial filter and the plurality of light sources. In an embodiment, the device further comprises a plurality of dichroic mirrors interposed between the plurality of spatial filters and the plurality of light sources. In an embodiment, each dichroic mirror of the plurality of dichroic mirrors is illuminated by a respective light source of the plurality of light sources, each dichroic mirror deflecting light to illuminate a respective spatial filter of the plurality of spatial filters. In an embodiment, the device further comprises a prism assembly interposed between the plurality of spatial filters and the camera port.

[0036] In an embodiment, the device further comprises a substrate surface for projecting the image thereon. In an embodiment, the substrate surface comprises at least one protein sample, wherein the projection of the image of the substrate surface provides for binding the protein sample to the substrate surface according to the pattern of the image projected thereon.

[0037] In an embodiment, the spatial filter comprises a liquid crystal display. In an embodiment, the spatial filter comprises a photography slide.

[0038] In an embodiment, the light source comprises a laser diode. In an embodiment, the light source comprises a lamp.

[0039] In an embodiment, the device further comprises a connector for coupling the device to the microscope.

[0040] In an embodiment, the device further comprises a housing for the spatial filter and the light source.

[0041] In an embodiment, the microscope further comprises a plurality of camera ports.

[0042] In an embodiment, the present disclosure relates to a method for adsorbing proteins with full control over their individual concentrations and spatial distributions. The method of the present disclosure provides for the generation of protein patterns while simultaneously simplifying and improving fabrication time. In an embodiment, the present disclosure relates to a method providing for the generation of a single protein pattern. Yet moreover, in an embodiment the present disclosure relates to a method providing for the generation of a multi-component protein pattern.

[0043] In an embodiment, the present disclosure relates to widefield illumination LAPAP. In yet a further embodiment, the present disclosure relates to a method for implementing widefield illumination LAPAP using any standard microscope equipped with a camera port.

[0044] In an embodiment, the present disclosure relates to a method for generating protein patterns using a standard microscope equipped with a camera port; the microscope being equipped with a light spatial filter positioned at the focal plane of the microscope; a sample to be patterned positioned at the focal plane of the microscope objective; and a light source, whereby illuminating the light spatial filter with the light source projects an image to be patterned onto the sample.

[0045] In yet a further embodiment of the present disclosure, the widefield illumination LAPAP method comprises the use of chemical reagents commonly found in cellular biology research laboratories.

[0046] In an embodiment, the present disclosure relates to a method for generating protein patterns using widefield illumination LAPAP and antibodies including fragments thereof. In a further embodiment, the present disclosure relates to a method for generating protein patterns using widefield illumination LAPAP and lyophilized antibodies.

[0047] In an embodiment of the present disclosure, the widefield illumination LAPAP method comprises the use of primary and fluorescently tagged antibodies to link the proteins of interest. Yet moreover, in an embodiment of the present disclosure, the widefϊeld illumination LAPAP method comprises the use of primary and fluorescently tagged secondary antibodies to link the proteins of interest. In yet a further embodiment of the present disclosure, the widefield illumination LAPAP method comprises the use of primary and phosphorescently tagged antibodies to link the proteins of interest. Yet moreover, in an embodiment of the present disclosure, the widefield illumination LAPAP method comprises the use of primary and phosphorescently tagged secondary antibodies to link the proteins of interest.

[0048] In an embodiment, the present disclosure relates to a device for optical protein patterning, the device comprising a light spatial filter and at least one light source; and a means for connecting the device to a camera port of a microscope. Connecting the device to the camera port of the microscope provides for positioning the spatial filter at the focal (i.e. image) plane of the microscope and whereby illuminating the light spatial filter with the light source provides for a reduced image to be projected at the focal plane of the microscope objective.

[0049] Finally, in an embodiment, the present disclosure relates to reproducible in vitro assays to create protein patterns.

