MULTI-FOCAL LIGHT-SHEET STRUCTURED ILLUMINATION FLUORESCENCE MICROSCOPY SYSTEM

Abstract

A multi-focal light-sheet structured illumination system that can be implemented as a part of a commercial fluorescence microscope or a module that is adaptable to fit a number of commercially available microscopes. The system provides simultaneous capture of 2D images from multiple planes within a 3D volume, which are resolved laterally and axially to provide improved resolution along the three dimensions (x,y,z). A Wollaston prism allows several axially-localized high-contrast structured illumination patterns to be generated.

Claims

1. A structured illumination system for a fluorescence microscope, comprising: an incoherent light source configured to produce a light beam; a first linear polarizer configured to receive the light beam from the incoherent light source; a plurality of equidistant parallel slits configured to be illuminated by the light beam after passing through the first polarizer; a Wollaston prism comprising two birefringent wedges, each birefringent wedge with an optical axis, configured to divide the light beam into two spherical waves to generate a plurality of light-sheet structured patterns; a first converging lens with a front focal plane, configured to receive the divided light beam after passing through the Wollaston prism, wherein the front focal plane is collinear with the plurality of equidistant parallel slits; and a second linear polarizer configured to receive the light beam after passing through the first converging lens.

2. The system of claim 1, wherein plurality of equidistant parallel slits are disposed in a binary mask with a vertical axis.

3. The system of claim 2, wherein the optical axes of the birefringent wedges are orthogonal to each other, and both optical axes are oriented at an angle of about 45 degrees with respect to the vertical axis of the binary mask.

4. The system of claim 3, wherein the first linear polarizer is oriented at an angle of about 45 degrees with respect to both optical axes.

5. The system of claim 1, wherein the incoherent light source comprises a quasi-monochromatic light-emitting diode, a white lamp with a narrow bandwidth filter, or a coherent light source with a rotating diffuser, wherein the rotating diffuser is configured to destroy a spatial coherence of the coherent light source.

6. The system of claim 2, the binary mask further comprising a horizontal axis, wherein displacement of the Wollaston prism orthogonal to the vertical axis of the binary mask and parallel to the horizontal axis of the binary mask produces a controlled shifting of the plurality of light-sheet structured patterns.

7. The system of claim 1, further comprising a quarter-wave plate disposed between the Wollaston prism and the second linear polarizer.

8. The system of claim 7, further comprising a polarization rotator disposed between the quarter-wave plate and the second linear polarizer.

9. The system of claim 2, further wherein alteration of the orientation of the plurality of light-sheet structured patterns is achieved by joint rotation of the binary mask, the first linear polarizer, a quarter-wave plate, and the Wollaston prism.

10. The system of claim 1, further wherein alteration of the orientation of the plurality of light-sheet structured patterns is achieved by a tunable image rotator positioned after the Wollaston prism.

11. The system of claim 1, wherein the plurality of light-sheet structured patterns further comprise a tunable spatial-period tuned by adjusting the axial separation between the plurality of equidistant parallel slits and the Wollaston prism.

12. The system of claim 1, wherein the plurality of light-sheet structured patterns comprises an axial confinement that is inversely proportional to the number of equidistant parallel slits.

13. The system of claim 1, further comprising a single objective lens; and a recording system, wherein the recording system comprises a plurality of cameras for the simultaneous detection of multiple focal planes.

14. The system of claim 1, wherein the illumination system is configured to retro-fit a plurality of fluorescent microscopes.

15. A method for obtaining super-resolved images with high optical-sectioning capability, comprising the steps of: producing a light beam from an incoherent light source; passing the light beam through a first linear polarizer and a plurality of equidistant parallel slits; subsequently splitting the light beam by a Wollaston prism into two spherical waves with orthogonal polarization for each of the equidistant slits to generate a light-sheet structured pattern; subsequently passing the beam through a converging lens and a second polarizer; illuminating a sample with the beam; capturing images from the sample through a recording system; and processing the captured images.

16. The method of claim 15, wherein the recording system comprises a plurality of cameras.

17. The method of claim 16, further comprising the steps of: simultaneously illuminating a plurality of transverse sections of the sample; simultaneously capturing a plurality of focal planes of the sample by the plurality of cameras.

18. The method of claim 17, further comprising the steps of: axially scanning the volume of the sample; capturing a plurality of two-dimensional images from the plurality of focal planes within the three-dimensional volume of the sample; and resolving the captured two dimensional images laterally and axially to create super resolved images along three dimensions.

