Patent Description:
Light microscopy is based on propagating light from an illuminated sample which is passed through a set of lenses resulting in an enlarged view of the desired object. This basic principle is used in a variety of microscopic techniques but suffers from a range of deficiencies such as limited resolution and reduced image clarity. The optical resolution in light microscopy is due to diffraction of light and therefore objects smaller than <NUM> are difficult to resolve. This is even worse in the z direction (optic axis), where this limit is extended to <NUM> or more. Nevertheless, many cellular structures and components are often smaller than this optical resolution limit and determining the properties of biomolecules such as proteins in their natural environment is important when analysing their function and elucidating cellular processes. The application of microscopes in life and material science is ever increasing and methods allowing the imaging of small objects under physiologically conditions are highly desirable. Resolution in live samples is generally lower than that in fixed specimens because of the size of the sample, the scattering of tissue, lack of pigmentation and the movement of cellular components.

Illumination techniques such as STimulated Emission Depletion (STED) microscopy, Structured Illumination Microscopy (SIM), or single-molecule-based (SM) techniques (PALM/STORM) have revolutionised microscopy and enabled so called ultra-high resolution. Although these techniques offer clear advantages in terms of spatial resolution over the traditional illumination methods, creation of these images require complex instrumentation and data analysis. These techniques suffer from deep imaging capabilities of live biological samples.

Fluorescence Light-Sheet microscopy techniques have become increasingly popular and are more suitable for imaging live cells. The idea behind light-sheet-based microscopy techniques is to illuminate only a thin layer of the sample from the side, vertical to the direction of observation in a well-defined volume around the focal plane of the detection optics. This technique does not require the use of strong lasers making it minimal invasive and reducing photobleaching.

In a widely adopted light-sheet technique Selective Plane Illumination Microscopy (SPIM) cylindrical optics or scanning through galvanometric mirrors are used to create a sheet of light of varying thickness and can be adapted to different sample sizes: for smaller samples (<NUM>-<NUM>), the light-sheet can be made very thin (~<NUM>), whereas for larger samples (<NUM>-<NUM>), the sheet has to be thicker (∼<NUM>-<NUM>) to remain relatively uniform across the field of view.

In contrast to the detection system used in epifluorescence microscopy, where a single objective lens is used to both illuminate the sample and to collect its fluorescence along the same path, SPIM comprises: (<NUM>) a detection lens horizontally aligned and immersed in a fluid-filled chamber, with a sample embedded in a transparent gel and immersed in the chamber medium held from the top; (<NUM>) an excitation lens to illuminate the sample perpendicularly to the optical axis of the detection lens; and (<NUM>) single cylindrical lens, or galvanometric mirrors, forming the light-sheet inside the chamber through the excitation lens. A stack of images is acquired by moving the sample in a stepwise fashion along the detection axis.

Although Fluorescence Light-Sheet microscopy addresses in principle some of the limitations encountered by other techniques, complex machinery and difficult set ups make this method unsuitable for routine laboratory practise. As described above, Fluorescence Light-Sheet microscopy requires <NUM> objectives to be placed perpendicularly and close to the sample, which besides the distinctive machinery requires also special sample holders and prevents using high NA objective and regular coverslips. It is apparent that there is no optimal solution which can address the issue of imaging in 3D an entire single cell with best possible nanometric resolution provided by SM-based super-resolution microscopy.

This disclosure relates to a device for containing a sample which is adapted to fit to any microscope assembly and provide light sheet microscopy of a sample or samples contained in the device. The device includes a sample well wherein one or more sides of the well are provided with an angled reflective surface adapted to reflect a light sheet transversely through a sample to provide a fluorescence image detectable by a single objective. The light sheet and fluorescence collection are performed through the same objective. The device provides a simplified and inexpensive solution to the aforesaid problems associated with high resolution fluorescence microscopy. The disclosure provides a single objective SPIM [soSPIM] approach and allows performing SPIM imaging on a standard inverted microscope by virtue of an array micro-mirrored chip. The detection and excitations are performed through the same and unique single objective. The device can be scaled to include variable size reflective surfaces (e.g. from <NUM> microns to <NUM>) and using the appropriate magnification objectives (e.g. from 100X to 10X), the soSPIM system allows 3D SPIM from 3D high- and super-resolution of a single cell, up to the whole organism level, [for example embryo imaging], on the same instrument.