[0050] The foregoing and other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non- restrictive description of illustrative embodiments thereof, given by way of example only.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In the appended drawings: [0052] FIG. Ia is a schematic illustration of widefield illumination

LAPAP in accordance with an embodiment of the present disclosure; FIG. Ib is an illustration of an Einstein protein pattern produced using widefield illumination LAPAP; FIG. Ic is a schematic illustration of widefield illumination LAPAP in accordance with another embodiment of the present disclosure

[0053] FIG. 2a is an illustration of the fluorescence intensity as a function of the spatial filter's pixel value using widefield illumination LAPAP; the results illustrate that the amount of bound protein can be increased by one order of magnitude. FIG. 2b is an illustration of the fluorescence intensity as a function of the exposure time using widefield illumination LAPAP; the results illustrate that the amount of bound protein can be increased about 35 fold in going from an exposure time of 5 seconds to about 12 minutes. FIG. 2c is an illustration of a modified USAF resolution target produced with widefield illumination LAPAP using a 6Ox objective (1.35 NA); the red rectangle shows a region where 6 lines were separated by 1 Dm (on the left-hand side) and by 1.5 Dm (on the right-hand side). FIG. 2d is an illustration of the average linear profiles of the aforementioned region showing that it is possible to resolve individual lines separated by 1 Dm and 1.5 Dm respectively. Regarding the resolution obtained (FIG. 2c and 2d), it is of note that a line separation of 1 Dm is considerably above the diffraction limit for a 1.35NA objective using 470 nm light source. The smallest feature that can be patterned using the present setup is essentially limited by the pixel size of the spatial filter.

[0054] FIG. 3a is an illustration of the fluorescence intensity as a function of the dwell time for FITC conjugated goat anti-rabbit IgGs bound and patterned to a glass substrate by a focused 473 nm laser and incubated by rabbit anti- laminin, biotin conjugated goat anti -rabbit and streptavidin-Cy5. The semi-log plot of antibody concentration as a function of dwell time shows a dynamic range of close to one order of magnitude. FIG. 3b is an illustration of a log-log plot of antibody concentration as a function of laser power. The plot shows a dynamic range of close to one order of magnitude. The inset shows one of the ten patterns used to obtain the plot.

[0055] FIG. 4 is a schematic illustration of two-component antibody patterns composed of photobleached FITC and Cy 5 conjugated antibodies illuminated simultaneously by 473 and 671 nm lasers (FIG. 4a and FIG. 4b respectively). The blue (473 nm) and red (671 nm) lasers were not superposed for better clarity. The red laser photobleaches and adsorbs Cy5 goat anti-mouse IgG which binds to mouse anti-myc (9El 0) (shown in yellow). Cy5 goat anti-mouse IgG was used to reveal the pattern. The blue laser photobleaches and adsorbs FITC goat anti-rabbit IgG which binds to rabbit anti-laminin (shown in pink). FITC goat anti- rabbit IgG was used to reveal the pattern. FIG. 4c is an illustration showing FITC (shown in green) and Cy5 (shown in red). The bottom-left pattern was obtained by scanning 50 lines by increasing the laser intensity from left to right (blue laser) and increasing the intensity from left to right (red laser). The top-right pattern was obtained by increasing the intensity for both lasers in the same direction giving precisely superposed red and green gradients, therefore appearing yellow. The top- left gradient was obtained using only the blue laser while the bottom-right gradient was obtained using only the red laser.

[0056] FIG. 5 is an illustration of a three-component protein pattern obtained using widefield illumination LAPAP. An image of a circle was positioned top-center of the spatial filter and a sample containing B4F was illuminated to adsorb it onto a glass surface. The sample was then rinsed and a drop of FITC goat anti-rabbit IgG was placed on the sample and illuminated by a bottom-right positioned circle. The sample was again rinsed and a drop of FITC goat anti-mouse IgG was placed on the sample and illuminated by a bottom-left positioned circle. The sample was again rinsed and incubated with a solution containing Streptavidin, mouse ant-myc (9E10) and rabbit anti-thyl. To reveal the pattern, the sample was incubated with B4F (blue circle), TRITC goat ant-mouse IgG (green circle) and Cy5 goat anti-rabbit IgG (red circle).

[0057] FIG. 6a is a schematic illustration of widefield illumination

LAPAP in accordance with a further embodiment of the present disclosure comprising a single light source. FIG. 6b is a schematic illustration of widefield illumination LAPAP in accordance with yet a further embodiment of the present disclosure adapted for the patterning of multi-component protein systems. The embodiment illustrated in FIG. 6b comprises a plurality of light sources and corresponding dichroic mirrors, each of the light sources being configured for the patterning of a specific component of the system. In this embodiment, each of the light sources shares a common spatial filter. FIG. 6c is a schematic illustration of yet a further embodiment of widefield illumination LAPAP adapted for the patterning of multi-component protein systems. The embodiment illustrated in FIG. 6c also comprises a plurality of light sources configured for the patterning of a specific component of the system. In this embodiment, each of the light sources is configured with its own spatial filter.