19. The method of claim 18, further comprising the steps of: capturing three separate two-dimensional images from each of the plurality of focal planes.

20. The method of claim 15, further comprising the steps of: capturing a plurality of three-dimensional forward images; decomposing the plurality of three-dimensional forward images; applying a Wiener filter to the decomposed images for deconvolution and shifting; and combining the filtered images to form restored super-resolved images.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A shows a view of the optical configuration of a conventional fluorescence microscope implemented in an upright scheme.

[0018] FIG. 1B shows a view of the optical configuration of a multi-focal light sheet structured illumination microscope implemented in an upright scheme in accordance with an embodiment of the present invention.

[0019] FIG. 2A shows a view of the optical configuration of a conventional fluorescence microscope implemented in an inverted scheme.

[0020] FIG. 2B shows a view of the optical configuration of a multi-focal light sheet structured illumination microscope implemented in an inverted scheme in accordance with an embodiment of the present invention.

[0021] FIG. 3 shows a schematic description of the steps required to retrieve restored super-resolved (SIM) images from the recorded structured-illumination microscopy images through the acquisition of 3 images with different phases.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0022] In various exemplary embodiments, the present system comprises a multi-focal light-sheet structured illumination module that can be implemented in any commercial fluorescence microscope. The present invention provides simultaneous capture of 2D images from multiple planes within a 3D volume, which are resolved laterally and axially to provide improved resolution along the three dimensions (x,y,z). The illumination method of this invention employs a Wollaston prism, thereby allowing several axially-localized high-contrast structured illuminations patterns.

[0023] FIGS. 1A and 2A show conventional fluorescence microscopes that are not equipped with the illumination system presently disclosed. FIGS. 1B and 2B show a schematic view of a multi-focal light-sheet structured illumination system in accordance with an embodiment of the present invention. As shown in FIGS. 1B and 2B, the conventional microscopes of FIGS. 1A and 2B can be readily adapted to include the illumination system in accordance with embodiments of the present invention.

[0024] In the upright configuration of FIG. 1B, incoherent light from an incoherent light source 10 is passed through a first polarizer 11a and a plurality of equidistant slits 12 that are parallel to one another (which may be in the form of a binary mask). Subsequently, the light is split by a Wollaston prism 13. The Wollaston prism 13 may be comprised of two birefringent wedges that are joined together along their respective hypotenuses. In general, a Wollaston prism comprises two orthogonal prisms jointed together along their base to form two right triangle prisms with perpendicular optical axes, so that outgoing light beams diverge with the angle of divergence determined by the prisms' wedge angle and the wavelength of the light. Upon exiting the Wollaston prism 13, the light beam is divided into two spherical waves with orthogonal polarization for each of the plurality of the slits 12. The light is sequentially transmitted through a first converging lens 2f, a quarter-wave plate (214) 14, and a second polarizer 11b. The second polarizer 11b, may be configured to rotate about an axis. In this way, rotation of the second linear polarizer 11b permits a controlled shifting of a structured pattern.

[0025] The light beam next is reflected via a folded mirror 7b and transmitted through a second converging lens 2b to be reflected downward via a dichroic mirror 3 and deflected off of a sample being held in a sample holder 5. The light beam can be focused on the sample by manipulation of a focusing screw 6 to move the sample holder relative to objective lens 2. The fluorescent light emitted by the sample is collected by an objective lens 4 and up through the dichroic mirror 3. A third converging lens 2c transmits the light through the eyepiece 9 imaging path or through the sensor 8 imaging path using a second folded mirror 7a. If the light is not reflected by the folded mirror 7a, then it is transmitted through a fourth converging lens 2d and fifth converging lens 2e, and, ultimately, the image is projected onto a sensor 8 for collection of the image data.

[0026] In the inverted configuration of FIG. 2B, light from an incoherent light source 10 is passed through a first polarizer 11a and a plurality of equidistant slits 12 that are parallel to one another (which, as noted above, may be in the form of a binary mask). Subsequently, the light is split by a Wollaston prism (as described above) 13. The light is sequentially transmitted through a first converging lens 2f, a quarter-wave plate (214) 14, and a second polarizer 11b. The second polarizer 11b may be configured to rotate about an axis. In this way, rotation of the second linear polarizer 11b permits a controlled shifting of a structured pattern.

