Device for improving performance in STED and RESOLFT microscopy using a single phase mask

Abstract

The present invention refers to a method for high spatial resolution imaging comprising a phase plate or a spatial light modulator (SLM) device for STimulated Emission Depletion (STED) microscopy and Reversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy, where a bivortex pattern is imprinted on the said phase plate or SLM to generate a beam. The bivortex pattern allows some freedom in shaping the STED beam to improve the microscope's axial performance and optical sectioning capacity. The present invention further refers to a method for STED and RESOLFT microscopy comprising the step of modulating the optical phase of a laser using a phase plate or a spatial light modulator device with a phase mask comprising a bivortex with a tunable radius. The disclosed phase masks and methods of STED and RESOLFT microscopy may advantageously be applied to provide a hybrid 2D/3D STED regime but one with a significant reduction in the degrees of freedom for alignment relative to what is found in incoherent beam superpositions, thus providing an improvement in beam quality, namely a minimized central intensity and lower sensitivity to aberrations, resulting in an increased signal level and axial performance.

Claims

1. A phase plate or spatial light modulator device for Stimulated Emission Depletion (STED) and Reversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy, comprising: at least two vortex sections, one inner disc-shaped vortex and one outer ring-shaped vortex, in a single phase mask, and a bivortex profile phase mask (ϕ) according to the mathematical function: $\varphi \left(r,\theta \right)=\{\begin{array}{cc}n\theta & \mathrm{if}r<r_1\\ n\theta +a\pi & \mathrm{if}{r}_2>r>r_1\end{array},$ where r is the perpendicular distance to the optical axis, θ is the azimuthal angle and r.sub.1 and r.sub.2 define the radius of the inner and the outer vortex, respectively, n is an integer which equals 1 for best resolution and, a, which defaults to 1, is a parameter between 0 and 2, which can be used to refine the distribution of the beam energy along the optical axis.

2. The phase plate or spatial light modulator device according to claim 1 characterized by, a phase shift between the inner vortex and the outer vortex, which occurs at a variable distance from the optical axis.

3. The phase plate or spatial light modulator device according to claim 1, comprising a phase step between the inner vortex and the outer vortex which can be chosen to be of a magnitude between 0 and 27c radians.

4. The phase plate or spatial light modulator device according to claim 1, comprising a smoothened inter-vortex transition.

5. The phase plate or spatial light modulator device according to claim 1, comprising more than two vortex structures.

6. The phase plate or spatial light modulator device according to claim 1, comprising another arbitrary phase masks within or outside the bivortex regions.

7. A method for STED and RESOLFT microscopy characterized by, comprising a step of: modulating the physical phase of a optical beam using compensation or corrective phase functions on a phase plate or a spatial light modulator device to generate an effective phase mask as described in claim 1.

8. A method for STED and RESOLFT microscopy, comprising: modulating the physical phase of an optical beam using a phase plate or a spatial light modulator device generating a phase mask comprising at least two vortex sections, one inner disc-shaped vortex and one outer ring-shaped vortex, in a single phase mask, wherein the step is characterized by, comprising a bivortex profile phase mask (ϕ) according to the mathematical function: $\varphi \left(r,\theta \right)=\{\begin{array}{cc}n\theta & \mathrm{if}r<r_1\\ n\theta +a\pi & \mathrm{if}{r}_2>r>r_1\end{array},$ where r is the perpendicular distance to the optical axis, θ is the azimuthal angle and r.sub.1 and r.sub.2 define the radius of the inner and the outer vortex, respectively, n is an integer which equals 1 for best resolution and a, which defaults to 1, is a parameter between 0 and 2 which can be used to refine the axial distribution of the beam.

9. The method for STED and RESOLFT microscopy according to claim 8 in which the step of modulating the physical phase of an optical beam using a phase plate or a spatial light modulator device generating a phase mask is characterized by, comprising a phase shift between the inner vortex and the outer vortex, according to the radius of each vortex, with the inner vortex radius being most preferably smaller or equal to the radius of the outer vortex.

10. The method for STED and RESOLFT microscopy according to claim 8 in which the phase step is characterized by, having an arbitrary value, most preferably 1 π radians.

11. The method for STED and RESOLFT microscopy according to claim 8 in which the step is characterized by, comprising a smoothened inter-vortex transition.

12. The method for STED and RESOLFT microscopy according to claim 8 in which the step is characterized by, comprising more than 2 vortex structures.

13. The method for STED and RESOLFT microscopy according to claim 8 in which the step is characterized by, comprising other arbitrary phase masks within or outside the bivortex regions.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Drawings of the default bivortex phase mask. a) The effective phase plate's mask function is defined as a disc-shaped vortex surrounded by a ring-shaped vortex. The line separating the two sections (at a radius r1) defines a phase shift of □. b) In practice, the (outer) ring vortex can have an arbitrary radius larger than r2, with the microscope lens geometry typically limiting the effective radius of the phase function to r2.