[0058] FIG. 7 is a schematic illustration of widefield illumination

LAPAP in accordance with an of the present disclosure comprising a single light source.

DETAILED DESCRIPTION

[0059] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this specification pertains. [0060] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0061] As used in this specification and claim(s), the words

"comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0062] The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0063] As used herein, the term "spatial filter" or "light modulator" refers to any device capable of altering or modulating the structure of a beam of light. Non-limiting examples of spatial filters include LCDs and slides. When an LCD is used as the spatial filter, a polarizer is typically associated therewith.

[0064] The present disclosure relates to widefield illumination LAPAP as a method for creating protein patterns with full control over their individual concentrations and spatial distributions. The widefield illumination set-up that was implemented comprised an add-on device that can be coupled to the camera port of any standard microscope. Such a device typically comprises a light spatial filter located at the focal plane of the microscope which is used as an illumination port rather than an imaging port. Illuminating this spatial filter using a laser (diode) produces an image of the spatial filter at the focal plane of the objective. As illustrated in FIG. Ia, a collimated blue diode 10 (470 nm) illuminates a spatial filter 20 which is positioned at the focal plane of the camera port lens 30 of a microscope 60 to produce an image of the spatial filter 20 at the focal plane of the objective 40, which is focused at the top surface 70 of a glass-bottom dish 50. Biotin-4-Fluorescin or FITC conjugated antibodies are then photobleached following the pattern represented in the image sent to the spatial filter 20. An Einstein protein pattern was produced on the top surface of the glass-bottom dish 50 using a 6Ox objective and B4F. The protein pattern was revealed using streptavidin- Cy5 and imaged by fluorescence microscopy (FIG. Ib). Thus, instead of scanning a laser to tailor a desired pattern, the same pattern can be achieved by widefield illumination. Such illumination modality speeds up the process, does not require automation software and requires substantially no optomechanical elements. In fact, the Einstein image was produced following an exposure time of only 5 minutes as opposed to 80 minutes of exposure typically required using standard LAPAP [10].

[0065] FIG. Ic, shows the device 100 coupled to a microscope 60 thereby defining an assembly 102. The device includes a light source 10 which illuminates the spatial filter 20 positioned at the focal plane of the camera port 30 to produce an image of the spatial filter 20 at the focal plane of the objective 40, which is projected on the substrate surface 50. A collimator 104 is interposed between the light source 10 and the spatial filter 20. The device 100 further includes a pair of polarizers 106a and 106b, polarizer 106a is interposed between the spatial filter 20 and the camera port 30 while polarizer 106b is interposed between the spatial filter and the light source 10. The device 100 is housed within a housing 108.

[0066] In order to obtain a perfectly focused image of the spatial filter

20 on the sample, the spatial filter 20 must be precisely placed at the position where the CCD (charge-coupled device) would normally be located if that port were equipped with a camera instead of a patterning setup (i.e. at the focal plane of the camera port lens 30). In an embodiment of the present disclosure, an extended light source is used for illumination the spatial filter 20. Other non-limiting examples of suitable light sources comprise light emitting diodes and lamps.

[0067] Several objects can be used as spatial filters. Non-limiting examples of suitable spatial filters comprise photography slides and liquid crystal displays (LCDs). An advantage achieved by the use of LCDs is that they can be controlled by using a standard VGA (Video Graphics Array) or SVGA (Super Video Graphic Array) computer output that allows for a very simple setup and no need for specialized software.

[0068] Using widefield illumination LAPAP, two options are readily available to vary the protein concentration: using either a grayscale image as a spatial filter or using binary black and white image series. In order to characterize the dynamic range of both possibilities, a pattern of 12 squares was fabricated by generating a greyscale image as a filter where each square had a linearly increasing intensity going from 21 to 255. It was shown that the amount of protein can be modulated by one order of magnitude by changing only the pixel value within the image on the spatial filter (FIG. 2a), where 0 is black and 255 is white. The reduced dynamic range is not a limitation of the new illumination method itself, but is mainly due to the poor polarization efficiency of the low-cost polarizers used, which reduced the contrast ratio of the spatial filter. Nevertheless, the dynamic range obtained is sufficient for most of envisaged biological applications.