[0027] The light beam passes sequentially through a second converging lens 2g, a first dichroic mirror 3a, and a third converging lens 2b, which directs light to the sample holder 4 with a sample being held therein. The light can be focused on the sample by manipulation of a focusing screw 6. The fluorescent light emitted by the sample is collected by an objective lens 5, reflected by two mirrors 7a, 7b, and transmitted through two converging lenses 2c, 2d. A folded mirror 3b projects the image through an eyepiece 9 or the sensor 8. Before forming the image on the sensor for collection of the image data, the light passes through a sixth converging lens 2e.

[0028] As seen by comparing FIG. 1A with FIG. 1B, or FIG. 2A with FIG. 2B, the above-described systems replace the simple light source 1 and converging lens 2a used in the prior art.

[0029] It should be noted that the arrangement and number of lenses 2, and mirrors 3, 7 may be flexible, and that the arrangements shown in FIGS. 1B and 2B represent exemplary embodiments. Alternate arrangements that do not affect the optical resolution of the images obtained are envisioned.

[0030] For a non-limiting example, the Wollaston prism can be replaced by any polarization-sensitive beam splitter. In another alternative, the binary mask could be generated using a programmable electro-optical device (e.g., a SLM), thereby providing control of a variable separation between the slits and the number of slits required.

[0031] As shown, the illumination system provides light-sheet structured patterns onto the sample space 4 of tunable spatial period in order to obtain high resolution images of fluorescent specimens under research. These light-sheet structured patterns are generated by illuminating the Wollaston prism 13 through the emerging wavefront from a set of equidistant and parallel slits 12. The slits 12 can be illuminated by a spatially-incoherent light 10. The light source 10 employed may comprise a quasi-monochromatic light emitting diode (LED) or a laser, wherein the spatial coherence of the light emission is broken using, for example, a rotating diffuser. Alternatively, the light source 10 may be a white-light lamp with a narrow bandwidth filter. As obvious to one of skill in the art, additional light sources may be appropriate for use with the present illumination system in various embodiments.

[0032] The presently disclosed illumination system provides the incoherent superposition of N high-contrast sinusoidal patterns. Each of these N structured patterns are axially-extended and there is a lateral displacement among them which produces axially-localized structured fringes. Because of this lateral displacement, the visibility of the structured pattern changes periodically, defined by the following Equation 1:

[00001]$V\left(z,N\right)=\frac{\sin \left(\pi N\frac{\left(u_e-u_o\right)\eta }{f}{x}_0\frac{z}{f}\right)}{\mathrm{Nsin}\left(\pi \frac{\left(u_e-u_o\right)\eta }{f}{x}_0\frac{z}{f}\right)},$

where u.sub.e=−u.sub.O=α.sub.W(n.sub.e−n.sub.O)/λ and αW are the apex angles of the birefringent wedges; ne and nO are an extraordinary and an ordinary refractive index of the Wollaston material, respectively; η is an axial separation between the slits and the Wollaston prism; f is the focal length of the converging lens inserted after the Wollaston prism; and z is an axial distance between the lens and the observation plane.

[0033] To provide a period-tunable structured illumination system, the incoherently-illuminated slits 12 are set at the front focal plane of a converging lens 2f, whose focal length is f, and the Wollaston prism 13 is inserted between them. In this orientation, axial displacement (i.e, orthogonal relative to the illuminating light beam) of the Wollaston prism 13, (η), produces a continuous variation of the spatial-period in the structured pattern, p, given by the following Equation 2:

[00002]$p=\frac{f}{\left(u_e-u_o\right)\eta }.$

with terms as defined above.

[0034] As previously mentioned, the structured pattern created by the Wollaston prism 13 after the converging lens is imaged onto the sample plane by means of an afocal telecentric imaging system (comprising 2b and 4 of FIG. 1B, and 2g and 2b of FIG. 2B). Inside the 3D volume of the specimen one can find different axial planes with high-contrast structured fringes. Thus, multi-focal light-sheet structured pattern of period-tunability can be obtained on the sample volume. While the lateral period of the fringes can be tuned by axially displacing the Wollaston prism 13, see Eq. 2, the axial positions (z.sub.max) and the separation of the different structured planes is mainly determined by the separation between the slits 12 (x.sub.0) and totally independent of the number of slits, as seen in the following Equation 3:

[00003]$z_{\max }=\left(2m+1\right)\frac{f^2}{\left(u_e-u_o\right)\eta x_0},$

where m is a positive integer. From this equation, one can derive that the axial separation between two planes of maximum visibility is determined by the following Equation 4:

[00004]$T=\frac{f^2}{\left(u_e-u_o\right)\eta x_0}.$

[0035] In this illumination system, the higher the number of slits 12, the narrower the axial confinement of the fringes, meaning that the axial confinement (Δz) of these patterns is inversely proportional to the number of incoherently-illuminated slits 12 (N). The axial extension of the planes with maximal contrast is defined by the following Equation 5:

[00005]$\begin{array}{c}\Delta z=\frac{f^2}{N\left(u_e-u_o\right)\eta x_0}\\ =T/N\end{array}.$

[0036] The illumination system may generate a light-sheet structured pattern in a sample plane of axial extension defined by the following Equation 6:

[00006]$\begin{array}{c}\Delta z^{\prime }=M_{\mathrm{ill}}^2\Delta z\\ =M_{\mathrm{ill}}^2\frac{f^2}{N\left(u_e-u_o\right)\eta x_0},\end{array}$

and a lateral period defined by the following Equation 7:

[00007]$\begin{array}{c}{p}^{\prime }=M_{\mathrm{ill}}p\\ =M_{\mathrm{ill}}\frac{f}{\left(u_e-u_o\right)\eta },\end{array}$

where M.sub.ill is the lateral magnification between an illumination plane and the sample plane.

[0037] As shown in FIGS. 1B and 2B, the illumination system disclosed herein is uniquely designed to be implemented in any commercial microscope as an external illumination module. The illumination system can be fitted to commercial microscopes in a transmission or a reflection configuration and in upright and inverted schemes. Since the disclosed multi-focal light-sheet SIM system requires minimal implementation, the illumination system maintains the advantages of any commercial microscope in terms of robustness, stability, and user-friendly operation while simultaneously offering the additional advantages disclosed herein.

[0038] Advantages of the multi-focal light sheet structured-illumination system include, but are not limited to, the following: (1) generation of high-contrast structured fringes with tunable spatial-frequency; (2) phase-shifting of the recorded structured images by rotating a polarizer when the light used to illuminate the Wollaston prism is circularly-polarized; (3) every 90-degree rotation of the analyzer providing a phase-shifting of 7E radians in the fringes, independent of the lateral modulation frequency of the structured pattern, and thus calibration is not needed; (4) creation of multi-focal light-sheet structured fringes inside the volume of the specimen which permits recording of the 3D structure of the sample without any movement of the sample, itself; (5) no coherence noise to distort the recorded images; (6) discrimination of different features of biological samples based on their response to a specific direction of light polarization because the Wollaston prism is a polarization-sensitive device; (7) accomplishing optical-sectioning capability comparable to that of LSFM without requiring two objectives lenses and overcoming the resolution limit; and (8) overcoming the resolution limit by recording three phase-shifted raw images instead of nine needed by other SIM systems (this reduction in data-acquisition is an advantage for live-cell imaging, which requires fast imaging and at lower light exposures).

[0039] The images recorded by the camera should be computationally processed in order to obtain super-resolved images with optical-sectioning capability. FIG. 3 shows the acquisition and processing steps of this technique. This process can be divided in four main steps, and with the proper code, the restored images can be obtained almost in real-time. The first step is the acquisition phase. In the acquisition phase, 3D forward images (D.sub.1, D.sub.2, D.sub.3) are captured with three different phases (ϕ.sub.1, ϕ.sub.2, ϕ.sub.3) by axially scanning the specimen's volume, with 3D irradiance distribution given by O(r). At each field of view, three images are acquired that correspond to three different phases of the structured pattern along the direction of the fringes. In the second step, the images are decomposed according to the following formulas, which includes computation of the fast 3D Fourier transform of the 3D images:

