Lightsheet z 1

Manufactured by Zeiss

Sourced in Germany, Italy

The Lightsheet Z.1 is a high-performance light sheet fluorescence microscope designed for advanced bio-imaging applications. It utilizes a unique illumination technique to provide optical sectioning and high-speed, low-phototoxicity imaging of large samples. The system is optimized for imaging of living organisms, tissues, and 3D cell cultures.

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Lightsheet Z.1 microscope (79 citations) Lightsheet Z.1 fluorescence microscope (3 citations) Lightsheet Z.1 LSFM system (3 citations) Lightsheet Z.1 5x/0.16 (2 citations) Lightsheet Z.1 Selective Plane Illumination Microscope (2 citations) Lightsheet Z.1 lightsheet microscope (2 citations)

105 protocols using lightsheet z 1

Live Embryo Imaging with Fluorescent Labeling

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Imaging of live embryos injected with fluorescent labelled MOs, fluorescent protein fusions or antibodies was performed on Zeiss Lightsheet Z1 or Zeiss 880 confocal microscope with Fast Airyscan Module.
For imaging on Zeiss Lightsheet Z1, embryos were mounted in 1% low melt agarose column using size three glass capillaries and incubated in E3 media at 28 °C during imaging. Z-stacks of ~200–300 slices in 0.5–1 µm steps were acquired every 30–60 seconds for 1.5–2.5 h, with ×20 objective.
For imaging on the Zeiss 880 confocal microscope in Fast Airyscan mode, embryos were mounted in 0.5% low melting agarose in glass bottom imaging dish and incubation chamber temperature was maintained at 28 °C during imaging. Z-stacks of ~100–200 slices were acquired (step size of 0.25 µm) every 30–60 seconds for 1.5–2.5 h using a ×25 or ×40 objectives. Post-processing of the acquired images was performed using Zeiss ZEN Blue Software.

Hadzhiev Y., Qureshi H.K., Wheatley L., Cooper L., Jasiulewicz A., Van Nguyen H., Wragg J.W., Poovathumkadavil D., Conic S., Bajan S., Sik A., Hutvàgner G., Tora L., Gambus A., Fossey J.S, & Müller F. (2019). A cell cycle-coordinated Polymerase II transcription compartment encompasses gene expression before global genome activation. Nature Communications, 10, 691.

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Lightsheet Microscopy of Gastruloids

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The gastruloids were imaged using a ZEISS Lightsheet LS Z1 microscope with appropriate filters for mCherry, Alexa 488 and DAPI. Prior to imaging, the gastruloids were embedded in 1.5% LMP agarose in a glass capillary and kept in the fridge for 5 min until the agarose was solidified. Subsequently, the capillary containing the samples in agarose columns were placed into the RIMS-filled sample chamber of the ZEISS Lightsheet Z1. The agarose column was slightly pushed out of the capillary into the RIMS solution and left overnight to clear the gastruloids. Post-processing of the images was performed using ZEN Blue/Black software (ZEISS).

López-Anguita N., Gassaloglu S.I., Stötzel M., Bolondi A., Conkar D., Typou M., Buschow R., Veenvliet J.V, & Bulut-Karslioglu A. (2022). Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids. Development (Cambridge, England), 149(20), dev200679.

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3D Light-Sheet Microscopy of Cleared Brain

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LSFM
was accomplished using a Zeiss Lightsheet Z.1. The microscope was
fitted with a 2.5× objective, solid-state lasers (excitation
wavelengths 488 nm for FAM and 640 nm for sensor), and a PCO.edge
16 bit sCMOS camera for detection. The cleared brain was embedded
in low-melt 2% agarose (ThermoFisher) then submerged in CUBIC reagent-2
overnight. The next day, magnetic staples were superglued to the agarose-embedded
tissue, and the sample was hung vertically in front of the LSFM objective
(2.5×) using a custom magnetic fixture. The brain was slowly
lowered into the imaging chamber filled with reagent-2 for at least
20 min prior to image collection. A z-stack (range
of 3.258 mm with a 3 μm step size) was obtained. The 3D area,
encompassing the injured and contralateral hemispheres, was approximately
9.7 mm (x-axis) by 4.5 mm (y-axis)
by 3.3 mm (z-axis). 3D images, clipping planes, and
videos were generated in Arivis Vision4D.

Kandell R.M., Kudryashev J.A, & Kwon E.J. (2021). Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based Nanosensor. ACS Nano, 15(12), 20504-20516.

