Transmission structured illumination microscopy with tunable frequency illumination using tilt mirror assembly

Abstract

We present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture (NA) objective in standard SIM provides super-resolution compromising with the field-of-view (FOV). Employing low NA (20X/0.4) objective lens detection, we experimentally demonstrate [Formula: see text] (0.56 mm[Formula: see text]0.35 mm) size single FOV image with [Formula: see text]1.7- and [Formula: see text]2.4-fold resolution improvement (exploiting various illumination by tuning tilt-mirrors) over the diffraction limit. The results are verified both for the fluorescent beads as well as biological samples. The tSIM geometry decouples the illumination and the collection light paths consequently enabling free change of the imaging objective lens without influencing the spatial frequency of the illumination pattern that are defined by the tilt-mirrors. The large and scalable FOV supported by tSIM will find usage for applications where scanning large areas are necessary as in pathology and applications where images must be correlated both in space and time.

Figure 1

Concept of tSIM: (a) scheme of the experimental setup for one orientation; (b) tunable frequency illumination patterns are generated by a pair of mirror facets corresponding to low and high frequency illumination respectively; (c) scheme of polarization conversion of the interfering beams where top, middle and below panel corresponds to the state of polarization above, on and below the vortex half wave plate respectively; (d) hexagonal arrangement of mirror-facets for three orientation angles required for isotropic resolution enhancement; (e) resolution limit represented by the optical transfer function (solid circle), maximal shift (dotted circle) in conventional SIM offers two-fold resolution enhancement. (f) maximum spectral support of tSIM governed by with periodicity $\frac{\lambda }{2sin{\theta }_1}$, tunable under different interfering angles $\theta$; (g, h) frequency support of a low-NA detection objective lens under similar illumination configurations, where tSIM support more than conventional 2X resolution enhancement of SIM. The 3D graphics shown in (a, b, d) are designed using a free and open-source software for 3D computer graphics, Blender (Version 3.2, https://www.blender.org/).

Figure 2

Experimental results of fluorescent beads: (a) single FOV tSIM reconstruction and widefield image of 200 nm fluorescent beads; (b) diffraction limited spectra; (c) reconstructed tSIM spectra; (d, f) magnified widefield images of white box in R${}_1$ and R${}_2$ regions, respectively; (e, g) closeup of the tSIM reconstruction in R${}_1$ and R${}_2$ regions, respectively; (h) intensity line-profiles of (d, e); (i) intensity line-profiles of (f, g).

Figure 3

tSIM imaging of actin filaments in U2OS cells: (a) single FOV tSIM reconstruction versus diffraction limited image with the excitation/emission wavelengths of 532 nm/560 nm and inset shows spectra of the reconstructed image; (b, d) diffraction limited images of regions R${}_1$ and R${}_2$; (c, e) reconstructed tSIM image of regions R${}_1$ and R${}_2$ respectively; (f, g) intensity profile of a line marked between the arrowheads in (c, e).

Figure 4

High-frequency tSIM imaging of actin-stained U2OS cell: (a) single FOV tSIM reconstruction and diffraction limited image with the excitation/emission wavelengths of 532 nm/560 nm and inset shows spectra of the reconstructed image; (b, d) widefield image of R${}_1$ and R${}_2$ regions respectively; (c, e) reconstructed image of R${}_1$ and R${}_2$ regions respectively; (f, g) intensity line profiles between the magenta and blue arrowheads.

Figure 5

Simulation result of high-frequency tSIM imaging using synthetic fluorescent particles: (a) single FOV tSIM reconstructed image and widefield image using 20X/0.65 detection objective lens and inset shows spectra of the reconstructed image; (b, f) ground-truth object of R${}_1$ and R${}_2$ regions; (c, g) widefield image of R${}_1$ and R${}_2$ regions; (d, h) reconstructed image of R${}_1$ and R${}_2$ regions; (e, i) intensity line profiles within the yellow box.

Figure 6

Experimental results of actin filaments of U2OS cell stained with alexa-fluor 532: (a) widefield image; (b) tSIM reconstruction image; (c, d) magnified widefield images of white box in R${}_1$ and R${}_2$ regions respectively; (e, f) closeup of the tSIM images in R${}_1$ and R${}_2$ regions respectively; (g) intensity line-profiles of (c, e) shown by magenta colour; (h) intensity line-profiles of (d, f) shown by blue colour.