WO2015030678A1 - Micro-textured surface with integrated micro-mirrors for 3d multi-scale microscopy - Google Patents
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- WO2015030678A1 WO2015030678A1 PCT/SG2014/000405 SG2014000405W WO2015030678A1 WO 2015030678 A1 WO2015030678 A1 WO 2015030678A1 SG 2014000405 W SG2014000405 W SG 2014000405W WO 2015030678 A1 WO2015030678 A1 WO 2015030678A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/168—Specific optical properties, e.g. reflective coatings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/068—Optics, miscellaneous
Definitions
- This disclosure relates to a device for containing a sample which is adapted to fit to any microscope assembly and provide transverse illumination of a sample or samples through a single objective; uses of the device in light/fluorescence microscopy, in particular but not exclusively Selective Plane Illumination Microscopy [SPIM]; and methods to fabricate the device.
- SPIM Selective Plane Illumination Microscopy
- 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 250 nm are difficult to resolve. This is even worse in the z direction (optic axis), where this limit is extended to 500nm 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.
- 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.
- SPIM Selective Plane Illumination Microscopy
- 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 (20-100 ⁇ ), the light-sheet can be made very thin ( ⁇ 1 pm), whereas for larger samples (1-5 mm), the sheet has to be thicker (-5-10 ⁇ ) to remain relatively uniform across the field of view.
- SPIM comprises: (1) 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; (2) an excitation lens to illuminate the sample perpendicularly to the optical axis of the detection lens; and (3) 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.
- 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 2 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 20 microns to 2 mm) 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.
- 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.
- 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.
- 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.
- sample or sub-components includes whole cells or sub-cellular parts and also whole organisms (or sub-organism parts) such as embryos.
- 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.
- 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.
- first and/or second angled reflective surface is angled between about 20° to 80°.
- said angled reflective surface has an angle selected from the group consisting of: 25°, 30°, 35°, 45°, 50°, 55°, 60°, 65°, 70°, 75° or 80° +/- 5%.
- said angled reflective surface is between about 44°-46° +/- 5%
- said angled reflective surface is angled at about 45° +/- 5%.
- said reflective surface is provided as a metal deposition on all or part of said angular surface[s].
- said reflective surface comprises gold.
- said reflective surface comprises chromium
- said reflective surface comprises a mixture of deposited metals.
- said mixture comprises chromium and gold.
- said support substrate is wholly or partly opaque.
- said support substrate is wholly or partly transparent.
- 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.
- said first part has a higher reflective index when compared to said second part.
- said angled reflective surface does not comprise a deposited reflective metallized surface and said transverse light beam is reflected by total internal reflection.
- the reflective index of said first part is between about 1.40 and 1.59 +/- 5%
- the refractive index of said second part is about 1.33 +/- 5%.
- said sample comprises a cell or cells.
- said cell or cells are live.
- said cell or cells are fixed.
- 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.
- said device is fabricated from a UV curable polymer.
- said device is fabricated from an acrylate based polymer.
- said acrylate based polymer is a polyacrylate.
- said device is fabricated from a polycarbonate base polymer.
- said device comprises a polystyrene polymer. In an alternative preferred embodiment of the invention said device is fabricated from an elastomeric polymer.
- said elastomeric material is an organic silicone based polymer.
- said organic silicone based polymer is polydimethylsiloxane.
- said device is fabricated from a polymeric material that has a refractive index matched to cell culture medium to provide an optically clear device.
- 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.
- the height, length and width of said sample well is at least 10pm. In a further preferred embodiment of the invention the height or length or width of said sample well is between 0pm and 2000pm.
- the height and/or length and/or width of said sample well is selected from the group: at least 50pm, 50 ⁇ , 100pm, 200 ⁇ , 300pm, 400pm, 500pm, 600pm, 700pm, 800pm, 900pm or 1000pm.
- the height and/or length and/or width of said sample well is selected from the group: at least 1 100pm, 1200pm, 1300pm, 1400pm, 1500pm, 1600pm, 1700pm, 1800pm, 1900pm or at least 2000pm.
- 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.
- a sample holding device for use in light microscopy.
- a sample holding device for use in fluorescence microscopy.