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Intact Heart Imaging with Light-Sheet

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The cleared hearts were scanned using light-sheet fluorescence microscopy, achieving an approximately 5-mm penetration depth. Optical images of clarified intact mouse hearts were mounted with the apex facing the objective lens. The hearts were imaged using a light-sheet fluorescence microscope (Lightsheet Z.1, Carl Zeiss Microscopy Co, Ltd. Germany; stack size, 4.823 mm; 2.283 µm/pixel × 2.283 µm/pixel in-plane resolution; step size 7.67 µm) equipped with a 5× objective (EC Plan-Neofluar 5×) at 638-nm excitation^{31 (link)}.

Lee S.E., Nguyen C., Yoon J., Chang H.J., Kim S., Kim C.H, & Li D. (2018). Three-dimensional Cardiomyocytes Structure Revealed By Diffusion Tensor Imaging and Its Validation Using a Tissue-Clearing Technique. Scientific Reports, 8, 6640.

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Visualizing Complex Tissue Samples

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Specimens were visualized either by conventional confocal or light sheet microscopy. For standard confocal imaging, samples were positioned in a glass bottom petri dish (Fluoro Dish, World Precision Instruments) submerged in a drop of 80% (v/v) glycerol in H₂O to minimize shifting and moving of the specimen during image acquisition. Human gut samples were mounted as described in Supplementary Figure 2. Image stacks were recorded on a LSM 5 Exciter attached to an AxioImager (Carl Zeiss, Microscopy GmbH, Jena, Germany) with a Plan-Neofluar 10x/0.3 objective or Acroplan 20x/0.4 Corr. For imaging on a light sheet microscope (Lightsheet Z.1, Carl Zeiss, Microscopy GmbH, Jena, Germany), samples were glued to a hook-shaped holder, submerged in 80% (v/v) glycerol in H₂O, and evaluated with an EC Plan-Neofluar, 5x/0.16 objective, and a Clr Plan-Neofluar 20x/1.0 Corr nd = 1.45 objective.
Images were processed and 3D-reconstructions were rendered using ZEN software (Carl Zeiss).

Neckel P.H., Mattheus U., Hirt B., Just L, & Mack A.F. (2016). Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure. Scientific Reports, 6, 34331.

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Light Sheet Imaging of Tumor Growth

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24 hpf embryos were embedded in 1% low melt agarose in a glass capillary and extruded into a sample chamber containing E3 medium supplemented with MS222. Images were acquired using a Light Sheet Z.1 (Carl Zeiss, Germany) with a water dipping 20X detection objective (W-Plan-APOCHROMAT-1.0NA) and dual side 10X illumination objectives (LSFM, 0.2NA). Samples were illuminated from a single side and Z-stacks were acquired every 15 min using a 1.2X optical zoom, 4.13 µm light sheet thickness and 2 µm Z-interval. Tumor volume was measured using the Countour Surface tool in Imaris 8.3.1 (Bitplane). For visualization, max intensity projections were produced and drift was corrected using a rigid body transformation in the ImageJ plugin, StackReg [48 (link)]

Pudelko L., Rouhi P., Sanjiv K., Gad H., Kalderén C., Höglund A., Squatrito M., Schuhmacher A.J., Edwards S., Hägerstrand D., Berglund U.W., Helleday T, & Bräutigam L. (2017). Glioblastoma and glioblastoma stem cells are dependent on functional MTH1. Oncotarget, 8(49), 84671-84684.

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Lymph Node Imaging with CUBIC Clearing

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Tumor draining and contralateral inguinal lymph nodes (from mice inoculated with 0.5 × 10⁶ of CTV stained CD8⁺ OT-I T-cells as described above) were dissected and briefly rinsed in 0.01 M PBS (pH 7.4)/heparin (5 IU/ml final concentration) and subjected to 24 hours (hrs) fixation with 4% paraformaldehyde at 4°C. After fixation, lymph nodes were rinsed 3 times for 1 hr with PBS and the residual connective and adipose tissues were gently removed under a stereomicroscope. Next, lymph nodes were prepared using CUBIC-R1-based tissue optical clearing for imaging as described previously.^{39 (link)} Briefly, lymph nodes were immersed in CUBIC-R1 solution for at least 1.5 days (with a longer, up to 7 days incubation time for the enlarged lymph nodes) and subjected to imaging using a Zeiss Lightsheet Z.1 equipped with two 5× objectives (N.A. 0.1) that project two independent co-axial lightsheets onto the lymph nodes from the left and right. Each lightsheet was aligned manually into the same imaging plane, according to the procedure provided by Carl Zeiss AG and the resulting images were automatically fused by ZEN software (Zeiss). Additional detail for tissue optical clearing is provided in supplementary materials.

