Optical Imaging Device
The optical imaging device addresses the challenge of non-uniform illumination by forming sub-beams and inducing controlled shifts to ensure consistent excitation patterns, improving image resolution and efficiency.
Patent Information
- Application Number
- JP2024541820
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2022-01-27
- Filing Date
- 2023-01-03
- Publication Date
- 2025-01-24
AI Technical Summary
Existing optical imaging techniques face challenges in achieving uniform structured illumination over a wide field of view, particularly due to the limitations of Gaussian-shaped lighting and high-density fluorescent molecule detection, which affect image quality and resolution.
The optical imaging device employs a system that forms multiple sub-beams from a spatially coherent light beam, interferes them with the sample surface to create an interference pattern, and uses a scanning element to induce controlled shifts, ensuring uniform illumination across the field of view.
This approach achieves uniform structured illumination over a wide field of view, enhancing image resolution and efficiency by maintaining consistent excitation patterns throughout the imaging process.
Smart Images
Figure 2025502215000001_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to the field of optical imaging devices. [Background technology]
[0002] Single-molecule localization microscopy (SMLM) is based on the delay of the stochastic emission of molecules to achieve the emission of single fluorescent molecules. A sample containing fluorescent molecules is illuminated with irradiance adjusted in such a way that on average there is only one active (i.e., emitting) molecule in the focal volume of the observation microscope each time. The images acquired by SMLM present image spots called point spread functions (PSFs), each spot representing the image of a molecule. From the PSF, the position of the molecule can be measured by determining the center of the spot using an algorithm. This position can be determined with an accuracy that can be much better than the diffraction-limited resolution of the microscope. This stochastic process is repeated for tens of thousands of images to obtain a final super-resolution image with high spatial resolution (up to 10 nm). For example, Lelek et.al, Single-molecule localization microscopy, Nature Reviews Method Primers, vol. 1, 39, 2021, Lelek et al. describe the basic principles of SMLM (e.g., different types of molecular labeling, determining the position of the PSF, and the process of reconstructing a super-resolution image from the identification of the PSF).
[0003] Further, Mailfert et.al, A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC, vol. 113, 3, 2018, disclose an algorithm used to determine the PSF. In particular, Mailfert et. al. relate the algorithm to be optimized to obtain higher spatial resolution for samples with a high density of fluorescent molecules. In particular, the algorithm evaluates the probability of detection and the identification uncertainty for each fluorescent molecule so that inaccurate identification can be eliminated when reconstructing a super-resolution image.
[0004] However, high spatial resolution depends on molecular detection and, as a result, on the density of single fluorescent molecules. In particular, the higher the density, the more difficult it is to detect and identify the molecules, and the lower the image quality (due to artifacts such as sample drift and overlapping PSFs). On the other hand, the lower the density of single fluorescent molecules, the higher the spatial resolution.
[0005] Moreover, most SMLM techniques use Gaussian shaped illumination to illuminate the sample. However, Gaussian shaped illumination provides non-uniform excitation and limits the available field of view. Mau et al. in "Mau et al., Fast wide field scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields, Nature Communications, vol. 12, 3077, 2021" disclose a method to achieve uniform illumination. The system is called ASTER (Adaptive Scanning for Adjustable Excitation Area) and is illustrated in FIG. 1. The system includes a laser source LS that emits a Gaussian light beam. The Gaussian beam is focused on two galvanometer scanning mirrors GM to control the illumination at the sample plane. The light beam is focused by a cylindrical lens LS on the back focal plane BFP of the objective lens MO of the microscope MSP. While the light beam maintains its position at the back focal plane, an angular shift applied to the mirror induces a similar angular shift of the back focal plane BFP and a position shift of the beam at the sample plane. Rapid scanning of the Gaussian beam position in a predefined pattern, such as a raster scan or Archimedean spiral, then produces an overall uniform illumination across the field of view when averaged over the camera frame exposure time. The system of FIG. 1 allows for increased accuracy in determining the center of the PSF, a larger field of view, and greater efficiency compared to systems using conventional Gaussian shaped illumination that provides non-uniform excitation.