- said device is for use in light sheet microscopy.
- said device is for use in Selective Plane Illumination Microscopy.
- a microscope assembly comprising a sample holding device according to the invention.
- said microscope assembly is a light microscope assembly.
- 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.
- said light sheet microscopy is Selective Plane Illumination Microscopy.
- said assembly includes a variable focus lens which when in use controls the focal point of a light sheet.
- a method to image a biological sample using a microscope assembly comprising the steps: i) providing a device according to the invention comprising one or more samples;
- said light source is selected from the group consisting of: a Gaussian beam, a Gaussian light sheet, a Bessel beam.
- said method is Selective Plane Illumination Microscopy.
- Selective Plane Illumination Microscopy uses a Gaussian light sheet light source.
- said microscope assembly comprises a variable focus lens which controls the focal point of a light sheet generated by said light source.
- a screening method to monitor the effect of a test agent on cell function comprising: i) providing a sample holding device according to the invention comprising one or more wells wherein said wells comprise one or more cell-types; ii) contacting the wellfs] with an agent to be tested; and
- said device comprises a cell array and is adapted to be read by an array reader.
- a method for the diagnosis or prognosis of disease comprising the steps: i) providing an isolated cell sample from a subject and placing the sample in a sample holding device according to the invention;
- a method for the fabrication of a sample holding device for use in light microscopy of a biological sample comprising the steps: i) providing a sample holding device comprising: a support substrate comprising one or more sample wells adapted to contain and be compatible with a sample wherein said well is provided on at least one wall with an angled surface adjacent said sample well; and ii) depositing on at least the angled surface of said device a metal to provide a reflective surface.
- 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.
- said acrylate based polymer is a polyacrylate.
- said device is fabricated from a polycarbonate base polymer.
- said device is fabricated from an elastomeric polymer.
- said elastomeric material is an organic silicone based polymer.
- said organic silicone based polymer is polydimethylsiloxane.
- said metal is gold or chromium.
- said deposited metal is a combination of chromium and gold.
- said metal is deposited by thermal evaporation.
- a sample holding device obtained or obtainable by the method according to the invention.
- kits comprising: i) a device comprising a support substrate comprising one or more sample wells adapted to contain and be compatible with a sample wherein said well is provided on at least one wall with an angled surface adjacent said sample well;
- a closure adapted to fit the opening of the device sample well.
- Figure 1 illustrates: (A) SEM images of micro-grooves displaying 45° surfaces realized by anisotropic KOH wet etching on a silicon wafer. (B) SEM images of a silicon wafer displaying 45° surfaces and two different sizes of micro-wells (60x300 ⁇ and 25x25 ⁇ ). (C) Transmission image of the final device in UV-curable polymer with metallized 45° surfaces and sealed in a 30 mm bottom free plastic dish;
- Figure 2 illustrates the fabrication process for the final polymeric device.
- opening windows are defined on a (100) oxidized silicon wafer by UV-photolithography process.
- (A-ii.) (100) oxidized wafer is structured with grooves defined by 45° slanted (110) crystal planes as reported in the text (a).
- (B-i.) Opening in the remaining oxide layer are then defined by UV lithography and RIE;
- (B-ii.) Micro-wells are then carved by plasma dry etching in the silicon using the structured oxide as a mask (b).
- B-iii. The photolithographic resist and the oxidized layer is then removed.
- Figure 3 illustrates the fabrication process and imaging procedure of biological sample.
- Etched silicon wafers were used as 45° micro-mirrors to create a light sheet that passed perpendicular to the optical axis of the objective.
- An anisotropic wet etching process is first used to create 45° surfaces on a silicon wafer (i). The wafer is then directly pressed and sealed on a clean coverslip where a drop of the biological sample to be imaged has been deposited (ii). The biological sample is ready for imaging (iii).
- B The fabrication process of a UV-curable polymer based device displaying micro-wells flanked with 45° metallized surfaces.
- Anisotropic and dry etching processes are used to defined 45° surfaces and micro-wells respectively within a silicon wafer (i).
- a PDMS replica of this wafer is realized (ii) and used to reproduce the wafer shape on a coverslip in a UV-curable polymer via a capillary filling process (iii).