[0069] Similarly, a white square filter was used to subsequently fabricate 12 square patterns using a 6Ox 1.35NA objective with various exposure times for each square ranging from 5 seconds to 12 minutes at a constant power of 15.5 DW. The results illustrate that the amount of bound protein can be increased about 35 fold in going from an exposure time of 5 seconds to about 12 minutes (FIG. 2b). To evaluate the amount of bound protein as a function of the exposure time, the bound B4F was revealed using streptavidin Cy5. [0070] In an embodiment, the present disclosure relates to a method for generating protein patterns using widefield illumination LAPAP and antibodies. The use of antibodies provides for specific proteins to be patterned. In an embodiment of the present disclosure, fiuorophores (e.g. fluorescently conjugated antibodies) are first bound to the substrate (e.g. glass surface) by either standard or widefield illumination LAPAP. First binding the fluorescently conjugated antibodies onto the substrate surface allows for a decrease in the number of incubation steps required to produce full protein patterns. In an embodiment of the present disclosure, fluorescently tagged secondary antibodies replace B4F as the molecules that are first bound onto the substrate surface by photobleaching. Primary antibodies and full proteins are subsequently attached to create the desired protein patterns. As illustrated in FIG. 3, patterns of primary rabbit anti-laminin antibodies were produced by first binding FITC (fluorescein isothiocyanate) conjugated goat anti-rabbit IgGs followed by incubation with rabbit anti-laminin, biotin conjugated goat anti-rabbit and streptavidin-Cy5. Different concentrations of antibody pairs were obtained by either changing the laser intensity or the dwell time of the laser. For both cases (fluorescence intensity as a function of laser intensity or dwell time), a dynamic range of close to one order of magnitude was obtained. Since the molecular mass of tagged antibodies is roughly 200 times the mass of B4F, an IgG concentration of 2mg/ml was typically used to obtain a similar amount of fluorescent molecules per volume unit.

[0071] In an embodiment of the present disclosure, multi-component protein patterns are obtained by combining multiple laser wavelengths and different fluorophores. Superposed gradients of two different antibodies can be obtained with full control over their individual concentrations. Provided that the absorption maxima of the two fluorophores are distinct (so that each laser line photobleaches only one specific molecule), the binding of each antibody can be controlled by the intensity of the corresponding wavelength. FIG. 4 provides a schematic illustration of two-component antibody patterns composed of photobleached FITC and Cy5 conjugated antibodies illuminated simultaneously by 473 and 671 nm lasers (FIG. 4a and FIG. 4b respectively). FIG. 4c provides an illustration of a two-component sample depicted in four different gradients. The top-right region appears in yellow as the FITC goat anti-rabbit IgG (green) and Cy5 goat anti-mouse IgG (red) gradients are precisely superposed. The bottom-left pattern shows gradients of opposing slopes, therefore appearing half-green and half-red with fading intensity toward the centre. The remaining two patterns are both obtained using one type of antibody and appear red and green respectively. A small amount of "cross-talk" from one antibody to the other can however be observed. In the locations where only the blue laser is used (FIG. 4c, top left corner) a small amount of Cy5 goat anti-mouse IgG is adsorbed; in the locations where only the red laser is used (FIG. 4c, bottom right corner) a small amount of FITC goat anti-rabbit IgG is adsorbed.

[0072] The width of the absorption spectra constitutes the only limitation to the number of molecules composing the multi-component system. However, the challenge often resides in finding more than two molecules whose absorption spectra do not significant overlap. Indeed, despite the visual results obtained (FIG. 4c), a quantitative analysis of the profile of each fluorescence channel showed some degree of "cross talk". Control experiments were performed in order to obtain an understanding of the binding of Cy 5 goat anti-mouse IgG with the 473nm laser and of the binding of FITC goat anti-rabbit IgG with the 671nm laser. The results indicated that the 671nm laser was not able to bind FITC goat anti-rabbit IgG when it was not mixed in solution with Cy5 goat anti-mouse IgG. The most probable explanation for the cross-talk binding of FITC-goat anti-rabbit IgG illuminated by 671nm laser is the non-specificity of the chemical reaction that allows single Cy5 goat anti-mouse IgG to covalently bind to the surface while also linking FITC-goat anti-rabbit IgGs. For the binding of Cy5-goat anti-mouse IgG with the 473nm laser, it was found that approximately one third of the cross talk is due to direct photobleaching of Cy5 by the 473nm laser. This cross-talk became significant when a third fluorescently tagged antibody was mixed and a 532nm laser was added to the setup. The undesirable mixing of molecular species when adding TRITC conjugated antibodies yielded serious cross talk with the two other tagged IgGs. The use of subsequent illuminations provided a solution to the "cross-talk" problem

