The Cellular Genomics and Imaging Core offers multiple instruments with unique capabilities on campus.
Cellular Genomics and Imaging Instruments
Zeiss Lightsheet Z.1
Light sheet fluorescence microscopy (LSFM) is an imaging method that uses one or more illumination objectives to excite fluorophores in a specimen with a thin sheet of light, which are then visualized with a separate imaging objective perpendicular to the sheet. LSFM has several advantages over confocal microscopy, including:
- Excellent sample penetration
- High speed
- Minimal phototoxicity and photobleaching
These properties make LSFM ideal for many biological imaging applications, including:
- Fluorescence imaging of thick samples
- Long-term imaging of live samples
The Zeiss Lightsheet Z.1 includes several additional features to optimize LSFM:
- Four-axis sample positioning permits rotation of the specimen for easy viewing from any angle.
- Multiview acquisition allows the same tissue to be imaged from multiple perspectives, then integrates these views for optimal resolution and accurate rendering.
- Two illumination objectives, one on each side of the sample chamber, enhance even illumination of the specimen.
- Pivot scanning slightly rotates the sample during acquisition to eliminate shadowing and striping artifacts observed in other systems
- Cleared tissue optics (5x objective) designed for CLARITY and similar clearing techniques (RI=1.45)
For more information about LSFM imaging methods, visit MicroscopyU and for details about the Lightsheet Z.1, visit the Carl Zeiss Microscopy. To discuss whether light sheet microscopy would be of benefit for your application, please contact us for a free consultation.
System specifications:
The Zeiss LightSheet Z.1 Dual Illumination Microscope System is ideally suited for imaging of thick and/or live specimens, because it uses separate illumination and detection light paths to minimize photobleaching and phototoxicity and maximize imaging depth. The 4-axis sample positioning system allows imaging from any perspective and Multiview acquisitions increase axial resolution. The system features two illumination objectives and uses ultrafast pivot scanning to reduce shadowing artifacts. The system includes temperature and CO2 environmental control of the sample chamber. Solid-state lasers provide excitation lines at 405, 445, 488, 515, 561, and 638nm, with simultaneous two channel detection using PCO.edge 16-bit sCMOS cameras. Imaging objectives include 5x dry and 20x and 40x water immersion lenses, with 2.5x system optical zoom, yielding effective magnifications of 2x-100x. Optics for cleared tissue imaging using a 5x objective are designed for a refractive index of 1.45, matching that used in CLARITY and similar techniques. Image acquisition and basic manipulation is performed with Zen software, that also integrates with Arivis Vision 4D for rendering and analysis. A separate workstation is available for these analyses.
Protocols
Guides for common applications using the Zeiss Lightsheet Z.1:
- Sample preparation guide (from Zeiss)- Detailed white paper describing a variety of sample types and mounting options. (also see video)
- Basic 4D acquisition (Z-series, time-lapse imaging)- Detailed hardware and software guide for setting up a time-lapse session.
- Mounting and imaging zebrafish in glass capillaries (JOVE)- Detailed and extensive guide for the entire process of mounting and imaging zebrafish in glass capillaries. Note that software and post-processing refers to ImageJ software.
- FEP mounting of zebrafish for long term imaging (JOVE, Huisken Lab)- Instructions for mounting and imaging zebrafish in low concentration agarose using FEP tubing to permit unrestricted development in long imaging sessions.
- Mounting of large cleared specimens- Description of flexible mounting devices constructed from LEGOs.
- Quick Guide to using the Zeiss Tiling Tool- Directions for using the standalone tiling app for setting acquisition of image tiles.
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Stitching tiled images in Arivis Vision4D- Guide to properly align tiled images
Leica SP8 DLS
The Leica TCS SP8 DLS is a dual function fluorescence microscope that can be used as a conventional laser scanning confocal microscope (LSCM) or as a lightsheet fluorescence microscope (LSFM). A fully automated inverted microscope platform supports both modes.
As a confocal microscope, the Leica SP8 DLS features:
- Up to 4 channel simultaneous detection
- Two high sensitivity hybrid detectors and two PMTs
- Four laser lines for excitation of most standard fluorophores
- Spectral detection permits complete flexibility in choosing emission detection wavelengths
- Integration with Huygens Deconvolution software optimizes images efficiently
Leica employs a novel mirror device and condenser-mounted objective to convert the SP8 DLS into a lightsheet microscope, providing ability to:
- Rapidly image even thick transparent fluorescent samples
- Conduct time-lapse imaging over long periods with low bleaching and toxicity
- Illuminate samples from both sides for clearer images and reduced shadowing
- Excite standard fluorophores with choice of four laser lines
For more information about the Leica SP8 DLS, please visit Leica Microsystems or contact us.
System specifications:
Leica TCS SP8 DLS hybrid laser scanning confocal and digital lightsheet microscope. The dual-mode motorized inverted DMi8 microscope has four laser excitation lines (405, 488, 552, and 638nm). In confocal mode, the Acousto-Optical Beam Splitter and Spectral Detection allow user selection of any desired emission detection window. Two hybrid detectors plus two PMTs permit either high or standard sensitivity fluorescence detection, plus transmitted light/DIC detector. Available confocal optics include 10x dry, 20x multi-immersion, 63x glycerol, and 40x, 63x, and 100x oil immersion objectives. The Digital LightSheet (DLS) mode excites samples in a plane at 90 degrees from the imaging objective, eliminating excitation out of the plane of focus. The high-sensitivity Hamamatsu Orca Flash 4.0 V2 sCMOS 16-bit camera, rapidly captures images with minimal phototoxicity and sample bleaching. DLS mode uses 10x and 25x long working distance water immersion imaging objectives. Image processing is performed using Leica LAS X software with integrated Huygens Deconvolution package.
Protocols
UnionBio BioSorter Pro
The Union Biometrica BioSorter Pro is a large object flow cytometer designed to sort objects beyond the capacity of standard FACS. The instrument is capable of sorting objects based on fluorescence or size and, with the 1000 micron flow cell, easily accommodates specimens up to approximately 700 microns in size. The BioSorter is ideal for sorting intact small animals, such as zebrafish embryos and larvae, Drosophila embryos and larvae, C. elegans, small tissues, and organoids.
Olympus LV200
The LV200 Bioluminescence Imaging System is a light-tight, environmentally controlled inverted microscope specifically designed for long-term imaging of bioluminescent cells and tissues. A highly sensitive camera permits imaging of even low levels of bioluminescence.
For more information about the LV200, visit Olympus Lifescience.
System specifications:
Olympus LV200 Bioluminescence Imaging System. The fully automated LV200 inverted microscope platform is contained in a light-tight environmental chamber with temperature, humidity, and gas controls. An Andor iXon 897D EMCCD camera provides high sensitivity and speed. System objectives range from 4x-100x. Bright field and fluorescence imaging capabilities are also included. Image acquisition and analysis are performed using cellSens software.