- the polymer surface is then metallized with a chrome layer under high vacuum (iv) and a final step enables protection of the 45° surfaces and to etch the metal deposited in the micro-wells (v).
- the UV-curable device is finally ready for cell culture and imaging (iv.);
- Figure 4 illustrates the soSPIM principle.
- A Schematic representation of the soSPIM principle composed of a micro-fabricated sample-holder displaying 45° micro-mirroring surfaces on a conventional coverslip, an excitation beam steering system and an optical detection path that allows the positioning of the mirror and the fluorescence signal detection.
- B Representation of the sectioning capabilities produced by the displacement ( ⁇ ) of the excitation beam along the mirror combined with the axial positioning of the objective ( ⁇ ) and the defocusing of the excitation beam ( ⁇ ) to maintain the position of the light sheet thinnest part on the biological samlpe.
- C Schematic representation of the beam steering system.
- XG x-axis galvanometric
- RL relay lenses
- YG y-axis galvanometric mirror
- VL focus tunable lens
- a cylindrical lens (CL) can be inserted into the optical path for direct light sheet creation.
- (XG), (YG), (VL) are all conjugated to the back focal plane (BFP) of the objective so that the excitation beam is always emitted parallel to the objective optical axis.
- BFP back focal plane
- TL enables the beam diameter to vary at the BFP;
- Figure 5 illustrates the characterization of the beam thickness and of the defocusing system.
- Dotted grey (resp. black) lines represent the field of view of the light sheet defined by 2 times de Rayleigh length for an excitation beam with 4 mm (resp. 2 mm) diameter at the BFP.
- B Axial beam profile for different divergence strength of the electrically driven variable focal lens (VL).
- the position of the focal point can be adjusted by dynamically changing the divergence of the laser beam without change in the width and length of the laser sheet.
- C Linear dependence of the position of the focalization point of the excitation beam, ie the thinnest part of the light sheet, with the current in the electrically driven variable focal lens;
- Figure 6 illustrates deviation compensation of the light sheet position.
- B Light sheet profiles at different depths within the sample without compensation for the axial movement of the objective.
- Figure 7 illustrates 3D volume imaging of whole Drosophilia Embryos.
- A Schematic representation of the soSPIM principle for imaging thick samples such as Drosophila embryos with millimetre sized microfabricated 45° mirrors. The Drosophila embryos are embedded, at different development stages, in 1 % low melting point agar gel between two metalized 45° mirrors and imaged with the soSPIM microsope.
- Figure 8 illustrates 3D high- and super-resolution capabilities of the soSPIM method.
- A Comparison between widefield acquisition and the sectioning capabilities of soSPIM and spinning disk methods on S180 cells expressing the membrane protein E-Cadherin-GFP. The inset represents the averaged line scans within the red boxes for comparison of sectioning capabilities.
- B Upper panel: soSPIM optical sections of a S180 cell doublet expressing the membrane protein E-Cadherin-GFP positioned within a 24x24 pm 2 microwells flanked by a 45° mirroring surface. The upper right panel represents the Maximum Intensity Projection (MIP) along the y-axis.
- MIP Maximum Intensity Projection
- Inset zoom on a single- molecule detected in the white box.
- Upper right panel single-molecule intensity profile (red line) of the single-molecule represented in the (E) inset along the white box, with its Gaussian fit (black line) given a FWHM of 2.51 pixels (402 nm).
- Lower panel histogram of the collected number of photons per localization during the PALM acquisition of panel (D).
- Figure 9 illustrates 3D volume imaging at the cellular level within micro-wells.
- A Simultaneous two colours soSPIM optical sections of three S180 cells expressing the membrane protein E-Cadherin-GFP (upper panel) and the cortical actin protein F- -Tractin-RFP (middle panel) positioned each in a different 24x24 ⁇ 2 micro-well.
- the scanning light-sheet illuminates only the zones defined by the red bars and the readout speed of the camera is equivalent to single well imaging.
- the lower panel is a phase contrast image of the three wells. Scale bars are 5 ⁇ . Materials and Methods
- 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 [70, 71].