[0006] The system of FIG. 1 provides a suitable solution for producing uniform illumination (ie, flat-top) over a given field of view.
[0007] Meanwhile, solutions to provide structured illumination techniques with uniform illumination over the entire field of view are still lacking. In fact, most structured illumination techniques provide illumination that is non-uniform due to the Gaussian nature of the initial light beam, or that loses most of the power of the initial light beam (e.g., the expansion of the center of a Gaussian beam). Other homogenization methods are not compatible with some specific imaging techniques. For example, beam homogenization using multimode fibers results in a large number of angles of incidence of the output light beam, which is not compatible with total internal reflection fluorescence (TIRF) imaging.
[0008] The present invention aims to alleviate certain problems of the prior art. To this end, the object of the invention is an optical imaging device similar to the device of figure 1, further comprising an optical component suitable for forming from an illumination light beam a number of sub-beams which are focused in the back focal plane of an objective lens and which interfere at the sample plane to form an interference pattern. The optical component together with the scanning element provides a structured illumination which is uniform over a wide field of view. [Prior art documents] [Non-patent literature]
[0009] [Non-Patent Document 1] Lelek et.al,Single-molecule localization microscopy,Nature Reviews Method Primers,vol.1,39,2021 [Non-Patent Document 2] Mailfert et.al,A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC,vol.113,3,2018 [Non-Patent Document 3] Mau et al.,Fast wide field scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields,Nature Communications,vol.12,3077,2021 Summary of the Invention [Means for solving the problem]
[0010] The object of the invention is to provide an optical imaging device suitable for analyzing a sample, comprising: a light source suitable for emitting a spatially coherent light beam; an optical relay element suitable for focusing a light beam on a focal plane; a lens system having a back focal plane at a focal plane, the lens system being suitable for collimating a light beam and forming a collimated light beam; a sample holder suitable for holding a sample at a sample plane such that a collimated light beam illuminates the sample and triggers emission of sample light; a pixelated photodetector adapted to acquire an image of the sample light at a given frame exposure time; Including, the device is optically positioned in the optical path of the light beam upstream of the optical relay element; an optical component suitable for forming from the light beam a number of sub-beams which are focused in a back focal plane and which interfere at the sample plane to form an interference pattern; a scanning element suitable for inducing, within a frame exposure time, a plurality of controllable shifts of the positions of the sub-beams in the sample plane such that each controllable shift is identical for the sub-beams and such that an average of the sub-beams in the sample plane over the frame exposure time corresponds to an interference pattern being uniformly projected over a predetermined field of view; The optical imaging device further comprises:
[0011] According to an embodiment of the invention, the optical component is a digital micromirror device or a spatial light modulator arranged optically downstream after the scanning element.
[0012] Alternatively, according to another embodiment, the optical component comprises: a beam splitter suitable for splitting the light beam into sub-beams; a phase delay element arranged in the optical path of at least one sub-beam, which is suitable for inducing a controlled phase shift in at least one sub-beam and thus forming a so-called phase-shifted sub-beam and for shifting the fringes of the interference pattern; Preferably, the phase delay element is a fiber optic element or a fiber optic phase shifter connected to a plurality of fiber optic elements directing the phase shifted sub-beams.
[0013] Preferably, in this embodiment, the optical component further comprises a plurality of optical fibre elements, each suitable for directing a respective sub-beam to a so-called downstream end of the optical fibre element, the downstream end being arranged in a pupil plane of a microscope device optically conjugated with the back focal plane. Preferably, the device comprises a double telecentric zoom in the optical paths of the sub-beams, suitable for forming a magnified image in the pupil plane, a controlled change of the zoom magnification altering the distance Δ between each sub-beam in the back focal plane.
[0014] According to an embodiment, the beam splitter is suitable for splitting the light beam into two sub-beams.