[0073] In an embodiment of the present disclosure, multi-component protein patterns are obtained by subsequent illuminations. FITC conjugated antibodies against different antigens were used to obtain three component patterns (FIG. 5). A glass-bottom culture dish was positioned on the microscope at the focal plane of the objective and three circles were patterned by widefield illumination. The dish was clamped to the microscope to avoid displacement during the subsequent illumination and rinsing steps. Subsequent illumination using LAPAP and different conjugated antibodies provided a multi component protein pattern. In an embodiment of the present disclosure, a three-component protein pattern was obtained by sequential photobleaching and by changing the solution between each exposition (mouse anti-myc; rabbit anti-thyl; and streptavidin). FIG. 5 is an illustration of a three-component protein pattern in which streptavidin is revealed by B4F (blue), mouse anti-myc (9E10) by TRITC-goat anti-mouse IgG (green) and rabbit anti-thyl by Cy5-goat anti-rabbit IgG (red). Seven regions are clearly distinguishable in the image.

[0074] In a further embodiment of the present disclosure, and as illustrated in FIG. 6a, the widefield illumination LAPAP set-up comprises a device including a light source and a spatial filter in between which may optionally be disposed one or more light beam expanders or lenses. The device is connected to the camera port of a microscope such that the spatial filter is positioned at the focal plane of the camera port lens. Indeed, in the case where the device comprises a plurality of lenses the position of the focal plane will be shifted. Illuminating the spatial filter using the light source projects the image of the spatial filter at a sample positioned at the focal plane of the microscope objective. Non-limiting examples of suitable light sources comprise lasers, light emitting diodes and any commercially available lamps. FIG. 6b is an illustration of a further embodiment of the widefield illumination LAPAP set-up as contemplated by the present disclosure. In this embodiment a plurality of light sources are used providing for the patterning of multi-component protein systems. Each of the light sources shares a common spatial filter. FIG. 6c is a schematic illustration of yet a further embodiment of widefield illumination LAPAP adapted for the patterning of multi-component protein systems. The embodiment illustrated in FIG. 6c comprises a plurality a light sources each of which is configured for the patterning of a specific component of the system. A plurality of prisms located downstream from the spatial filter combines the plurality of light sources into a multi-wavelength single beam adapted for the patterning of the multi-component protein system. In this embodiment, each of the light sources is configured with its own spatial filter such that a plurality of patterns can be produced on the sample.

[0075] FIG. 7 is a schematic illustration of an assembly 200 for optical protein patterning including a device 202 for optical protein patterning and a microscope 204. The microscope 204 includes an objective 206 and a camera port 208 defining a focal plane 210. A substrate surface such as a dish 212 including a protein sample is positioned at the focal plane of the objective 206. The device 202 includes a light source 214 for illuminating a spatial filter 216. A beam expander 218 can be positioned between the spatial filter 216 and the light source 214. The spatial filter 216 includes an image for being projected at the focal plane 210 of the camera port 208. A magnifying lens 220 shown here interposed between the camera port 208 and the spatial filter 216 provides for projecting the image at the focal plane 210. Therefore, when the light source 214 illuminates the spatial filter 216, the image is projected by the magnifying lens 220 at the focal plane 210 of the camera port 208 to be then projected on the substrate surface 212 via the objective 206.

[0076] EXPERIMENTAL [0077] Widefield LAPAP

[0078] A 470 nm 380 mW blue collimated light-emitting diode (LED)

(Thorlabs, NJ) was used as the light source. An 800 x 600 translucent liquid crystal microdisplay (LCD) (Holoeye, CA) combined with two linear polarizing filters placed on each side with a polarizing efficiency of 95% was used as the spatial filter.

[0079] The LCD was positioned at the right-side port on an Olympus

1X71^® microscope and since it followed the C-mount standard, the LCD was placed at a distance of 17.5 mm from the flange of the camera port. Patterns of 12 squares were fabricated by generating an 8-bit greyscale image as a filter where each square had a linearly increasing intensity going from 21 to 255. The image filter was generated by a custom program in Matlab (Math Works, MA). A B4F solution was exposed for 30 minutes using a 2Ox 0.75NA objective and the pattern subsequently revealed by streptavidin-Cy5.

[0080] An 800 x 600 pixels image of the USAF resolution target was produced on the LCD spatial light modulator. The resolution target was modified by adding 12 white lines of a single pixel thickness, 6 of them separated by 1 black pixel and the remaining six by 2 black pixels.