- 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 [48, 72, 73] .
- Our application requires the definition of a slanted mirror, with an angle of 45° 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.
- a suitable wet etching process is selected.
- Silicon wafer with (100) orientation, single side polished and thermally oxidized (300 nm thick) were bought from commercial provider (Bonda Technology Pte Ltd 10 Anson Road, #18-18 International Plaza, Singapore 079903). Opening in the oxide layer were defined by optical lithography and Reactive Ion Etching (RIE) as follows.
- AZ 5214E MicroChemicals GmbH, Nicolaus-Otto-Str. 39 D-89079 Ulm, Germany
- resist was spin coated at 3000 rpm and soft baked for 1 min at 125° C on a hot plate, for a final thickness of ⁇ 1.1 m. The resist was exposed to the Mine of a mercury arc lamp, with an energy dose of 100 mJ/cm 2 .
- v hM The etching rate of different crystal planes
- the alkaline agent e.g. KOH or TMAH
- wet etching A was 3M KOH + 1M IPA alcohol (water solution) at 75° C; wet etching B was TMAH 30% in water + 200 ppm Triton ® surfactant at 75° C.
- Planes (1 1) are always the slowest etched, thus for very prolonged etching time, eventually all the initial structures will collapse to a rectangular groove delimited by (111 ) planes (forming and angle of 54.7° toward (100) planes).
- Figure 1 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.
- Silicon (100) wafer, structured as previously reported, are used as starting substrate ( Figure 2A).
- 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 [75] 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.
- switched processes also known as Bosch process
- a second generation of intermediate molds is then produced by PDMS casting and curing.
- an anti-stick layer is introduced to silicon surface by mean of vapor phase silanization [76].
- 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.
- the soSPIM excitation beam steering system ( Figure 4A-C) was adapted on a conventional inverted microscope (inverted Nikon Ti-E). Illumination lasers (405 nm@200 mW, 488 nm@200 mW, 561 nm@200 mW and 635 nm@ 175 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.
- AOTF acousto-optic tunable filter
- An achromatic reflective collimator (Thorlabs RC02APC- P01) was used to produce a collimated 2 mm wide laser beam at the output of the optical fiber for all lasers.
- a laser beam telescopic expander, (Thorlabs AC254-050-A, focal length 50 mm and Thorlabs AC254-100-A, focal length 100 mm) 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 AC254-050-A, focal length 50 mm both).
- the laser beam is then imaged on a focus tunable lens (VL) (Optotune, Custom EL-30-10 focal lens from -80 mm to +1000 mm) by relay lenses (Thorlabs, AC245-050-A, focal length 50 mm 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 Wl N.A.
- a sample holder with 45° micro-mirroring surfaces on top of the objective enab'es 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-736 Plnano - 200 ⁇ ) that enables the objective focal plane to be positioned according to the depth of the light sheet ( Figure 4B).
- a cylindrical lens (Thorlabs ACY254-150-A, focal length 150 mm) 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 512, Photometries).
- EMCCD camera Evolve 512, Photometries
- 100x oil objective we used the 100x oil objective. This allowed us to optimize the pixel size in the imaging plane to 60 nm.
- a second CCD camera (Hamamatsu, Orca Flash 2.8) coupled with a 0.45x magnification lens (Nikon), which provides a large field of view, was used to position and image the 45° micro-mirror according to the sample ( Figure 4A).
- 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 2 ⁇ a0x2ZRxl surrounding the focalization point of the excitation beam, where d0 and ZR are the waist and the Rayleigh length of the excitation beam respectively, and 1 the width given either by the scanning properties or by the cylindrical lens 78 .
- 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 45° micro-mirror.
- a defocusing system 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-10-30-C-VIS-LD). The focal range of this system is from +1000 mm to -80 mm conjugated to the BFP of the objective. This system enables the position of the light sheet to vary up to 280/260 ⁇ 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.
- 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 ( Figure 5A-B).
- the displacement of the objective by focusing the excitation beam according to the movement of the objective ( Figure 5C- D). This compensation ensures the light sheet displacement is less than 5% for imaging planes ranging from 0 to 40 ⁇ .