[0015] According to another embodiment, the beam splitter is adapted to split the light beam into at least three sub-beams. Preferably, in this embodiment, at least two of the optical fiber elements each have: a primary optical fiber guiding each of the sub-beams; a controllable fiber switch connected to a primary optical fiber; a plurality of secondary optical fibers each having a downstream opening arranged in the pupil plane, the controllable fiber switch being suitable for coupling each sub-beam into one of the plurality of secondary optical fibers, called a selection fiber, the downstream opening of the selection fiber being connected to the plurality of secondary optical fibers forming the downstream end of the optical fiber element; Includes.
[0016] Preferably, the scanning element is located in a plane that is optically conjugate with the back focal plane.
[0017] According to an embodiment, the sample fluoresces and the collimated light beam triggers emission of fluorescence by fluorophores in the sample, the fluorescence being the sample light.
[0018] Another object of the present invention is to provide a method for analyzing a sample, comprising the steps of: - emitting a spatially coherent light beam; - forming a plurality of sub-beams from a light beam; - focusing the sub-beams on a focal plane; - collimating the sub-beams to form a collimated light beam; - illuminating the sample with a collimated light beam and triggering emission of sample light, the sub-beams interfering at the sample plane to form an interference pattern; - inducing a plurality of controllable shifts of the positions of the sub-beams in the sample plane, the angle between each of the sub-beams in the sample plane being the same for the plurality of controllable shifts; - acquiring an image of the sample light with a pixelated photodetector at a predetermined frame exposure time, inducing a plurality of controllable shifts within the frame exposure time; The method includes:
[0019] The method of the invention preferably comprises a subsequent step H, which consists in modifying the fringes of the interference pattern a number of times, and each time repeating steps F and G and obtaining an image of the sample light associated with the modified fringes.
[0020] Other characteristics, details and advantages of the invention will become apparent from the description given with reference to the attached drawings, given by way of example and respectively illustrating: [Brief description of the drawings]
[0021] [Figure 1] A prior art ASTER device. [Figure 2A] 1 is an optical imaging device according to a first embodiment of the present invention. [Figure 2B] 4 is an optical imaging device according to a second embodiment of the present invention. [Figure 3A] 1 is a device according to an embodiment of the present invention. [Figure 3B] 2A-2D show front (top) and side (bottom) views of a pupil plane of a device according to an embodiment of the present invention. [Figure 3C] 13 is an arrangement of sub-beams at the back focal plane and an interference pattern at the sample plane in a device according to an embodiment of the present invention. [Figure 3D] 1 shows the scanning steps required to cover the entire field of view with a device according to an embodiment of the present invention. [Figure 4A] 1 is a device according to an embodiment of the present invention. [Figure 4B] 13 is an arrangement of sub-beams at the back focal plane and an interference pattern at the sample plane in a device according to an embodiment of the present invention. [Figure 4C] 1 shows the scanning steps required to cover the entire field of view with a device according to an embodiment of the present invention. [Diagram 5] 1 is a device according to an embodiment of the present invention. [Figure 6] This is the method according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] In the drawings, elements are not drawn to scale unless otherwise indicated.
[0023] FIG. 2A illustrates an optical imaging device 1 according to a first embodiment of the invention, suitable for analyzing a sample Ech. This device 1 is an advancement of the prior art device of FIG. 1 (the ASTER device). Thus, like the ASTER device of FIG. 1, the device 1 of the invention comprises a light source LS suitable for emitting a spatially coherent light beam L. The light source is typically a laser emitting a light beam L having a wavelength corresponding to the excitation wavelength of a fluorophore used to label molecules contained in the sample. According to a preferred embodiment, the light beam L has a Gaussian-shaped illumination profile in order to simplify the scanning required to achieve a uniform structured illumination (see further).
[0024] Furthermore, the device 1 includes an optical system including an optical relay element OR and a lens system MO having a back focal plane BFP including at least one lens. The optical relay element OR (typically a cylindrical lens) is suitable for focusing the light beam L on the back focal plane BFP of the lens system MO such that the lens system MO collimates the light beam and forms a collimated light beam CL. In a preferred embodiment, the microscope is an inverted microscope.
