Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: 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 provides 3D reconstructions. The graph compares the signal for a central and a peripheral region of interest. Scale bars: 100 μm

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Microscopy, Imaging, Confocal Microscopy, Comparison, Staining

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: 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 : 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 : 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 : Figure S5 provides details. f The average signal of tiles in the order of acquisitions. Note that no significant photo-bleaching trend is observed

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Refractive Index, Comparison, Imaging

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: LSTM optical sectioning. a x - z maximum intensity projections of an image stack acquired from human brain tissue, shown in ( b ), stained with DAPI. The camera rolling shutter exposure time determines the effective slit (rolling shutter) width (0.1–1 ms, i.e., 66–665 μm on the sCMOS sensor and 6–60 μm on the sample. The images were acquired using two different scanning modes: LSTM 1-axis scan (1-AS) and LSTM 2-axis scan (2-AS, default). Total frame exposure was 20 ms for all the images. As evident, the 2-AS mode allows for uniform planar illumination for achieving quantitative imaging, and the axial resolution decreases with increased rolling shutter exposure. All scale bars are 100 μm. b LSTM imaging of a large thick section of cleared human brain tissue (~ 10.5 mm × 14.1 mm × 3 mm) stained with DAPI. We used 0.5-ms rolling shutter exposure settings and 20 ms for entire frame exposure to acquire this dataset. Scale bar is 1 mm

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Staining, Imaging

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: Rapid uniform high-resolution imaging of mouse central nervous system. a A CLARITY-cleared Thy1-eYFP transgenic mouse brain with attached spinal cord was imaged with LSTM microscopy using 10×/0.6NA/8mmWD detection objective (correction collar adjusted to 1.45 refractive index). A rolling shutter exposure of 0.5 ms and a full frame exposure of 20 ms were used. High-resolution 3D rendering was generated after 2 × 2-fold down-sampling. The bounding boxes are 11.8 mm × 27.6 mm × 5.2 mm for the whole sample and 5.1 mm × 3.1 mm × 3.5 mm for the subvolume ( magenta ). Images were acquired with 5-μm z -spacing using an effective light sheet thickness of ~ 5 μm. Lateral pixel sampling was 0.585 × 0.585 μm. A detailed volume rendering is shown in Additional file 8 : Video 3 . b A large thick coronal slice of a Thy1-eYFP transgenic mouse brain was imaged with LSTM using 488 nm excitation wavelength. A rolling shutter exposure window of 0.5 ms and a full frame exposure of 20 ms were used. The volume rendering was performed using 4 × 4 fold down-sampled data. The bounding box is 9.6 mm × 13.5 mm × 5.34 mm. Images were acquired with 5-μm z -spacing using an effective light sheet thickness of ~ 5 μm. Lateral pixel sampling was 0.585 × 0.585 μm. Additional file 9: Video 4 shows volumetric rendering. c The same sample as shown in b was imaged with a high-NA 25×/1.0NA/8mmWD objective. A rolling shutter exposure window of 0.4 ms and a full frame exposure of 20 ms were used. The volume rendering was performed after 2 × 2-fold down-sampling. The bounding box is 6 mm × 9.6 mm × 0.5 mm. Images were acquired with 5-μm z -spacing using an effective light sheet thickness of ~ 3 μm. Lateral pixel sampling was 0.234 × 0.234 μm

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Imaging, Transgenic Assay, Microscopy, Refractive Index, Generated, Sampling