[0081] Patterns from fluoresccntly tagged antibodies

[0082] In order to minimize non-specific protein adsorption, a blocking solution (1% goat serum and 1% BSA in PBS) was incubated for 30 minutes on a microwell culture dish (MatTek Corporation, MA). A 20 Dl drop of 2 mg/mL of FITC goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA) in 2% BSA and 80% goat serum was placed onto the coverglass of the dish. Patterning was then performed by moving the focus of a 473 nm laser diode across the sample. Rabbit anti-laminin (5 Dg/mL), biotinylated goat anti-rabbit IgG (5 Dg/mL) and streptavidin-Cy5 (5 Dg/mL), all in 3% BSA, were subsequently incubated over a 30 minute period to reveal the pattern obtained by FITC goat anti-rabbit IgG.

[0083] The dynamic range obtained with the antibody was characterized in terms of laser dwell time and power using a standard LAPAP setup [10]. For the dwell time characterization, lines at beam focus velocities ranging from 30 Dm/sec to 1 Dm/sec were scanned at a constant laser power of 160 DW. Following the incubation steps, streptavidin-Cy5 fluorescence was measured and assumed to be proportional to the bound FITC goat anti-rabbit IgG concentration. The laser power characterization was performed by scanning lines at a constant 1 Dm/sec velocity and by increasing the laser power from 1.8 μW to 277.2 μW followed by measuring the streptavidin-Cy5 fluorescence after the last incubation step.

[0084] Single step multi-component antibody patterns

[0085] A blocking solution (1% goat serum and 1% BSA in PBS) was incubated for 30 minutes on a 14 mm micro well culture dish. A drop of FITC goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA) and Cy5 goat anti- mouse IgG (2 mg/mL; each in 4% BSA and 60% goat serum) was placed onto the coverglass of the dish. Patterning was then performed by simultaneously moving the focal point of a pair of laser diodes (473 nm and 671 nm) across the sample (FIG. 4a). A total of four patterns were produced by scanning 50 lines of 25 μM width and by increasing the laser intensity every next line from 0.2 μW to 90.2 μW for the 473 nm laser and by increasing the laser intensity every next line from 0.2 μW to 430 μW for the 671 nm laser. One pattern was made by increasing the intensities of both laser lines in the same direction, giving two superposed gradients. A further pattern was made by increasing the intensity of a first laser line while decreasing the intensity of the second laser line, providing gradients of opposing slopes. The remaining two patterns were made by scanning only one laser line (either the 473 nm or the 671 nm laser) and the presence of non-specific binding assessed. Following photobleaching (i.e. illumination), two incubation steps of 30 minutes were performed in order to reveal the patterns: rabbit anti-laminin (5 μg/mL) and mouse anti-myc (9El 0) (5 μg/mL) in 3% BSA followed by FITC goat anti -rabbit IgG (5 μg/mL) and Cy5 goat anti-mouse IgG (5 μg/mL) in 3% BSA (FIG. 4b).

[0086] Multi-component patterns using subsequent illuminations

[0087] A solution of BSA (3%) was incubated on a coverglass of a micro well culture dish over a period of 30 minutes followed by rinsing with PBS. The culture dish was subsequently positioned on the microscope stage with the top surface of the coverglass positioned at the focal plane of the objective. The dish was clamped to the microscope to avoid displacement during the subsequent illumination and rinsing steps. A drop of B4F (50 μg/mL) in BSA (3%) was placed on the coverglass and exposed by a top-center circle over a period of 15 minutes using widefield LAPAP (blue LED). The sample was then rinsed several times with PBS followed by the deposition of a drop of FITC goat anti-rabbit IgG (2 mg/mL in BSA 2% and goat serum 80%) and illuminated by a bottom-right positioned circle over a period of 30 minutes using widefield LAPAP (blue LED). The sample was again rinsed several times with PBS followed by the deposition of a drop of FITC goat anti-mouse IgG (2 mg/mL in BSA 2% and goat serum 80%) and illuminated by a bottom-left positioned circle over a period of 30 minutes using widefield LAPAP (blue LED). The sample was again rinsed and incubated over a period of 30 minutes with a solution containing Streptavidin (5 μg/mL), mouse ant-myc (9E10) (5 μg/mL), and rabbit anti-thyl (5 μg/mL). Streptavidin is revealed by B4F (blue) (5 μg/mL), mouse anti-myc (9E10) by TRITC-goat anti -mouse IgG (green) (5 μg/mL), and rabbit anti-thyl by Cy5-goat anti-rabbit IgG (red) (5 μg/mL).