[0025] The sample holder SH (typically a cover slip) is suitable for holding the sample Ech on the sample surface SP so that the collimated light beam illuminates the sample. The sample then re-emits the sample light FL. Depending on the nature of the imaging sample, the sample light FL is different. According to a first embodiment, the sample fluoresces and the collimated light beam triggers the emission of fluorescence FL (corresponding to the sample light) by the fluorophores of the sample. In a first embodiment, the lens system is a microscope objective. According to a second embodiment, the sample partially reflects or backscatters the collimated light beam, forming the sample light FL. In a second embodiment, the device 1 is suitable for example for characterizing the surface of a sample.
[0026] For simplicity, the remainder of the specification will be described according to the first embodiment of the invention, however, those skilled in the art will appreciate that the features described below are also included in the second embodiment of the invention.
[0027] The pixelated photodetector Det is suitable for acquiring an image of the fluorescence with a given frame exposure time. As an example shown in FIG. 2A, the photodetector Det is optically arranged downstream of the sample holder with respect to the light beam L. In another embodiment, the photodetector Det is arranged such that the fluorescence is collected via an inverted objective lens MO and collimated by an optical relay element OR. In this embodiment, the separation between the light beam L and the fluorescence is performed, for example, via a dichroic mirror.
[0028] Furthermore, the device 1 includes a scanning element SE (typically two galvanometer scanning mirrors) arranged in a plane optically conjugated with the back focal plane BFP of the objective lens MO. To achieve this optical conjugation, by way of example, the device 1 includes a first lens L1 for focusing the light beam leaving the light source LS in the plane of the scanning element, and a second lens L2 for collimating the light beam leaving the scanning element. Other configurations obvious to those skilled in the art can be implemented for conjugating the plane of the back focal plane BFP to the plane of the scanning element. As mentioned above, an angular shift applied to the galvanometer scanning mirror SE induces a similar angular shift of the back focal plane BFP of the objective lens MO and a position shift of the light beam L at the sample plane SP.
[0029] As a difference with respect to the ASTER device of FIG. 1, the device of the invention comprises an optical component LSE arranged optically upstream of the optical relay element OR, which is suitable for forming from the light beam L a number of sub-beams SB1, SB2, SB3 focused in the back focal plane BFP. These sub-beams are not shown in FIG. 2A but can be seen for example in FIG. 3A, FIG. 3C, FIG. 4A, FIG. 4B or FIG. 5. In an embodiment, the optical component is designed to split the light beam L into at least two sub-beams (see for example FIG. 3A, FIG. 4A). In another embodiment, the optical component is designed to split the light beam L into more than two sub-beams (see for example FIG. 3A, FIG. 4A). These sub-beams interfere at the sample plane SP and form an interference pattern IF at this plane SP. In a first embodiment of the invention, the optical component LSE is arranged optically upstream of the scanning element SE. In a second embodiment of the invention illustrated in FIG. 2B, the optical component is arranged optically downstream of the scanning element SE.
[0030] Given the presence of the optical component, the scanning element SE is suitable for inducing, within a frame exposure time of the photodetector Det, a number of controllable tilts of the sub-beams in the back focal plane BFP and thus a number of shifts of the positions of the sub-beams in the sample plane. The optical component LSE is designed to generate a specific controlled interference pattern IF in the sample plane SP.
