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Fig. 1 | BMC Biology

Fig. 1

From: Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

Fig. 1

Light sheet theta microscopy (LSTM) for high-resolution quantitative imaging of large intact samples. a Light sheet microscopy (LSM) employs orthogonally illumination-detection optics, which limits the lateral dimensions of imaging volume. iSPIM, SCAPE/OPM and line scan confocal microscopy are partially effective in alleviating this limitation, however at the cost of reduction in usable working distance (magenta arrowheads) and image quality (e.g., SCAPE collects low-quality signal from non-native focal planes, and line scan confocal results in lower axial resolution and high photo-bleaching.). The proposed LSTM uses non-orthogonal (< 90°) illumination light sheets to effectively image very large samples, while maintaining high imaging speed and depth and uniform high resolution. b One or two light sheets intersect with the detection plane in a line illumination profile, which is synchronously scanned with the rolling shutter detection of an sCMOS camera to achieve optical sectioning. c Two scanning approaches: 1-axis scanning (1-AS) by perpendicular translation and simultaneous 2-axis scanning (2-AS, default LSTM) by translation along and perpendicular to the illumination axis such that the thinnest part is utilized for uniform planar illumination. d Comparison of point spread function (PSF) in 1-AS, default LSTM, and LSM configurations. Left: x-z maximum intensity projections of ~ 1 μm fluorescent microbeads imaged using the same detection (10×/0.6NA/8mmWD) and illumination (4×/0.28NA/28.5 mmWD) objectives. Axial full width at half maximum values (FWHM) across the field of view (blue LSTM in default 2-AS mode, green LSTM in 1-AS mode, red LSM). LSTM achieves uniform axial resolution (~ 4–6 μm FWHM) over the entire field of view, whereas both the 1-AS and LSM provide lower peripheral resolution (1-AS ~ 5–13 μm; LSM ~ 4–11 μm). Right: x-z projections (20 μm) of an image volume from a DAPI-stained human brain tissue. Additional file 5: Video 2 [29] provides 3D reconstructions. The graph compares the signal for a central and a peripheral region of interest. Scale bars: 100 μm

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