Structured oblique illumination microscopy for enhanced resolution imaging of non-fluorescent, coherently scattering samples

Abstract

Many biological structures of interest are beyond the diffraction limit of conventional microscopes and their visualization requires application of super-resolution techniques. Such techniques have found remarkable success in surpassing the diffraction limit to achieve sub-diffraction limited resolution; however, they are predominantly limited to fluorescent samples. Here, we introduce a non-fluorescent analogue to structured illumination microscopy, termed structured oblique illumination microscopy (SOIM), where we use simultaneous oblique illuminations of the sample to multiplex high spatial-frequency content into the frequency support of the system. We introduce a theoretical framework describing how to demodulate this multiplexed information to reconstruct an image with a spatial-frequency support exceeding that of the system's classical diffraction limit. This approach allows enhanced-resolution imaging of non-fluorescent samples. Experimental confirmation of the approach is obtained in a reflection test target with moderate numerical aperture.

Keywords: (030.0030) Coherence and statistical optics; (100.6640) Superresolution; (180.0180) Microscopy.

Fig. 1

Numerical simulation showing the extended transfer function and its enhanced-resolution reconstruction ability. (a) The extended transfer function given by Eq. (4) and the intensity of the associated structured illumination field given by Eq. (7). The transfer function’s axes are given in multiples of ${𝝎}_c$. The green dashed circle outlines the frequency support of the original diffraction limit. (b,c,d) True, orthogonally illuminated, and enhanced-resolution images, respectively, of a sample USAF test chart. (e,f,g) Magnified view of Group −1 El 4 set of bars at 0.71 lpmm from (b,c,d), respectively. Note the enhanced-resolution capabilities shown in (d,g).

Fig. 2

Schematic structured illumination imaging system with moderate numerical aperture. SM: single mode fiber at 405 nm; CL: collimating lens; DLP: pixel-addressable diffractive element; L1, L2, L3, L4: lens (f = 120, 200, 150, 50 mm); M: mask; BS: beam splitter; LA: limiting aperture; S: coherently scattering sample.

Fig. 3

Experimental data showing (a) an orthogonally-illuminated (BF) image and (b) an enhanced-resolution (SI) reconstruction. (c) Horizontal cross-sectional profiles taken from (a),(b). (d) Vertical cross-sectional profiles taken from (a),(b). (e) Intensity modulations vs bar freq compared between BF and SI.

Fig. 4

Top: (a) Experimental orthogonally-illuminated (BF) image and (b) enhanced-resolution (SI) reconstruction of 20 µm polystyrene beads. (c) and (d) represent enlarged regions of (a) and (b). (e) shows a comparison of the cross-sectional intensity profiles between the BF and SI images at the locations marked in yellow in images (c) and (d) Bottom: Same as above, but for a histological sample of a mouse joint.