Enlarged Field of View in Spatially Modulated Selective Volume Illumination Microscopy

Abstract

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.

Development of structured illumination microscope using transmission diffraction grating obtained by analogue microfilming method

“…Commonly, diffraction gratings are employed to generate the pattern on the sample, whereas two diffraction orders interfere in the focal plane of the objective [9], [10], [11], [12], [13]. In many applications, the beam is shaped using diffractive optical elements (DOE) [14], a spatial light modulator (SLM) [15], or a digital micromirror device (DMD) [16,17]. The fiber coupler method has also been utilized to generate two [18] or three beams [19,20] that interfere in the sample plane, effectively producing the pattern.…”