[0031] Importantly, the scanning element SE tilts the sub-beams, inducing identical angular shifts for the sub-beams and the back focal plane BFP, and identical position shifts in the sample plane for the sub-beams. Thus, tilting by the scanning element does not change the angle between each sub-beam in the sample plane SP. This means that a shift in the position of the sub-beams in the sample plane SP does not modify the fringes of the interference pattern IF, resulting in identical structured illumination shifting over the entire field of view at the sample plane SP. Thus, via the scanning element, the sub-beams scan over a part of this field of view, called the full field of view or a predetermined field of view, within the frame exposure time of the photodetector Det. This scanning gives rise to a time-averaged excitation profile in the sample plane. This average excitation profile corresponds to an interference pattern uniformly projected over the field of view of the device, defined by the XY area scanned by the sub-beams via the scanning element. The scanning induced by the scanning element SE is typically a raster scan or an Archimedes spiral, or any type of scanning known to a person skilled in the art to give a full uniform structured illumination.
[0032] The device 1 of the present invention provides an elegant solution for generating uniform structured illumination over a large field of view.
[0033] In the example of Fig. 2A, the device 1 includes an optional number of mirrors for the feasibility and compactness of the system. Furthermore, the device includes an optional double telecentric zoom DTZ in the optical path of the light beam L, which is suitable for changing the diameter of the light beam L via the magnification of the double telecentric zoom DTZ. More precisely, given the presence of the optical component, the controlled change of the magnification of the zoom DTZ changes the distance Δ between each sub-beam in the back focal plane and thus the fringe distance of the interference pattern in the sample plane (see further Fig. 3C or Fig. 5).
[0034] FIG. 3A illustrates a preferred embodiment of the device 1 of the invention. In this preferred embodiment, the light component LSE comprises a beam splitter BS suitable for splitting this light beam into sub-beams. As a non-limiting example, the beam splitter is formed by two opposing half prisms. Furthermore, the light component LSE comprises a phase delay element PS arranged in the optical path of at least one sub-beam. This phase delay element PS is suitable for inducing a controlled phase shift in at least one sub-beam (hereinafter shifted sub-beam) and thus shifting the fringes of the interference pattern. As a non-limiting example, the phase delay element PS is a fiber stretcher, a piezoelectric element, an electro-optical modulator or an acousto-optical modulator which shifts the phase of the shifted sub-beam or sub-beams by a controllable amount. The shift of the phase of the shifted sub-beam only shifts the fringes of the interference pattern IF in the sample plane, and not the structure of the interference pattern itself. In the present invention, the phase delay element PS does not shift the fringes of the interference pattern before achieving homogenization of the structured illumination. In other words, the sub-beams are scanned over a given field of view with a given interference pattern with fixed fringes, then the fringes of the interference pattern are shifted and the scan is repeated. This can be repeated several times. This allows the illumination of different areas of the sample and thus the excitation of different fluorophores of the sample. As a non-limiting example, in the preferred embodiment illustrated in FIG. 3A, the device 1 includes a phase delay element PS for each sub-beam.
[0035] Preferably, in the embodiment of Fig. 3A, the optical component further comprises a number of optical fibre elements F1, F2, F3, which are suitable for directing each sub-beam SB1, SB2, SB3 to so-called downstream ends DE1, DE2, DE3 of the optical fibre elements. To ensure focusing of each sub-beam in the back focal plane BFP, the downstream ends are arranged in a pupil plane PP of the device 1 optically conjugated with the back focal plane BFP. In the embodiment of Fig. 3A, preferably, the phase delay elements are fibre phase shifters connected to each optical fibre element directing a phase-shifted sub-beam. This embodiment allows for a simpler control of the interference pattern IF in the sample plane and a more robust architecture.
[0036] Preferably, in the embodiment of FIG. 3A, fiber elements F2 and F3 each comprise: a primary optical fiber PF2, PF3 guiding each sub-beam SB2, SB3; a controllable fiber switch FS connected to the primary optical fiber; a plurality of secondary optical fibres 2a, 2b, 2c; 3a, 3b, 3c, each having a downstream opening arranged in the pupil plane PP; Includes.
