Imaging for light sheet fluorescence microscopy (LSFM) was performed using a LaVision BioTec Ultramicroscope II (LaVision BioTec GmbH; Bielefeld, Germany) as previously described 71 (link), with the following modifications. Cleared agarose-tissue blocks mounted in a custom sample holder were submerged in 100% DBE. Artery images were acquired at 1.26× mag (0.63× zoom) with a 10 ms exposure, using the two-light-sheet configuration with both left and right light sheets, with the horizontal focus centered in the middle of the field of view, a numerical aperture of 0.039 (sheet thickness at horizontal focus = 19 μm), and a light sheet width of 100%. Pixel size was 4.96 μm and Z-slice spacing was 9.5 μm. Two channels were imaged: tissue autofluorescence with 488 nm laser excitation and a Chroma ET525/50m emission filter at 70% laser power and TAMRA channel with 561 nm laser excitation and a Chroma ET600/50m emission filter at 40% laser power. Focus in both channels was ensured by use of the chromatic correction module on the instrument. To minimize sample bleaching during initial imaging setup we lowered laser power and increased exposure times. Tissues studied for targeting and biodistribution include aorta, thymus, heart, lung, liver, kidney, spleen, skeletal muscle, gastrointestinal tract (stomach/small intestine/colon), brain, spinal cord, and tail.
Lavision biotec ultramicroscope 2
Manufactured by Miltenyi Biotec
Sourced in Germany
The LaVision BioTec Ultramicroscope II is a high-performance light sheet microscope system for large-volume, high-resolution imaging of cleared and optically transparent samples. It utilizes advanced optical sectioning techniques to capture detailed 3D images with minimal phototoxicity and photobleaching.
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5 protocols using lavision biotec ultramicroscope 2
1
Light Sheet Fluorescence Microscopy of Cleared Tissues
2
Quantitative Imaging of Spinal Cord Microglia
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The iDisco protocol was followed as described in (Renier et al., 2014 (link)) and https://idisco.info. Rabbit anti-Iba1 (Wako #019–19741) was used at 1:100 in Figure 3 at 1:200 in Figure 5 during a 3 day incubation at 37°C. Secondary antibody AlexaFluor594-conjugated donkey anti-rabbit (ThermoFisher #A21207) was used at 1:200 in Figure 3 , and an AlexaFluor647-conjugate at 1:600 in Figure 5 . Spinal cords were embedded in 1% agarose prior to clearing to facilitate handling for imaging. Imaging of iDisco samples was done using a two-photon microscope (Olympus MPE-RS; representative images in Figures 3 and 5 ) as well as a light sheet microscope (LaVision Biotec UltraMicroscope II; quantification of all spinal cord samples in Figures 3 and 5 , and videos). Imaging on the LaVision Biotec UltraMicroscope II was performed using their 4x objective. All images were acquired using six light-sheets with dynamic focusing (16 positions) enabled. For the Dynamic Focus Processing and Merge Lightsheet calculations, the Contrast Adaptive and Contrast algorithms were respectively used.
3
Large-Scale 3D Imaging of Cleared Samples
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Optically cleared samples were imaged on a LaVision BioTec Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) with an Olympus MVX10 Zoom Microscope Body (Olympus, Tokyo, Japan) with an optical magnification range from 1.26x to 12.6x and an NA of 0.5. An NKT SuperK (Power SK PP485) supercontinuum white light laser served as excitation light source. For excitation and emission detection of specific fluorophores custom band-pass filters (excitation 470/40, 577/25 or 640/30 nm; emission 525/50, 632/60 or 690/50 nm) in combination with an Andor Neo sCMOS Camera with a pixel size of 6.5 x 6.5 μm2 were used. For image acquisition, Z-steps 3 μm were chosen and tile scanning was performed with an overlap of 20%.
4
Ultramicroscopy Imaging of Optically Cleared Samples
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Optically cleared samples were imaged on a LaVision BioTec Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) equipped with an Olympus MVX10 Zoom Microscope Body (Olympus, Tokyo, Japan) allowing an optical magnification range from 1.26x to 12.6x and an NA of 0.5. An NKT SuperK (Power SK PP485) supercontinuum white light laser served as excitation light source. For excitation and emission detection of specific fluorophores custom band-pass filters (excitation 470/40, 577/25 or 640/30 nm; emission 525/50, 632/60 or 690/50 nm) in combination with an Andor Neo sCMOS Camera. For image acquisition, Z-steps 3 μm were chosen. 3D reconstruction and analysis of ultramicroscopy stacks were performed by using the volume rendering software Voreen [39 (link), 56 (link)].
5
Visualizing SARS-CoV-2 Infection in Ferret Lungs
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Light sheet micrographs of optically clear and immunostained respiratory tissues from SARS-CoV-2-infected ferrets were acquired with a LaVision BioTec Ultramicroscope II (LaVision BioTec, Bielefeld, Germany). The microscope was equipped with an Olympus MVX-10 zoom body (magnification range: 0.63×–6.3×, total magnification: 1.26×–12.6×; Olympus, Shinjuku, Tokyo, Japan), an Olympus MVPLAPO 2× objective (NA = 0.5), a LaVision laser module with four laser lines (488 nm, 561 nm, 639 nm, and 785 nm), and a Andor Zyla 5.5 sCMOS Camera (Andor Technology, Belfast, Northern Ireland) with a pixel size of 6.5 µm2. To visualize tissue morphology, non-specific autofluorescence was excited with the 488 nm laser. Excitation lines 561 nm and 639 nm were used to excite Alexa Fluor™ 568 and Alexa Fluor™ 647, respectively. Channels of a high-volume 3D image were acquired sequentially with a z-step size of 2 µm, a light sheet width of 100%, and a light sheet thickness of 3.89 µm (NA = 0.156). Acquisition was done with ImSpector (v7.0.124.0).
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