- Shear plate SI035, Thorlabs shearing interferometer
- 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 45° 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 45° micro-mirroring surfaces and micro-wells is represented in Figure 3.
- the micro-mirroring surfaces are produced in oriented silicon wafers by anisotropic etching in alkaline solutions (such as KOH or TMAH).
- alkaline solutions such as KOH or TMAH.
- the 45° 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 48, 9, 79 .
- a surfactant acts as a preferential protection layer 48, 9, 79 .
- the silicon wafer displaying 45° grooves could then be directly used as a mirroring device, as represented in Figure 3A.
- a drop of suspended cells was deposited on a clean #1.5 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 45° micro-mirrors, as described in Figure 3B and represented in Figure 3C-D. After producing the 45° surfaces as discussed, micro-wells were realized by dry etching (Figure 3B-i). The silicon wafer is then replicated in PD S (184 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 (Figure 3B-ii and iii).
- PD S 184 Sylgard, Dow Corning
- a Cr layer is deposited by thermal evaporation in an ultra-high vacuum chamber (Figure 3B- vi).
- 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 ( Figure 3B-v).
- the device is then washed several times with ultra-pure water and incubated overnight with 0.2x Pluronic solution (F127, Sigma) for surface passivation.
- the coverslips are finally sealed in a bottom free 35 mm plastic- dish that allows easy cell culture within the device (Figure 1C).
- CM-Mc CM-Mc medium
- jetPRIME DNA transfection reagent Polyplus Transfection
- CM-Mc containing 0.4 mg/mL of G418.
- clones were chosen under a fluorescence microscope, to ensure reliable and proper fluorescence localization. Selected clones were then isolated and transferred to 24 well dishes for expansion and frozen in culture medium containing 10% DMSO. Further selection with G418 was omitted after the next thawing without any loss of fluorescence.
- S180 cells were cultured in High-Glucose DM EM (Sigma) supplemented with 10% FBS (Sigma), 1% GlutaMAX (Sigma) and 1% penicillin/streptomycin (Sigma).
- U2-OS cells were cultured in CM-MC medium composed of McCoy's 5A medium (Life Technologies), supplemented with 10% FBS (Sigma), 1% GlutaMAX (Sigma), 1% non-essential amino acids (Life technologies), and 1% penicillin/streptomycin (Sigma).
- S180 cells were cultured in 35 mm plastic dishes to ensure they reached 70% confluency the day of imaging.
- the cells were then washed two times with 1x PBS (Sigma) and immersed in 1 mL C02 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 10 min.
- a drop of the suspended cells was then deposited on a clean coverslip and a silicon mask displaying 45° micro-mirroring surfaces was pressed and sealed onto the coverslips with varnish.
- the cells were directly imaged on the microscope.
- microwells For experiments using microwells, a drop of suspended cells was deposited in the microwells and the device was placed in the incubator for 10 to 20 min, allowing the cells to fill the microwells. The microwells were then washed one time with imaging medium and filled with 2 mL of new imaging medium before being placed on the microscope.
- U2-OS cells were fixed in -20°C methanol on the day of imaging. Once detached by a 0.2x Trypsin solution (Sigma diluted in PBS) the cells were allowed to round up in the incubator for 10 min in complete medium. The cells were then centrifuged for 3 min at 1000 rpm and re-suspended in PBS for washing. They were centrifuged again for 3 min at 1000 rpm and re-suspended in -20°C methanol for 5 min at -20°C. The cells were then washed 2 times in PBS and re-suspended in PBS for imaging as described earlier.
- Image acquisition and processing for super-resolution imaging 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 512, Photometries). 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 405 nm photoactivation laser and a 561 nm excitation laser were used and directed toward the objective with a custom dual band cube filter (Exc: ZET 405/488/561x triple band laser, Dichroique: ZT 405/488/561 rpc triple band laser, Em: ZET 488/561 double band laser tirf, Chroma).
- Single-molecule localization and super-resolution image reconstruction was achieved using the WaveTracer module 20 and a wavelet-based analysis method 21.
- TNF-alpha influences the lateral dynamics of TNF receptor I in living cells. Biochimica et biophysica acta 1823, 1984 (Oct, 2012).
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