[0037] In the embodiment of Fig. 3A, the controllable fiber switch FS is suitable for coupling each sub-beam into one of the secondary optical fibers, called the selective fiber. The downstream opening of the selective fiber thus forms the downstream end of the optical fiber element. The downstream opening is located at a predefined position in the pupil plane. Fig. 3B illustrates a schematic front view (top) and a side view (bottom) of the pupil plane PP with the downstream openings of the secondary optical fibers 2a, 2b, 2c; 3a, 3b, 3c and the downstream end of the optical fiber element F1. This illustration is given as an example, other configurations of the downstream openings can be selected according to the invention.
[0038] By means of the controllable fiber switch FS and the multiple secondary optical fibers, different arrangements of the sub-beams can be freely selected in the vertical and horizontal axes of the pupil plane PP, and therefore in the back focal plane BFP. This means that the controllable fiber switch FS can easily change the interference pattern IF in the sample plane SP. The fiber switch can also "block" selected sub-beams or multiple sub-beams in order to change the number of interference patterns IF in the sample plane that are focused in the back focal plane BFP. This further changes the interference pattern IF in the sample plane SP. This configuration is much more robust and insensitive to misalignments, since it uses an arrangement of optical fibers.
[0039] In the embodiment of Fig. 3A, device 1 includes an optional optical system RS, which in this example is a double telecentric system with a magnification of -1. This system RS forms an inverted image of the pupil plane PP in a secondary image plane. A polarizer Pol located in the secondary image plane allows the sub-beams to be polarized in a predetermined direction before they pass through any other optical components of device 1.
[0040] Furthermore, the device of the embodiment of Fig. 3A includes an optional double telecentric zoom DTZ as mentioned above in the optical path of the sub-beams. In the embodiment of Fig. 3A, the zoom DTZ forms a magnified image of the pupil plane PP relayed by the system RS on the scanning element SE (e.g. a galvanometer mirror). The zoom DTZ is therefore suitable for changing the distance Δ between each of the sub-beams at the back focal plane BFP via the control of the magnification. The scanning element SE reflects or transmits the sub-beams leaving the zoom DTZ such that they interfere at a plane optically conjugated with the sample plane, called the conjugate sample plane SPC, and form an interference pattern IF at said plane SPC. This conjugate sample plane SPC is imaged by a number of lenses and / or mirrors on the sample plane SP. As illustrated by the arrows in Fig. 3A, the scanning element SE is suitable for shifting the position of the sub-beams at the plane SPC (and therefore the sample plane SP) and scanning them over the entire field of view to generate a time-averaged structured uniform illumination.
[0041] Alternatively, in another embodiment, the scanning element SE is arranged optically upstream of the zoom DTZ, but the configuration illustrated in Fig. 3A in which the scanning element SE is arranged optically downstream of the zoom DTZ is preferred to prevent some of the sub-beams from not being collected by the zoom lens, e.g. due to tilting of the galvanometer mirror.
[0042] Figures 3A, 3C and 3D respectively illustrate the optical paths of the sub-beams, their positions at the back focal plane BFP and their interference patterns at the sample plane SP in an embodiment of the invention in which the distance Δ between the two sub-beams SB1, SB2 in the back focal plane is relatively long (i.e. high zoom DTZ). Similarly, Figures 4A, 4B and 4C respectively illustrate the same elements as Figures 3A, 3C and 3D in an embodiment of the invention having a relatively short distance Δ (i.e. low zoom DTZ) and thus a long inter-fringe distance.
[0043] More specifically, Figures 3C and 4B illustrate diagrammatically the arrangement of the sub-beams at the back focal plane BFP and the interference pattern IF at the sample plane SP.
[0044] Because of the relatively long distance Δ, in the embodiment of Figure 3C, the interference pattern IF at the sample plane SP has a diffraction-limited inter-fringe distance and the area of illumination is relatively small. In the embodiment of Figure 4B, the distance Δ is relatively shorter, so the inter-fringe distance of the interference pattern IF is relatively longer (i.e., not diffraction-limited) and the area of illumination is relatively larger. In both embodiments, if the light beam has a Gaussian intensity profile, the fringes of the interference pattern have a Gaussian envelope.