[0088] It is to be understood that the disclosure is not limited in its application to the details of construction and parts as described hereinabove. The disclosure is capable of other embodiments and of being practiced in various ways. It is also understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present disclosure has been described hereinabove by way of illustrative embodiments thereof, it can be modified without departing from the spirit, scope and nature as defined in the appended claims.

REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A method for optical protein patterning using a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the method comprising:

positioning a substrate surface with a sample protein at the focal plane of the microscope objective;

providing at least one image within the focal plane of the camera port; and

illuminating the image so as to project the image on the substrate surface via the microscope objective thereby binding the protein sample on the substrate surface according to the pattern of the image projected thereon.

2. A method according to claim 1, further comprising treating the substrate surface with a blocking solution prior to adding the sample protein thereon.

3. A method according to any one of claims 1 or 2, further comprising revealing the protein pattern formed on the substrate surface by adding fluorophores that bind to the protein patterned on the substrate surface.

4. A method according to claim 3, wherein the fluorophores comprise fluorescently conjugated antibodies.

5. A method according to any one of claims 3 or 4, wherein the protein pattern on the substrate surface is imaged by fluorescence microscopy.

6. A method according to any one of claims 1 to 5, further comprising modulating the concentration of protein that binds to the substrate surface.

7. A method according to claim 6, wherein the image comprises pixels, wherein modulating the concentration of protein that binds to the substrate surface is provided by modulating the pixel value within the image.

8. A method according to any one of claims 6 or 7, wherein modulating the concentration of protein that binds to the substrate surface is provided by modulating the time period of illumination.

9. A method according to any of claims 6 to 8, wherein modulating the concentration of protein that binds to the substrate surface is provided by modulating the intensity of illumination.

10. A method according to any one of claims 1 to 9, further comprising binding fluorophores to the substrate surface prior to adding the sample protein thereon.

11. A method according to claim 10, wherein the fluorophores comprise fluorescently conjugated antibodies.

12. A method according to any one of claims 10 or 11, wherein the fluorophores are bound to the substrate surface by standard illumination.

13. A method according to any one of claims 10 or 11, wherein the fluorophores are bound to the substrate surface by widefield illumination.

14. A method according to any one of claim 1 to 13, wherein at least two protein samples are added on the substrate surface.

15. A method according to claim 14, wherein multiple wavelengths are used for illuminating the image.

16. A method according to claim 15, further comprising controlling the binding of each protein sample by the intensity of a corresponding wavelength.

17. A method according to any one of claims 14 to 16, wherein the protein samples comprise distinct fluorophores.

18. A method according to claim 17, wherein the distinct fluorophores comprise respective fluorescently conjugated antibodies.

19. A method according to any one of claims 17 or 18, wherein the distinct fluorophores have distinct absorption spectra maxima.

20. A method according to any one of claims 1 to 19, further comprising adding at least one other protein sample on the substrate surface following patterning of the initial protein sample during the illumination step.

21. A method according to claim 20, further comprising at least another illumination step so as to bind the other protein sample on the substrate surface according to the pattern of the image projected thereon.

22. A method according to claim 21, wherein another image is illuminated for patterning of the other protein sample.

23. A method according to any one of claims 21 or 22, wherein the wavelength used to illuminate the image for the other protein sample is distinct from the wavelength used to illuminate the image for the initial protein sample.

24. A method according to any one of claims 1 to 23, wherein a plurality of images are provided within the focal plane of the camera port.

25. A method according to claim 24, wherein each image of the plurality is illuminated by a respective light source.

26. A method according to any one of claims 1 to 23, wherein illumination is provided by a plurality of light sources.

27. A substrate surface having a protein pattern obtained by the method as defined in any one of claims 1 to 26.

28. A biosensor obtained by the method as defined in any one of claims 1 to 26.

29. A device for optical protein patterning adapted for use with a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the device comprising:

at least one spatial filter comprising an image for being projected in the focal plane of the camera port; and

at least one light source for illuminating the spatial filter,

wherein the illumination of the spatial filter provides for projecting the image at the focal plane of the objective.

30. A device according to claim 29, further comprising a magnifying lens providing for projecting the image at the focal plane of the earner al port.

31. A device according to claim 30, wherein the magnifying glass is interposed between the spatial filter and the camera port.