The disclosure demonstrates 3D imaging capabilities using 100X, 60X, 40X 20X and 10X objectives with excellent resolution and SM-based super-resolution microscopy. Advantageously, 3D optical sectioning using the device does not require moving the sample, but only the objective and the light sheet, allowing acquisition speeds comparable with other imaging techniques such as spinning-disc microscopy. Moreover, the use of arrayed devices allows simultaneous imaging of multiple cells. This provides the capability to image multiple single cells simultaneously to dramatically reduce the acquisition time and improve imaging throughput. The arrayed devices can contain thousands of single cell wells facilitating sample processing of cells and even whole organisms, such as embryos.

According to an aspect of the invention there is provided a sample holding device for use in transverse illumination of a sample or sub-components of a sample comprising: a support substrate comprising a sample well adapted to contain and be compatible with said sample wherein said well is provided on at least one wall with an angled reflective surface adjacent said sample well which when in use directs a transverse light beam from a light source through a sample contained within said sample well to provide substantially transverse illumination of a sample contained therein and imaging the sample using a single objective.

Reference to "sample or sub-components" includes whole cells or sub-cellular parts and also whole organisms (or sub-organism parts) such as embryos.

In a preferred embodiment of the invention said well comprises at least two angled reflective surfaces wherein said surfaces are positioned substantially opposite each other and defining a space in which said sample is placed.

In a preferred embodiment of the invention said well is a channel comprising two angled reflective surfaces wherein said surfaces are positioned substantially opposite each other and defining a space in which said sample is placed.

In a further preferred embodiment of the invention the first and/or second angled reflective surface is angled between about <NUM>° to <NUM>°.

In a preferred embodiment of the invention said angled reflective surface has an angle selected from the group consisting of: <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>° or <NUM>° +/- <NUM>%.

In a preferred embodiment of the invention said angled reflective surface is between about <NUM>°-<NUM>° +/- <NUM>%.

In a preferred embodiment of the invention said angled reflective surface is angled at about <NUM>° +/- <NUM>%.

In a preferred embodiment of the invention said reflective surface is provided as a metal deposition on all or part of said angular surface[s].

In a preferred embodiment of the invention said reflective surface comprises gold.

In an alternative preferred embodiment of the invention said reflective surface comprises chromium.

In a preferred embodiment of the invention said reflective surface comprises a mixture of deposited metals. Preferably said mixture comprises chromium and gold.

In a preferred embodiment of the invention said support substrate is wholly or partly opaque.

In an alternative preferred embodiment of the invention said support substrate is wholly or partly transparent.

In a preferred embodiment of the invention said support substrate is a composite comprising at least first and second parts comprising at least first and second polymers wherein said first part forms a body of the support substrate and comprising said first polymer and said second part forms a sample well and comprising said second polymer.

In a preferred embodiment of the invention said first part has a higher reflective index when compared to said second part.

In preferred embodiment of the invention said angled reflective surface does not comprise a deposited reflective metallized surface and said transverse light beam is reflected by total internal reflection.

In a preferred embodiment of the invention the reflective index of said first part is between about <NUM> and <NUM> +/- <NUM>%.

Preferably the refractive index of said second part is about <NUM> +/- <NUM>%.

In a preferred embodiment of the invention said sample comprises a cell or cells. Preferably said cell or cells are live.

In an alternative preferred embodiment of the invention said cell or cells are fixed.

In a preferred embodiment of the invention said device comprises a plurality of sample wells of similar or identical dimensions and arranged in an array and adapted for sequential or simultaneous analysis of samples contained within said sample wells.

In a preferred embodiment of the invention said device is fabricated from a UV curable polymer.

In a preferred embodiment of the invention said device is fabricated from an acrylate based polymer.

In a preferred embodiment of the invention said acrylate based polymer is a polyacrylate.

In a preferred embodiment of the invention said device is fabricated from a polycarbonate base polymer.

In an alternative preferred embodiment of the invention said device comprises a polystyrene polymer.

In an alternative preferred embodiment of the invention said device is fabricated from an elastomeric polymer.

In a preferred embodiment of the invention said elastomeric material is an organic silicone based polymer.

In a preferred embodiment of the invention said organic silicone based polymer is polydimethylsiloxane.

In a preferred embodiment of the invention said device is fabricated from a polymeric material that has a refractive index matched to cell culture medium to provide an optically clear device.

In a preferred embodiment of the invention said device is further provided with a removable lid contacting the opening of the device sample well and when in use creating a contained sample well to contain a sample.

In a preferred embodiment of the invention the height, length and width of said sample well is at least <NUM>.

In a further preferred embodiment of the invention the height or length or width of said sample well is between <NUM> and <NUM>.