[0045] Figures 3D and 4C illustrate, by means of arrows drawn across the field of view of the device, the steps of scanning required to cover the entire field of view within a frame exposure time and achieve time-averaged uniform structured illumination. Given the smaller area of illumination, the number of steps required to cover the entire field of view in the embodiments of Figures 3A, 3C and 3D is greater than the number of steps in Figures 4A, 4B and 4C.
[0046] Thus, the embodiments of Figures 4A, 4B, and 4C allow for faster scanning of the field of view, while the embodiments of Figures 3A, 3C, and 3D allow for smaller stripes and therefore higher spatial resolution in structured illumination imaging.
[0047] FIG. 5 illustrates diagrammatically a variant of the preferred embodiment of FIG. 3A, in which the fiber switch FS transmits three sub-beams at the pupil plane PP. In this embodiment, the interference pattern at the plane SPC and at the sample plane is an array of spots. This embodiment is of interest for realizing depth confocal microscopy with structured multi-spot illumination. The effect of the magnification of the zoom DTZ is illustrated in FIG. 5. The dashed sub-beams SB1, SB3 represent the positions of the sub-beams under a larger magnification of the zoom DTZ (i.e. at a more significant distance Δ) than the solid sub-beams SB1, SB3. The change in magnification modifies the interference pattern IF. The arrows drawn near the plane SPC represent the displacements of the sub-beams induced by the scanning element SE, which is used for homogenization of the structured illumination over a given field of view.
[0048] In another embodiment of the invention, which differs from the embodiment illustrated in FIG. 3A, the optical component is a digital micromirror device or a spatial light modulator that is optically positioned downstream after the scanning element.
[0049] FIG. 6 illustrates a method for analyzing a sample according to the invention. The method is suitable to be implemented by a device 1 according to the invention. The method comprises a first step 1, which is to emit a spatially coherent light beam L. This step is realized by a light source LS. Then, in step B, the light component element LSE forms a number of sub-beams SB1, SB2, SB3 from the light beam. Then, in step C, the optical relay element OR focuses the sub-beams in the back focal plane. In step D, the microscope objective lens MO collimates the sub-beams and forms a collimated light beam CL. In step E, the collimated light beam illuminates the sample and triggers the emission of the sample light FL, the sub-beams interfere in the sample plane and form an interference pattern IF. Then, in step F, the scanning element SE induces a number of controllable shifts of the positions of the sub-beams in the sample plane. In this step F, the angle between each sub-beam in the sample plane is identical for the number of controllable shifts. In the final step G of the method of FIG. 6, the pixelated light detector Det acquires an image of the sample light with a predefined frame exposure time. A controllable shift is induced in this frame exposure time in step F. As mentioned above, both steps B and F are necessarily performed between steps A and G, although step B can be performed before or after step F can be performed in a different order.
[0050] The method of the present invention provides an elegant solution for generating uniform structured illumination over a large field of view.
[0051] According to a preferred embodiment, the method of the invention comprises a subsequent step H, which consists in modifying the fringes of the interference pattern IF a number of times, and each time repeating steps F and G and obtaining an image of this sample light associated with the modified fringes.
Claims
1. An optical imaging device (1) suitable for analyzing a sample (Ech), comprising: a light source (LS) suitable for emitting a spatially coherent light beam (L), an optical relay element (OR) suitable for focusing said light beam (L) in a focal plane (BFP); a lens system (MO) having a back focal plane (BFP) at said focal plane, said lens system (MO) being suitable for collimating said light beam and forming a collimated light beam (CL); a sample holder (SH) suitable for holding said sample at a sample plane (SP) such that said collimated light beam illuminates said sample and triggers the emission of a sample light (FL); a pixelated photodetector (Det) suitable for acquiring an image of said sample light with a given frame exposure time; Including, the device is optically positioned upstream of the optical relay element in the optical path of the light beam (L); an optical component (LSE) suitable for forming from said light beam a number of sub-beams (SB1, SB2, SB3) focused in said back focal plane, which interfere at said sample plane and form an interference pattern (IF); a scanning element (SE) suitable for inducing, within said frame exposure time, a plurality of controllable shifts of the positions of said sub-beams in said sample plane such that each controllable shift is identical for said sub-beams and such that an average of said sub-beams in said sample plane over said frame exposure time corresponds to said interference pattern being uniformly projected over a given field of view; The optical imaging device (1) further comprises:
2. 2. The optical imaging device according to claim 1, wherein the optical component is a digital micromirror device or a spatial light modulator arranged optically downstream after the scanning element.