32. A device according to claim 29, wherein the spatial filter is positioned at the focal plane of the camera port.

33. A device according to any one of claims 29 to 32, further comprising at least one beam expander interposed between the spatial filter and the light source.

34. A device according to any one of claims 29 to 32, further comprising at least one collimator interposed between the spatial filter and the light source.

35. A device according to any one of claims 29 to 32, further comprising at least one polarizer interposed between the spatial filter and the light source.

36. A device according to any one of claims 29 to 35, further comprising at least one polarizer interposed between the spatial filter and the camera port.

37. A device according to any one of claims 29 to 36, further comprising at least one dichroic mirror interposed between the spatial filter and the light source.

38. A device according to any one of claims 29 to 32, further comprising a plurality of light sources for illuminating the same spatial filter.

39. A device according to claim 38, further comprising at least one beam expander interposed between the spatial filter and the plurality of light sources.

40. A device according to claim 38, further comprising at least one collimator interposed between the spatial filter and the plurality of light sources.

41. A device according to any one of claims 38 to 40, further comprising at least one polarizer interposed between the spatial filter and the plurality of light sources.

42. A device according to any one of claims 38 to 41, further comprising at least one dichroic mirror interposed between the spatial filter and the plurality of light sources.

43. A device according to any one of claims 38 to 42, further comprising a plurality of dichroic mirrors interposed between the spatial filter and the plurality of light sources, each dichroic mirror of the plurality of dichroic mirrors being illuminated by a respective light source of the plurality of light sources, each dichroic mirror deflecting light to illuminate the spatial filter.

44. A device according to any one of claims 29 to 32, further comprising a plurality of spatial filters and a plurality of light sources.

45. A device according to claim 44, wherein each light source of the plurality of light sources illuminates a respective spatial filter of the plurality of spatial filters.

46. A device according to any one of claims 44 or 45, further comprising at least one beam expander interposed between the plurality of spatial filters and the plurality of light sources.

47. A device according to any one of claims 44 or 45, further comprising at least one collimator interposed between the plurality of spatial filters and the plurality of light sources.

48. A device according to any one of claims 44 to 47, further comprising at least one polarizer interposed between the plurality of spatial filters and the plurality of light sources. '•

49. A device according to any one of claims 44 to 48, further comprising at least one polarizer interposed between the plurality of spatial filters and the camera port.

50. A device according to any one of claims 44 to 49, further comprising at least one dichroic mirror interposed between the spatial filter and the plurality of light sources.

51. A device according to any one of claims 44 to 48, further comprising a plurality of dichroic mirrors interposed between the plurality of spatial filters and the plurality of light sources.

52. A device according to claim 51, wherein each dichroic mirror of the plurality of dichroic mirrors is illuminated by a respective light source of the plurality of light sources, each dichroic mirror deflecting light to illuminate a respective spatial filter of the plurality of spatial filters.

53. A device according to any one of claims 44 to 47, further comprising a prism assembly interposed between the plurality of spatial filters and the camera port.

54. A device according to any one of claims 29 to 53, further comprising a substrate surface for projecting the image thereon.

55. A device according to claim 54, wherein the substrate surface comprises at least one protein sample, wherein the projection of the image of the substrate surface provides for binding the protein sample to the substrate surface according to the pattern of the image projected thereon.

56. A device according to any one of claims 29 to 55, wherein the spatial filter comprises a liquid crystal display.

57. A device according to any one of claims 29 to 56, wherein the spatial filter comprises a photography slide.

58. A device according to any one of claims 29 to 57, wherein the light source comprises a laser diode.

59. A device according to any one of claims 29 to 57, wherein the light source comprises a lamp.

60. A device according to any one of claims 29 to 59, further comprising a connector for coupling the device to the microscope.

61. A device according to any one of claims 29 to 60, further comprising a housing for the spatial filter and the light source.

62. An assembly for optical protein patterning comprising:

a microscope having an objective and at least one camera port, the objective and the camera port defining respective focal planes; and

a device in accordance with any one of claims 29 to 61,

wherein the illumination of the spatial filter when positioned at the camera port focal point provides for projecting the image at the focal plane of the objective.

63. An assembly according to claim 62, wherein the microscope further comprises a plurality of camera ports.

64. A kit for optical protein patterning adapted for use with a microscope having an objective and a camera port, the objective and the camera port defining respective focal planes, the kit comprising:

- the device of any one of claims 29 to 61,