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Fig. 3 | BMC Biology

Fig. 3

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

Fig. 3

LSTM characterization. a Geometric constraints in LSTM. Specific example of using Olympus 4×/0.28NA/29.5WD and 10×/0.6NA/8mmWD for illumination-detection. Note that the working distance of the air illumination objective is elongated in high refractive index immersion media (Additional file 3: Figure S3). Angular separation of ~ 60° was used for all experiments. b Comparison of maximum illumination path length (MIPL) required for full sample coverage in LSTM and LSM. The illumination light sheets need to penetrate the entire width (w) of the sample (or half width for two-sided illumination) in LSM, whereas MIPL depends on the angular arrangement and the tissue thickness (t) in LSTM. Bottom left: dependence of LSTM MIPL on θ and sample thickness (t, arrow indicates increasing t). Bottom right: MIPL dependence on the sample width and thickness: magenta and cyan highlight LSTM < LSM and LSTM > LSM respectively, assuming θ = 60° (see Additional file 6: Figure S4 for full θ range). c Effective planar illumination thickness can be approximated as b/sin(θ), where b is the actual light sheet thickness. The right graph plots the effective light sheet thickness as a function of θ and b (arrow points to increasing b). d Comparison of redundant illumination in LSTM and LSM for imaging of a single plane (top row) and an image stack (bottom row). e Ratios of total illumination energy loads (LSTM/LSM) as a function of sample width (w), angular configuration (θ), sample thickness (t), and objective magnification (10× and 25×). Illumination energy load is higher in LSTM for smaller samples and similar to LSM for larger samples. The energy load ratio also decreases with increased angular separation (60° is marked) and the magnification of detection objective. Additional file 7: Figure S5 provides details. f The average signal of tiles in the order of acquisitions. Note that no significant photo-bleaching trend is observed

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