[00008]$\left(\begin{array}{c}{\tilde{D}}_{-1}\left(f\right)\\ {\tilde{D}}_0\left(f\right)\\ {\tilde{D}}_{-1}\left(f\right)\end{array}\right)=\left(\begin{array}{c}\tilde{O}\left(f_{\mathrm{xy}}+f_{p;x},f_z\right)\mathrm{FT}\left\{H\left(r\right)\times V\left(z\right)\right\}\\ \tilde{O}\left(f\right)\tilde{H}\left(f\right)\\ \tilde{O}\left(f_{\mathrm{xy}}-f_{p;x},f_z\right)\mathrm{FT}\left\{H\left(r\right)\times V\left(z\right)\right\}\end{array}\right)={\left(\begin{array}{ccc}{e}^{+i{\varphi }_1}& 1& e^{-i{\varphi }_1}\\ e^{+i{\varphi }_2}& 1& e^{-i{\varphi }_2}\\ e^{+i{\varphi }_3}& 1& e^{-i{\varphi }_3}\end{array}\right)}^{-1}\left(\begin{array}{c}{\tilde{D}}_1\left(f\right)\\ {\tilde{D}}_2\left(f\right)\\ {\tilde{D}}_3\left(f\right)\end{array}\right),$

where H(r) stands for the impulse response of the imaging system, V(z) is the axial modulation of the visibility of the illumination pattern, and f.sub.p;x is the lateral spatial frequency of this pattern in the sample's volume. In this equation, {tilde over (X)}=FT{X} represents the 3D Fourier transform of the function X.

[0040] After decomposition, the deconvolution and shifting set occurs, wherein three new 3D components are calculated by demodulating each SIM image using a Wiener filter. During shifting, the positions of the new components are centered in the Fourier domain. Finally, in the fourth step, the centered, deconvoluted, 3D images are combined to form the restored images. This step is accomplished by way of a linear sum, calculating the inverse fast 3D Fourier transform of the combination. Alternatively, different processing methods applicable to any SIM approach can be used to process the data acquired from this multi-focal light sheet structured-illumination system

[0041] Note that isotropic improvement in 3D resolution is achieved by rotating the structured pattern and acquiring data in 3 different orientation angles. This fringes' rotation can be produced by rotating jointly the first polarizer, the binary mask, the Wollaston prism and the quarter-wave plate. Commonly, one should acquire 3 phase-shifted SIM images per orientation (a total number of 9 images per transverse section) and apply the reconstruction method described above to achieve the final 3D super-resolution image. However, taking advantage of information redundancy, the total number of recorded images can be reduced to four images (two taken in the first orientation and one for each of the remaining two orientations).

[0042] In some embodiments, the system further comprises a polarization rotator (which may be mechanically, electrically, or optically driven) disposed between the quarter-wave plate and the second linear polarizer. In additional embodiments, alternation of the orientation of the plurality of light-sheet structured patterns is achieved by a tunable image rotator after the Wollaston prism, as (but not limited to) a mirror combination or a Dove prism.

[0043] Embodiments of the illumination system disclosed herein provide a number of novel aspects that are not currently available in the prior art. The novel aspects include, but are not limited to: (1) continuous variation of the spatial-period of the structured pattern fringes; (2) axial localization of the structured fringes (light-sheet structured patterns); (3) simultaneous illumination of multiple axial plane within the specimen; (4) polarization-sensitive illumination system; and (5) an external illumination module adaptable to any commercial fluorescent microscope.

[0044] The illumination system disclosed herein also offers numerous distinct advantages over current technology. Importantly, the embodiments of the present invention permit tunability of the structured fringes with the axial displacement of the Wollaston prism 13. This advantage circumvents the use of a spatial light modulator (SLM), which is currently used by many researchers and manufacturers. Unlike the presently disclosed illumination system, SLMs are extremely expensive and are not designed to be easily implemented in a commercial microscope. In addition, embodiments of the present invention use incoherent light, which is a significant advantage because the coherent light used in commercially available microscopes produces coherent noise that interferes with and distorts the ultimately recorded images. Further, in the present invention, there is no reduction of the contrast of the structured fringes for high spatial-frequency as in commercially-available modules.

[0045] Aspects of the presently disclosed invention provide high optical-sectioning capability while simultaneously overcoming the resolution limit. The high optical-sectioning is somewhat comparable to conventional light-sheet microscopy; however, unlike light-sheet microscopy, the present invention accomplishes such high-optical sectioning through the use of only a single objective lens. The present illumination is also capable of simultaneous illumination of different transverse sections of a specimen. In this way, multiple focal planes can be detected through the use of multiple cameras.

[0046] Thus, it should be understood that the embodiments and examples described herein have been chosen and described in order to best illustrate the principles of the invention and its practical applications to thereby enable one of ordinary skill in the art to best utilize the invention in various embodiments and with various modifications as are suited for particular uses contemplated. Even though specific embodiments of this invention have been described, they are not to be taken as exhaustive. There are several variations that will be apparent to those skilled in the art.