In a preferred embodiment of the invention the height and/or length and/or width of said sample well is selected from the group: at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

Alternatively, the height and/or length and/or width of said sample well is selected from the group: at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or at least <NUM>.

It will be apparent that the device according to the invention can be fabricated and adapted to receive samples such as single cells, or larger whole tissue or organism samples which can be imaged by soSPIM using reflective surfaces according to the invention.

According to an aspect of the invention there is provided a sample holding device according to the invention for use in light microscopy.

According to a further aspect of the invention there is provided a sample holding device according to the invention for use in fluorescence microscopy.

In a preferred embodiment of the invention said device is for use in light sheet microscopy.

Preferably said device is for use in Selective Plane Illumination Microscopy.

According to a further aspect of the invention there is provided a microscope assembly comprising a sample holding device according to the invention.

In a preferred embodiment of the invention said microscope assembly is a light microscope assembly.

In an alternative preferred embodiment of the invention said microscope assembly is a fluorescence and/or light microscope assembly.

In a preferred embodiment of the invention said microscope assembly is adapted for light sheet microscopy.

Preferably, said light sheet microscopy is Selective Plane Illumination Microscopy.

In a preferred embodiment of the invention said assembly includes a variable focus lens which when in use controls the focal point of a light sheet.

According to a further aspect of the invention there is provided a method to image a biological sample using a microscope assembly comprising the steps:.

In a preferred method of the invention said light source is selected from the group consisting of: a Gaussian beam, a Gaussian light sheet, a Bessel beam.

In a preferred method of the invention said method is Selective Plane Illumination Microscopy.

Preferably Selective Plane Illumination Microscopy uses a Gaussian light sheet light source.

In a preferred method of the invention said microscope assembly comprises a variable focus lens which controls the focal point of a light sheet generated by said light source. According to a further aspect of the invention there is provided a screening method to monitor the effect of a test agent on cell function comprising:.

In a preferred embodiment of the invention said device comprises a cell array and is adapted to be read by an array reader.

A number of methods are known which image and extract information concerning the spatial and temporal changes occurring in cells expressing markers of gene expression. Moreover, <CIT> discloses optical systems for determining the distribution or activity of fluorescent reporter molecules in cells for screening large numbers of agents for biological activity. The systems disclosed in the above patents also describe a computerised method for processing, storing and displaying the data generated. The screening of large numbers of agents requires preparing arrays of cells for the handling of cells and the administration of agents. The sample holding device according to the invention can be used for compatibility with automated loading and robotic handling systems. Typically, high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, bioluminescence) followed by integration of the signals from each well containing the cells, agent and indicator compound.

According to a further aspect of the invention there is provided the use of a sample holding device according to the invention in the diagnosis or prognosis of disease. According to a further aspect of the invention there is provided a method for the diagnosis or prognosis of disease comprising the steps:.

According to a further aspect of the invention there is provided a method for the fabrication of a sample holding device for use in light microscopy of a biological sample comprising the steps:.

In a preferred method of the invention said metal is gold or chromium.

In a preferred embodiment of the invention said deposited metal is a combination of chromium and gold.

In a preferred method of the invention said metal is deposited by thermal evaporation.

According to an aspect of the invention there is provided a sample holding device obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided a kit comprising:.

"Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

An embodiment of the invention will now be described by example only and with reference to the following figures;.

Silicon wet chemical etching is a commonly used method for the fabrication of Optical Micro Electro-Mechanical Systems (MOEMS), for it requires low-cost equipment and it allows high throughput production of structures with a fine definition of spatial geometries [<NUM>, <NUM>]. The etching rate of Si depends strongly on crystallographic orientation and etching conditions: composition and temperature of the chemical solution, among others parameters, allow to select the emerging crystal planes. By selecting the wafer orientation, the geometry of the opening in the masking layer and the chemistry of the etching, many different structures were shown to be achievable [<NUM>, <NUM>, <NUM>].

Our application requires the definition of a slanted mirror, with an angle of <NUM>° toward the plane surface. The mirroring surface needs to be as smooth as to create a uniform illumination sheet when enlightened with a scanning laser beam. A roughness of the surface is then acceptable, provided that its rms value is well below the wavelength of the light used. Both these conditions are granted if a suitable wet etching process is selected. In the following (see <NUM>° slanted mirror fabrication in Methods) one possible process is described and examples of the obtained structures are shown. Finally, in Fabrication of structured polymeric sheets, we show how these structures could be used as a template for the micro-fabrication of our final device.