Sosnowska A., Chlebowska-Tuz J., Matryba P., Pilch Z., Greig A., Wolny A., Grzywa T.M., Rydzynska Z., Sokolowska O., Rygiel T.P., Grzybowski M., Stanczak P., Blaszczyk R., Nowis D, & Golab J. (2021). Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma. Oncoimmunology, 10(1), 1956143.

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3D Imaging of Hippocampal Slices

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The periphery of hippocampus was coronally sectioned into slices of 1–3 mm-thickness for 3D imaging. After treating the slices for different lengths of times according to their thickness, they were embedded in 2% agarose, trimmed, put into AICI reagent again, and placed on the shaker for 1–2 h at 25 °C. The excitation source was a λ = 488 nm laser for GFP detection. 3D-images of brain slices were taken by light sheet fluorescence microscopy (LSFM, Lightsheet Z.1, Carl Zeiss, Germany). The illumination lens was a 5x, 0.1 NA at air and the objective of emission part was a 5x, 0.16 NA, EC Plan-Neofluar by Zeiss. The stitched 3D-images were 3 × 3 tiles with field of view of 2.47 × 2.47 mm (1920 × 1920 pixel), z-step size was a 5 μm. All acquisitions were controlled by ZEN (Carl Zeiss) software. The experiment for fine neuronal structure preserving was implemented by confocal microscopy (A1Rsi, Nikon, Tokyo, Japan) under the control of NIS (Nikon) software ver 5.0. The objective was a 60x, Plan Apochromat from Nikon. A field of view of images was a 212 × 212 μm (1024 × 1024 pixel) with 0.2 μm z-step varying focus depth.

Ryu Y., Kim Y., Lim H.R., Kim H.J., Park B.S., Kim J.G., Park S.J, & Ha C.M. (2022). Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging. International Journal of Molecular Sciences, 23(12), 6826.

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Multi-scale Imaging of Reproductive Organs

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Bright field images of the pregnant uterus and detached placenta were taken using a stereomicroscope (MZ16F, Leica). Immunohistochemically stained tissue sections were observed with an epifluorescence confocal microscope (LSM510, Carl Zeiss). Three-dimensional images of transparent organs were acquired using a light-sheet microscope (Lightsheet Z.1, Carl Zeiss). Images of whole reproductive organs were obtained using a 5x/0.16 NA objective lens, and detailed single-cell resolution images were acquired using a 20x/1.0 NA objective lens for the clearing method. Three-dimensional images were analyzed using ZEN software (Carl Zeiss). An excitation wavelength of 638 nm was used for visualizing autofluorescence signals.

Kagami K., Shinmyo Y., Ono M., Kawasaki H, & Fujiwara H. (2017). Three-dimensional visualization of intrauterine conceptus through the uterine wall by tissue clearing method. Scientific Reports, 7, 5964.

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Zebrafish Brain Tissue Preparation

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Larval and adult zebrafish were anesthetized using tricaine methanesulfonate (Sigma-Aldrich), and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. For tissue sectioning, samples were washed three times in PBS/0.1% Tween (PBST), placed in embedding media (1.5% agar, 5% sucrose), incubated in 30% sucrose solution overnight at 4°C, and 20-μm sections were cut using a Leica CM 1950 cryostat. Larval brains were dissected after fixation and mounted in 25% glycerol. Imaging was performed using a Zeiss Axioskop 2 Apotome or LSM 700 laser scanning confocal microscope. Images are single optical planes except where noted. For lightsheet imaging, adult brains were fixed overnight in the cranium, dissected, embedded in acrylamide gel, and cleared as in Isogai et al. (2017) (link). Lightsheet images were collected as optical sectioned z-stacks using a Zeiss Lightsheet Z1. Cell numbers and fluorescent intensity were quantified manually using ImageJ software (NIH) on single optical sections. Cells were outlined and the mean fluorescent intensity for each cell was calculated by drawing a circle of defined area through the brightest plane containing the nucleus.

Male I., Ozacar A.T., Fagan R.R., Loring M.D., Shen M.C., Pace V.A., Devine C.A., Lawson G.E., Lutservitz A, & Karlstrom R.O. (2020). Hedgehog Signaling Regulates Neurogenesis in the Larval and Adult Zebrafish Hypothalamus. eNeuro, 7(6), ENEURO.0226-20.2020.

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