3. The optical component comprises: a beam splitter (BS) suitable for splitting said light beam into said sub-beams (SB1, SB2, SB3); a phase delay element (PS) arranged in the optical path of at least one sub-beam, which is suitable for inducing a controlled phase shift in said at least one sub-beam and thus forming a so-called phase-shifted sub-beam and for shifting the fringes of said interference pattern; The optical imaging device of claim 1 .
4. The optical imaging device according to any one of claims 1 to 3, wherein the optical component further comprises a plurality of optical fibre elements (F1, F2, F3), each suitable for directing a respective sub-beam to a so-called downstream end (DE1, DE2, DE3) of the optical fibre element, the downstream end being arranged in a pupil plane (PP) of the microscope device which is optically conjugated with the back focal plane.
5. 5. The optical imaging device of claim 1, further comprising a double telecentric zoom (DTZ) in the optical path of the sub-beams suitable for forming a magnified image in the pupil plane, wherein a controlled change in the magnification of the zoom changes the distance Δ between each sub-beam in the back focal plane.
6. The optical imaging device of claim 3 , wherein the phase delay element is a fiber phase shifter connected to the fiber optic element or elements that direct the phase shifted sub-beams.
7. The optical imaging device according to any one of claims 3 to 6, wherein the beam splitter is suitable for splitting the light beam into two sub-beams.
8. The optical imaging device according to any one of claims 3 to 6, wherein the beam splitter is suitable for splitting the light beam into at least three sub-beams.
9. At least two of the optical fiber elements each include - primary optical fibres (PF2, PF3) directing each sub-beam; a controllable fiber switch (FS) connected to said primary optical fiber; a plurality of secondary optical fibres (2a, 2b, 2c; 3a, 3b, 3c) each having a downstream aperture arranged in the pupil plane, the controllable fibre switch being suitable for coupling each of the sub-beams into one of the plurality of secondary optical fibres, called a selection fibre, the downstream aperture of the selection fibre being connected to a plurality of secondary optical fibres (2a, 2b, 2c; 3a, 3b, 3c) forming the downstream end of the optical fibre element; The optical imaging device according to any one of claims 1 to 8, comprising:
10. The optical imaging device according to any one of claims 1 to 9, wherein the scanning element (SE) is arranged in a plane optically conjugate with the back focal plane (BFP).
11. 11. The optical imaging device of claim 1, wherein the sample emits fluorescence and the collimated light beam triggers emission of fluorescence by fluorophores in the sample, the fluorescence being the sample light.
12. 1. A method for analyzing a sample, comprising the steps of: A. Emitting a spatially coherent light beam; B. forming a plurality of sub-beams from the light beam; C. focusing said sub-beam (L) at a focal plane; D. Collimating the sub-beams to form a collimated light beam (CL); E. Illuminating the sample with the collimated light beam and triggering emission of a sample light (FL), the sub-beams interfering at the sample plane to form an interference pattern; F. inducing a plurality of controllable shifts in the positions of the sub-beams at the sample plane, where the angle between each sub-beam at the sample plane is the same for the plurality of controllable shifts; G. acquiring an image of said sample light with a pixelated photodetector at a predetermined frame exposure time, wherein said plurality of controllable shifts are induced within said frame exposure time; The method includes:
13. 13. The method of claim 12, comprising a subsequent step H of modifying the fringes of the interference pattern a plurality of times, and each time repeating steps F and G, and obtaining an image of the sample light associated with the modified fringes.