Silicon wafer with (<NUM>) orientation, single side polished and thermally oxidized (<NUM> thick) were bought from commercial provider (Bonda Technology Pte Ltd <NUM> Anson Road, #<NUM>-<NUM> International Plaza, Singapore <NUM>). Opening in the oxide layer were defined by optical lithography and Reactive Ion Etching (RIE) as follows. AZ 5214E (MicroChemicals GmbH, Nicolaus-Otto-Str. <NUM> D-<NUM> Ulm, Germany) resist was spin coated at <NUM> rpm and soft baked for <NUM> at <NUM>° C on a hot plate, for a final thickness of ~ <NUM>. The resist was exposed to the i-line of a mercury arc lamp, with an energy dose of <NUM> mJ/cm<NUM>. Development is done by immersion in AZ400 developer diluted to <NUM>:<NUM> in water for <NUM>, rinsing in water and final drying with gentle nitrogen blow. The lithographed resist layer was then used as a mask for a RIE step; CF<NUM> (<NUM> sccm)+ O<NUM> (<NUM> sccm) gas mixture at <NUM>*<NUM>-<NUM> mbar with <NUM> W applied power generating a plasma with a bias of <NUM> V was applied for <NUM>, removing the oxide layer from the cleared areas. Stripping of the resist led to the wafer ready for wet etching of Si.

Mirroring surfaces with a <NUM>° slant angle toward the (<NUM>) surface are obtained if (<NUM>) planes emerge during the wet etching process. The etching rate of different crystal planes (referred as vhkl in the following) could be tailored by selecting the appropriate chemistry and geometry. Two conditions needs to be fulfilled: <NUM>) the grooves opened in the oxide masking layer should be oriented with an angle of <NUM>° (in the plane) by respect to the <<NUM>> main flat on the (<NUM>) wafer; <NUM>) the chemical composition of the etching solution should comprise in addition to the alkaline agent (e.g. KOH or TMAH) an organic surfactanti. We used two different chemistries, in which v<NUM> > v<NUM> > v<NUM>. Wet etching A was <NUM> KOH + <NUM> IPA alcohol (water solution) at <NUM>° C; wet etching B was TMAH <NUM>% in water + <NUM> ppm Triton© surfactant at <NUM>° C. Planes (<NUM>) are always the slowest etched, thus for very prolonged etching time, eventually all the initial structures will collapse to a rectangular groove delimited by (<NUM>) planes (forming and angle of <NUM>° toward (<NUM>) planes). <FIG> shows typical results of an etching prolonged for 1h30 min. In both cases, the quality of the mirroring surfaces was good enough for optical application.

The fabrication process leading to a structured polymeric sheet is detailed in the following. Briefly, the silicon device produced with wet etching is used as a master for the replica (with inverted tone) of a PDMS intermediate mold, which is again replicated in the final polymeric sheet exploiting capillarity filling and UV curing. This double-step replication process allows for the sequential multiplication of the obtained devices: at every replica-step one "mother mold" is reproduced with inverted tone in many "daughter devices". <FIG> is a schematic of the process.

Silicon (<NUM>) wafer, structured as previously reported, are used as starting substrate (<FIG>). The remaining oxide layer, after wet etching, is again used as the masking layer for an etching process. Spin coating of positive photo-resist (same as before, i.e. AS 5214E) and UV lithography will produce after development aligned opening in between two consecutive mirrors. A switched plasma etching process [<NUM>] in an ICP reactor will then transfer the pattern into the silicon, without affecting the mirroring surfaces due to the protective resist layer: switched processes (also known as Bosch process) are optimized for high selectivity of silicon toward the protective resist layer. After removing of both resist and oxide, the structured silicon mould presents micro-wells enclosed by mirrors. From this first generation mould, a second generation of intermediate molds is then produced by PDMS casting and curing. In order to increase the quality of the PDMS replica and the life-time of the silicon mould, an anti-stick layer is introduced to silicon surface by mean of vapor phase silanization [<NUM>]. Many replicas could be produced with a single silicon mould, allowing for a fabrication scheme targeting high throughput (2c). The PDMS intermediate mould is then used to produce the final device. PDMS is pressed in contact to a glass cover slide, to which it sticks enough to define connected cavities, accessible by the open ending of each groove. Capillarity is then exploited to fill these cavities by a UV-curable liquid polymer. Exposure to UV through the glass cover slide ends with the curing of the polymer and thus the achievement of a self-standing polymeric sheet with micro-wells laterally enclosed by <NUM>° slanted surfaces. The last fabrication step is the coating of this sheet with a metallic layer. The sheet is inserted into an UHV metal evaporator, with the mirroring surfaces oriented to the evaporation source. Different metals could be used, that produce the final required reflectivity, but Cr is normally the preferred one because of its good adhesive properties also with plastic materials. A layer of <NUM>-<NUM> thick Cr is deposited by mean of thermal or E-gun evaporation (<FIG>). Since the sheet does not present top or bottom capping walls, Cr does not deposit inside the wells: vertical walls are usually very well preserved from deposition by the geometry of a UHV deposition chamber. The device is subsequently flipped on a coverslip and the groves interstices are filled with a UV curable resist and cured. It provides a protective layer to the mirrors. The wells are left empty since they do not reach out of the device. A final metal etching step is used to remove the potential metallic coatings inside the wells. Proper standard etching reagent is used depending on the coating metal used.