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: LSTM enables rapid uniform high-resolution imaging of very large samples. a For an unbiased comparison of the imaging performance of LSTM and LSM a highly cleared large rat brain tissue (~ 2 cm wide and ~ 5 mm deep; vasculature stained with tomato lectin) was imaged using the exact same detection (10×/0.6NA/8mmWD, correction collar adjusted to 1.45 refractive index) and illumination objectives (4×/0.28NA/28.5WD). Maximum intensity projections are shown. The bottom graph profiles the mean intensity across the length of the specified ( dashed rectangles ) regions of interest. In LSM ( cyan ), the intensity signal is progressively degraded towards the interior of the sample, whereas LSTM ( magenta ) allows uniform quality across the entire sample. The scale bars are 1 mm. b An image stack from the sample shown in a . Maximum intensity projections (50 μm) are shown at three different depths ( orange ). The bounding box is 1 mm × 1 mm × 5 mm. The scale bars are 100 μm. A detailed volume rendering is shown in Additional file 10: Video 5 . c Uniformly expanded (~ 4-fold in all three dimensions) slice of Thy1-eYFP transgenic mouse was imaged using LSTM with 10×/0.6NA/8mmWD detection objective. A rolling shutter exposure window of 0.2 ms and a full frame exposure of 20 ms were used. The resulting dataset consists of 723,200 images (2048 × 2048 pixels) and required ~ 22 h of acquisition time. The volume rendering was performed with 8 × 8 fold down-sampled dataset. Zoomed-in images are marked. d An image stack from the dataset shown in c . The bounding box size is 1.2 mm × 1.2 mm × 1 mm. Note that the dendritic spines can be unambiguously identified. Detailed volume rendering in Additional file 11: Video 6

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Imaging, Comparison, Staining, Refractive Index, Transgenic Assay

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: LSTM enables rapid volumetric imaging of highly motile animals. Live samples can undergo substantial non-isomorphic rearrangements in their body shape and cellular density, resulting in continuously changing local optical properties. LSM is particularly susceptible to misalignments and other aberrations because of the use of orthogonal light sheet illumination. LSTM is uniquely suitable for rapid volumetric live imaging of such difficult samples, as demonstrated by imaging of highly motile Hydra . a Hydra image is shown at different time points to highlight the non-isomorphic changes in freely moving animal. b LSTM was used to perform long-term (> 1 h demonstrated, Additional file 12: Video 7 ) high-resolution live imaging of an adult Hydra expressing GCaMP6s . Each volume consists of 17 z -planes. Manual tracking and analyses of calcium signaling were performed for the first ~ 500 s of recording. Maximum intensity projections covering the two halves are shown. Representative neuronal traces are shown for cells marked in corresponding colors. As shown in Additional file 13: Video 8 , the neuronal traces correlate with the rapid longitudinal contraction behavior of Hydra , and the other two traces are part of rhythmic potential circuits, in excellent agreement with the observations reported recently . Scale bars are 100 μm

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Imaging, Expressing

Journal: BMC Biology

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

doi: 10.1186/s12915-018-0521-8

Figure Lengend Snippet: LSTM microscopy implementation. a LSTM optical path. Two symmetric light sheets are generated by using a cylindrical lens ( CL ), scan lens ( SL ), tube lens ( TL ), and illumination objectives. The galvo scanners are used to translate the light sheets perpendicular to their propagation direction, and the electrically tunable lens ( ETL ) for translating the thinnest part of the light sheets along the propagation direction. An input beam of ~ 10 mm diameter is then trimmed through an iris. A slit is placed after the ETL to control the effective numerical aperture of the illumination. An additional iris is placed between the SL and TL to control the light sheet width. The illumination axes are arranged at ~ 60° to the detection axis. A custom 3D-printed cap with a quartz coverslip is attached to the illumination objective to allow dipping in the immersion oil to ensure that the low NA illumination rays from an air illumination objective enter perpendicularly to the oil. The detection arm consists of a detection objective (Olympus 10×/0.6NA/8mmWD or 25×/1.0NA/8mmWD, both with correction collars), a tube lens, and an sCMOS camera. b 3D model of the LSTM microscope. A vertical breadboard was used to mount the caged optical assemblies via x - y manual translation stages to allow fine adjustments. A sample chamber was attached to a 3-axis ( x , y , z ) motorized stage assembly. See also Additional files , , , and Additional file 4: Video 1 for further details and Table for complete parts list

Article Snippet: The 3D model of the LSTM microscope was made with Autodesk Inventor 2017.

Techniques: Microscopy, Generated, Control