(2) FIG. 2: CH-STED depletion beam generated by a phase plate comprising a bivortex phase mask. Images show signal scattered by gold nanobeads when scanned by the STED depletion laser. These images are thus representations of the STED beam geometry cross-section. Transformation of the CH-STED depletion beam is observed by decreasing the parameter c below 1. Morphological changes are mainly characterized by a widening of the dark central spot, which occurs selectively at the focal plane along with a global elongation of the beam's focal structure.

(3) FIG. 3: CH-STED employing the bivortex phase mask for imaging of a mitotic spindle. Top half mitotic spindle is imaged with 2D STED (which corresponds to CH STED with c=1), which is switched to CH STED in the bottom half (c<1). All other acquisition parameters remain unchanged. It can be seen that, although some xy resolution loss is expected when decreasing c below 1, the redistribution of the depletion beam becomes more effective at exposing the filaments composing the object due to an improved axial performance, with an elongated depletion volume. When required, the loss in xy resolution is compensated by an increase of the depletion laser power.

(4) FIG. 4: Illustrative embodiments comprising the bivortex phase function imprinted into the phase plate. a) Example of aCH-STED phase mask where arbitrary functions are added inside and/or outside the bivortex phase function. b) Example of a mask function where the phase transition between two sections of the bivortex is smoothened by convolution. c) Example of the phase plate's bivortex phase mask when the two vortices are shifted by a value aπ with a different from 1.

EXAMPLES

Example 1: Production of a Phase Plate Generating the Bivortex Phase Mask

(5) In an example, the bivortex phase mask of the present invention is generated by a phase plate device. Such device can be produced by crafting the negative of the bivortex phase mask of the present invention's phase plate onto a mold and by developing the mold to obtain a bivortex phase plate. The manufacture by molding may be achieved by methods known in the art, for example those described by Oemrawsingh et al, 2004, with the addition of comprising a step of: designing the mold as a negative of the present invention's phase mask. After release of the mold, a solid phase plate with a bivortex configuration is obtained.

Example 2: Production of the Bivortex Phase Mask by a Spatial Light Modulator

(6) In another example, the bivortex phase mask of the present invention can be generated by a spatial light modulator device (SLM). Such SLM can be produced by integrating complementary metal-oxide-semiconductor (CMOS) in the SLM, by methods such as the ones described in the art (ZHU, et al 2004.), with the addition of comprising a step of: imprinting the present invention's bivortex phase mask onto the SLM, through electronic control of its subunits.

Example 3: STED Microscopy Method Employing a Bivortex Phase Mask Through Compensation or Correction Functions

(7) Another example of an embodiment of the present invention refers to a method for STED microscopy that can be developed through comprising a step of: modulating the physical phase of a radiation beam on an existing phase plate or a spatial light modulator using compensation or corrective phase functions to ultimately generate the present invention's byvortex phase mask as the effective phase mask.

REFERENCES

(8) ANTONELLO, J., KROMANN, E. B., BURKE, D., BEWERSDORF, J. & BOOTH, M. J. 2016. Coma aberrations in combined two- and three-dimensional STED nanoscopy. Opt Lett, 41, 3631-4. BOOTH, M., ANDRADE, D., BURKE, D., PATTON, B. & ZURAUSKAS, M. 2015. Aberrations and adaptive optics in super-resolution microscopy. Microscopy (Oxf), 64, 251-61. HARKE, B., ULLAL, C. K., KELLER, J., & HELL, S. W. 2008. Three-dimensional nanoscopy of colloidal crystals. Nano letters, 8(5), 1309-1313. HELL, S. W. & WICHMANN, J. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett, 19, 780-2. KELLER, J., SCHONLE, A. & HELL, S. W. 2007. Efficient fluorescence inhibition patterns for RESOLFT microscopy. Opt Express, 15, 3361-71. KLAR, T. A., JAKOBS, S., DYBA, M., EGNER, A., & HELL, S. W. 2000. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proceedings of the National Academy of Sciences, 97(15), 8206-8210. OEMRAWSINGH SS1, VAN HOUWELINGEN J A, ELIEL E R, WOERDMAN J P, VERSTEGEN E J, KLOOSTERBOER J G, 'T HOOFT G W. Production and characterization of spiral phase plates for optical wavelengths. Appl Opt. 2004 Jan. 20; 43(3):688-94. TOROK, P. & MUNRO, P. 2004. The use of Gauss-Laguerre vector beams in STED microscopy. Opt Express, 12, 3605-17. WESTPHAL, V. & HELL, S. W. 2005. Nanoscale resolution in the focal plane of an optical microscope. Phys Rev Lett, 94, 143903. ZHU, H. BIERDEN, P., CORNELISSEN, S., et al. 2004. Advanced Wavefront Control: Methods, Devices, and Applications II, edited by John D. Gonglewski, Mark T. Gruneisen, Michael K. Giles, Proceedings of SPIE Vol. 5553 (SPIE, Bellingham, Wash. Lisbon, May 17, 2019