The soSPIM excitation beam steering system (<FIG>) was adapted on a conventional inverted microscope (inverted Nikon Ti-E). Illumination lasers (<NUM>@<NUM> mW, <NUM>@<NUM> mW, <NUM>@<NUM> mW and <NUM>@<NUM> mW) were collimated and collinearly combined via dichroic beam splitters and coupled into a single mode optical fiber for spatial filtering and convenient alignment. An acousto-optic tunable filter (AOTF) was used to select one or more wavelengths, control intensities and provide on-off modulation. An achromatic reflective collimator (Thorlabs RC02APC-P01) was used to produce a collimated <NUM> wide laser beam at the output of the optical fiber for all lasers. A laser beam telescopic expander, (Thorlabs AC254-<NUM>-A, focal length <NUM> and Thorlabs AC254-<NUM>-A, focal length <NUM>) providing a 2x magnification of the excitation beam may be inserted into the optical path to vary the diameter, or numerical aperture, of the excitation beam.

The laser beam is sent to an x-axis galvanometric mirror (XG), which is imaged onto a conjugated y-axis galvanometric mirror (YG) by relay lenses (Thorlabs AC254-<NUM>-A, focal length <NUM> both). The laser beam is then imaged on a focus tunable lens (VL) (Optotune, Custom EL-<NUM>-<NUM> focal lens from -<NUM> to +<NUM>) by relay lenses (Thorlabs, AC245-<NUM>-A, focal length <NUM> both). The focus tunable lens is finally imaged and centered onto the back focal plane (BFP) of a high numerical aperture microscope objective (CFI Plan Apochromat VC 60x WI N. <NUM>, or CFI Plan Apochromat VC 100x Oil N. <NUM>, Nikon) by a third telescope (Thorlabs, AC254-<NUM>-A and the tube lens of the microscope, focal length <NUM> both). These successive conjugations and centering steps enable a laser beam to be obtained that is collinear to the optical axis of the microscope objective when imaged through the objective, regardless of its radial position in the image plane.

A sample holder with <NUM>° micro-mirroring surfaces on top of the objective enables the excitation beam to reflect perpendicular to the optical axis of the objective. Scanning the excitation beam along the Y- direction enables the creation of a light sheet that penetrates the sample perpendicular to the optical axis of the microscope objective. Displacing the excitation beam along the X-direction in turn enables the depth at which the light sheet penetrates into the sample to vary. The sample holder is placed on an axial translation piezo stage (Physik Instrument, P-<NUM> Plnano - <NUM>) that enables the objective focal plane to be positioned according to the depth of the light sheet (<FIG>).

For super-resolution acquisition a cylindrical lens (CL) (Thorlabs ACY254-<NUM>-A, focal length <NUM>) is inserted into the excitation path. This enables the laser beam to focus in a single direction onto the BFP of the objective, creating a continuous illumination light sheet without scanning the laser beam on the mirror. The cylindrical lens is mounted in a rotational mount in order to align the large dimension of the light sheet with the long axis of the mirror, if needed.

The fluorescence signal is collected by the microscope tube lens, through the same high numerical aperture objective and captured with an EMCCD camera (Evolve <NUM>, Photometrics). For super resolution acquisition, we used the 100x oil objective. This allowed us to optimize the pixel size in the imaging plane to <NUM>.

A second CCD camera (Hamamatsu, Orca Flash <NUM>) coupled with a <NUM>. 45x magnification lens (Nikon), which provides a large field of view, was used to position and image the <NUM>° micro-mirror according to the sample (<FIG>). This camera was also used to define both the scanning direction of the excitation beam, in order to create the light sheet depending of the orientation of the mirror, and the movement of the light sheet along the perpendicular axis of the mirror, to vary its final depth into the sample. The position of the micro-mirror outside the field of view of the EMCCD allows for both an increase in the available field of view for imaging, and a decrease in the background noise created by the reflection of the excitation beam onto the micro-mirror.

The light sheet, created by scanning a focussed Gaussian beam, or by the focussing of a Gaussian beam through a cylindrical lens, could be considered as the volume <NUM>ω0x2Zx1 surrounding the focalization point of the excitation beam, where □<NUM> and ZR are the waist and the Rayleigh length of the excitation beam respectively, and <NUM> the width given either by the scanning properties or by the cylindrical lens <NUM>. In a common SPIM architecture, the light sheet is positioned on the focal plane of the excitation objective. However, in the soSPIM architecture, this would mean the light sheet is localized on the reflection point of the excitation beam on the <NUM>° micro-mirror. In order to displace the light sheet away from the micro-mirror and position it on the biological sample, a defocusing system has been implemented. It is composed of a divergent lens with a fast, electrically driven tunable focusing mechanism. (Optotune, Custom EL-<NUM>-<NUM>-C-VIS-LD). The focal range of this system is from +<NUM> to -<NUM> conjugated to the BFP of the objective. This system enables the position of the light sheet to vary up to <NUM>/<NUM> from the micro-mirror position, which is in agreement with the field of view of a 60x/100x magnification objective respectively. Such a defocusing system enables the light sheet to be positioned on the biological sample regardless of its position in the field of view of the EMCCD camera. The visualisation of the excitation beam through a fluorescent solution enabled the precise calibration of the beam in relation to the position of the light sheet, according to the micro-mirror, and the focal length of the tunable lens.

Moreover, the focus tunable lens is used to compensate for the displacement of the light sheet position, which may result from the axial movement of the objective when changing the imaging plane depth. Indeed, without compensation, the radial displacement of the light sheet position will be equal to the axial displacement of the objective (<FIG>). In order to ensure the thinnest part of the light sheet is always positioned on the biological sample, we compensate the displacement of the objective by focusing the excitation beam according to the movement of the objective (<FIG>). This compensation ensures the light sheet displacement is less than <NUM>% for imaging planes ranging from <NUM> to <NUM>.

Precise conjugation and centering of the XG and YG galvanometers, as well as the focus tunable lens, was carried out according to the BFP of the objective. This is essential in ensuring the light sheet is perpendicular to the optical axis of the microscope objective after reflection on the <NUM>° micro-mirrors, regardless of its reflection position on the micro-mirror. Conjugation was achieved by collimating the laser beam after each relay lens with a shearing interferometer (Shear plate SI035, Thorlabs) mounted in place of the microscope objective. Aligning the optical elements with the center of the objective BFP was achieved by iterative centering steps between the BFP and the image of the beam reflected off a flat mirror that was positioned perpendicular to the microscopes optical axis at the BFP. Slight deviations from the <NUM>° angle of the micro-mirror with respect to the optical axis of the microscope objective was compensated for by slightly decentering the laser beam on the BFP without modifying the conjugations.

The fabrication process of the silicon chips displaying <NUM>° micro-mirroring surfaces and micro-wells is represented in <FIG>. The micro-mirroring surfaces are produced in oriented silicon wafers by anisotropic etching in alkaline solutions (such as KOH or TMAH). The <NUM>° surfaces are achieved by preventing a fast etching of oriented crystal planes with the use of a surfactant, which acts as a preferential protection layer <NUM>, <NUM>, <NUM>. By tuning the etching conditions (i.e. alkaline and surfactant concentration and temperature of the etching bath), it is possible to reduce the etching rate of planes to be slower than for <<NUM>>, while the <<NUM>> always remain as the slowest. If the open areas on the wafer, cleared for the etching, are oriented on the plane <NUM>° away from the <<NUM>> reference flat of the wafer, then the exposed <<NUM>> planes are preserved and the progressive etching of <<NUM>> planes reveals a cavity flanked by exactly <NUM>° slanted surfaces.

The silicon wafer displaying <NUM>° grooves could then be directly used as a mirroring device, as represented in <FIG>. In this case, a drop of suspended cells was deposited on a clean #<NUM> coverslip and the silicon device was pressed in close contact onto the coverslip and sealed with common varnish.

A more sophisticated approach consists of designing a polymer-based device with micro-wells flanked by <NUM>° micro-mirrors, as described in <FIG> and represented in Figure 3C-D. After producing the <NUM>° surfaces as discussed, micro-wells were realized by dry etching (<FIG>). The silicon wafer is then replicated in PDMS (<NUM> Sylgard, Dow Corning), which can be used tens of times for the production of plastic devices with a UV-curable polymer coated onto standard coverslips (NOA polymer, Norland product), when a capillary filling process is implemented (<FIG>). To increase the reflectivity of the plastic surface, a Cr layer is deposited by thermal evaporation in an ultra-high vacuum chamber (<FIG>). The micro-mirrors are finally protected by a second layer of UV-curable polymer and the metal coating within the micro-wells is removed by wet etching (<FIG>). The device is then washed several times with ultra-pure water and incubated overnight with <NUM>. 2x Pluronic solution (F127, Sigma) for surface passivation. The coverslips are finally sealed in a bottom free <NUM> plastic- dish that allows easy cell culture within the device (<FIG>).

S180 cells stably expressing E-Cadherin-GFP were a kind gift of Jean-Paul Thiery (Institute of Molecular Cell Biology, A*STAR). A clonal U2-OS stable cell line expressing pDendra2-Fibrillarin (Evrogen, Cat#FP826-d, dendra2 fused to N-terminus of fibrillarin) was established with the U2-OS osteosarcoma cell line (ATCC HTB-<NUM>). This was maintained at <NUM> in a <NUM>% CO2-humidified incubator. One day prior transfection, <NUM> well dishes were plated at <NUM> × <NUM> cells/dish. Cells were transfected in CM-Mc medium using jetPRIME DNA transfection reagent (Polyplus Transfection) in a <NUM>:<NUM> ratio. The following day, transfected cells were transferred into <NUM> diameter culture dishes. Clone selection was then performed in CM-Mc containing <NUM>/mL of G418. Ten to fifteen days after transfection, clones were chosen under a fluorescence microscope, to ensure reliable and proper fluorescence localization. Selected clones were then isolated and transferred to <NUM> well dishes for expansion and frozen in culture medium containing <NUM>% DMSO. Further selection with G418 was omitted after the next thawing without any loss of fluorescence.

S180 cells were cultured in High-Glucose DMEM (Sigma) supplemented with <NUM>% FBS (Sigma), <NUM>% GlutaMAX (Sigma) and <NUM>% penicillin/streptomycin (Sigma). U2-OS cells were cultured in CM-MC medium composed of McCoy's 5A medium (Life Technologies), supplemented with <NUM>% FBS (Sigma), <NUM>% GlutaMAX (Sigma), <NUM>% non-essential amino acids (Life technologies), and <NUM>% penicillin/streptomycin (Sigma).

The day before imaging, S180 cells were cultured in <NUM> plastic dishes to ensure they reached <NUM>% confluency the day of imaging. The cells were then washed two times with 1x PBS (Sigma) and immersed in <NUM> CO2 independent cell culture medium, which was used as imaging medium. The cells were detached mechanically by pipetting the culture medium several times and placed in an incubator for <NUM>. A drop of the suspended cells was then deposited on a clean coverslip and a silicon mask displaying <NUM>° micro-mirroring surfaces was pressed and sealed onto the coverslips with varnish. The cells were directly imaged on the microscope. For experiments using microwells, a drop of suspended cells was deposited in the microwells and the device was placed in the incubator for <NUM> to <NUM>, allowing the cells to fill the microwells. The microwells were then washed one time with imaging medium and filled with <NUM> of new imaging medium before being placed on the microscope.

U2-OS cells were fixed in -<NUM> methanol on the day of imaging. Once detached by a <NUM>. 2x Trypsin solution (Sigma diluted in PBS) the cells were allowed to round up in the incubator for <NUM> in complete medium. The cells were then centrifuged for <NUM> at <NUM> rpm and re-suspended in PBS for washing. They were centrifuged again for <NUM> at <NUM> rpm and re-suspended in -<NUM> methanol for <NUM> at -<NUM>. The cells were then washed <NUM> times in PBS and re-suspended in PBS for imaging as described earlier.

Images were acquired on a regular inverted microscope (Nikon TiE) adapted for soSPIM illumination. Images were collected in streaming mode with an EMCCD camera (Evolve <NUM>, Photometrics). The acquisition was steered via the MetaMorph software (Molecular Devices). The beam steering system, described in the soSPIM set-up section, was synchronized using custom software within MetaMorph. A <NUM> photoactivation laser and a <NUM> excitation laser were used and directed toward the objective with a custom dual band cube filter (Exc: ZET <NUM>/<NUM>/561x triple band laser, Dichroique: ZT <NUM>/<NUM>/<NUM> rpc triple band laser, Em: ZET <NUM>/<NUM> double band laser tirf, Chroma). Single-molecule localization and super-resolution image reconstruction was achieved using the WaveTracer module <NUM> and a wavelet-based analysis method <NUM>.

We used this device to perform selective plane illumination through a single objective to image a full drosophila embryo at various magnifications (10X, 20X, 40x) (<FIG>). The transverse illumination is performed either by a scanning a single beam or a static sheet light that is steered in the horizontal plane. Due to the <NUM>° reflection it results in an horizontal plane illumination of the sample placed in the well by the selective plane illumination. The fluorescence signal is collected through the same objective. This device allows us to transform any standard microscope into a SPIM microscope provided that the right illumination shaping is implemented. In particular the control of the dynamic de-focalisation of the laser beam is crucial to ensure the focalisation of the laser sheet onto the optical axis of the objective. The implementation is illustrated in <FIG>.

As a second example we demonstrate how 3D imaging of single cell doublets can be achieved using wells of appropriate sizes (<FIG>). We demonstrate how a single high numerical aperture objective can be used to produce 3D optical sectioning by SPIM with disposable coverslips as well as a specific beam steering add-on unit. The light sheet diameter was set to obtain the best optical contrast depending of the size of the cells.

The axial extension of the light sheet was modified by adjusting the beam size at the back focal plane of the objective (<FIG>). In this study, we used light sheets ranging from <NUM>×<NUM>×<NUM> (length × width × thickness) to <NUM>×<NUM>×<NUM> with a 60x WI <NUM> NA objective at a distance of <NUM> to <NUM> from the micro-mirror. The light sheet thickness compares favorably with those obtained for single cell SPIM (IML-SPIM <NUM>: <NUM> to <NUM> ; Bessel beam <NUM>: <NUM> to <NUM> ; iSPIM <NUM>: <NUM>; RSLM <NUM>: <NUM>). Images of a single cell or cell doublets is illustrated on <FIG>. Sectionning capabilities equal that of Spinning disc microscopy in terms of resolution and imaging speed. Simultaneous imaging of <NUM> cells in <NUM> different wells can also be achieved and is demonstrated in <FIG>.

We further demonstrated the capability of the device to perform super resolution imaging deep in the sample (<FIG>). Live S180 cell doublets labeled with E-Cadherin-GFP, and U2-OS cells stably expressing fibrilarin-Dendra2 were used. We demonstrated that soSPIM is capable of achieving single-molecule-based super-resolution microscopy in 3D, up to <NUM> above the coverslips, using PALM <NUM>,<NUM>. In this case, Fibrilarin- Dendra2 photoconvertible proteins were simultaneously converted at <NUM> and excited at <NUM> by the multi-wavelength light sheet.

Due to the high numerical aperture objective, as well as the perpendicular illumination, which permitted a specific and confined activation, single-molecule detection with high signal to noise ratio was achieved. We successfully reconstructed super-resolution intensity images of nucleoli in suspended U2- OS cells ten microns above the coverslip. <FIG> were taken on planes (<NUM> and <NUM>) above the coverslip. The super-resolution images were reconstructed from <NUM>,<NUM> (resp. <NUM>,<NUM>) single-molecule localizations extracted from <NUM>,<NUM> frames of 80x80 pixels acquired at <NUM> fps (<FIG>). This revealed sub-nuclear structures below the diffraction limit. The median number of photons above the background per localization event was measured to <NUM> leading to a theoretical lateral resolution of <NUM> <NUM> (<FIG>). This resolution compares to that of other super-resolution methods, including SPIM <NUM>,<NUM> but is lower than conventional PALM when imaging close to the coverslip. This is mainly due to optical aberrations arising from imaging in 3D, deep in the sample. It is not inherently stemming from the illumination scheme.

Claim 1:
A microscope assembly for imaging a biological sample comprising:
an objective;
a light source configured to illuminate the sample through said objective and generate a light sheet;
a sample holding device comprising:
a support substrate comprising a sample well adapted to contain and be compatible with said sample, wherein said sample well is provided on at least one wall with an angled reflective surface; wherein:
said angled reflective surface is configured to reflect said light sheet through a part of said sample contained within said sample well to provide substantially transverse illumination of said sample contained therein and
said objective is configured to collect light emitted by said sample.