Waitt Advanced Biophotonics Core

The BPHO Core provides training and equipment for light microscopy and full-service sample processing and imaging for electron microscopy.

Training Materials, Protocols, and Manuals

Instrumentation

The Biophotonics Center Core Facility exists to provide Salk Faculty with technical support for and logistical access to state-of-the-art imaging technologies to enable the next generation of biological breakthroughs at the Institute.

Available equipment for core use:

Super-Resolution Microscopy

Zeiss Elyra PS.1 Super-Resolution Microscope


This inverted microscope is equipped with the following laser lines (405, 488, 561 and 635nm) and four color filtersets for both PAL-M and SR-SIM super-resolution imaging. It is equipped with two EM-CCD cameras, an Andor iXon3 897 512×512 35fps camera for PAL-M imaging in TIRF mode and an Andor iXon3 888 1024×1024 9 fps camera for 3D SR-SIM. The microscope also has a dedicated off-line processing workstation for the reconstruction of super-resolution imaging datasets.

Zeiss LSM 880 Rear Port Laser Scanning Confocal and Airyscan FAST Microscope

This inverted microscope is equipped with the following laser lines (405, 458, 488, 514, 561, 594 and 633nm). It is also equipped with a liquid-cooled 34 element internal GAsP detector for single-molecule sensitive full spectral imaging over the visible spectrum and a transmitted light detector for DIC imaging. It is also equipped with a Definite Focus module and incubation for extended time-lapse imaging of living cells. Finally, this microscope boasts the Airyscan FAST module for fast, superresolution microscopy with ~1.7x the resolution of standard confocal microscopes.

Oxford Nanoimaging 3D STORM and TIRF system

This inverted microscope is configured for TIRF, epifluorescence, HILO illumination, and PALM/STORM superresolution imaging. It is equipped with the following laser lines (405nm/150mW, 488/200mW, 561/500mW, and 640/1000mW) and 1.40 and 1.49 Olympus TIRF objectives. Two simultaneous imaging bands and laser filter sets. Large 50×80 mm field of view. TIRF auto alignment, autofocus, and drift/vibration compensation are all built-in. Software includes smFRET analysis, single-particle tracking, real-time localization, laser programming, python scripting interface, and colocalization analysis.

Confocal Microscopy

Zeiss LSM 710 Laser Scanning Confocal Microscope

This inverted microscope is equipped with the following laser lines (405, 458, 488, 514, 561, 594 and 633nm). It is also equipped with a 34 element internal detector for full spectral imaging over the visible spectrum and a transmitted light detector for DIC imaging. It is also equipped with a Definite Focus module for extended time-lapse imaging of living cells.

Zeiss CSU Spinning Disk Confocal Microscope

This inverted microscope is equipped with the following laser lines (405, 488, 561 and 635nm) and a Yokogawa spinning disk scan head with an EM-CCD camera and a stage-top incubation system for the high-speed confocal imaging of living cells in culture.

Yokogawa Cell Voyager 1000 Spinning Disk Confocal Microscope

This live cell imaging system is equipped with the following laser lines (405, 488 and 561) and a Yokogawa dual-pinhole spinning disk scan head with an EM-CCD camera and a stage-top incubation system for the high-speed confocal imaging of living cells in culture.

Lightsheet Microscopy

Zeiss Lightsheet Z.1 Dual Illumination Microscope

This microscope is ideal for large 3D or 4D volume imaging. It is a standalone sealed box system with temperature and CO2 control. 5x, 10x, and 20x objectives for both CLARITY and water-immersion. 405, 445, 488, 515, 561, and 638nm laser lines. Two PCO.Edge sCMOS cameras.

Two-photon Microscopy

Olympus FV1000MPE Two-photon / CARS Microscope


This upright microscope is equipped with the following single-photon laser lines (405, 440, 488, 514, 559, 633nm) as well as a Spectra-Physics MaiTai DeepSee infrared laser for two-photon excitation. It is equipped with 3 internal descanned detectors as well as 4 non-descanned two-photon fluorescence detectors and 2 forward scanning fluorescence detectors for Second Harmonic Generation (SHG) and Coherent Anti-Stokes Raman Microscopy (CARS).

TIRF Microscopy

Nikon Ti:E PFS TIRF Microscope

This inverted microscope is equipped with the following laser lines (405, 488, 561 and 635nm), an Andor EM-CCD camera and the Nikon patented PerfectFocus module for TIRF imaging. It is also equipped with a stage-top incubation system for maintaining living cells in culture.

Slide Scanning Microscopy

Olympus VS-120 Virtual Slde Scanning Microscope

This virtual slide scanner is equipped with an automated 100 slide loader and multiple imaging modalities. Fluorescent filter cubes include: DAPI, FITC, TRITC, and CY5. The system also takes advantage of an additional emission filter wheel allowing the use of a quad band exciter. In addition, this system can image with Bright field (histology).

Laser Capture Microdissection

Zeiss PALM Microbeam V4 Capture microdissection System

This microdissection microscope takes advantage of specialized membrane slides to cut and collect microscopic sections of tissue, or single cells. Capturing the sample in Eppendorf tubes, the user is then able to perform further analysis on their chosen sub-regions or cells.

Basic Wide-field/Epi-fluorescence Microscopy

Zeiss StereoLumar V12. Fluorescence Stereoscope

The V12 stereo scope is equipped with three different lenses allowing ideal imaging for dissection or large sample fluorescence imaging.

Incucyte ZOOM

Incucyte is an in-incubator live cell imaging platform capable of interval imaging in phase contrast or green/red fluorescent channels. The platform is equipped with a 4x and 10x objective and is able to image up to six tissue culture plates simultaneously (6 well to 384 well formats). Ideal imaging applications include: growth rate monitoring, kill curves, scratch wound assays (migration or infiltration), neurite outgrowth or neural network formation, phagocytosis assays, lineage tracing, and spheroid/organoid imaging. Incucycte is housed in the Stem Cell Core Facility (STEM). Please contact STEM Asst. Dir. Ken Diffenderfer (kdiffenderfer@salk.edu) for training and scheduling.

Transmission Electron Microscope

Carl Zeiss Libra 120kV PLUS Energy Filtered Transmission Electron Microscope (TEM) that is equipped with a 2kx2k fiber-optically coupled YAG CCD camera capable of montage imaging at high sensitivity and has an in-column energy filter which yields High-Contrast Imaging, which improves signal to noise ratios in images, as well as Electron Energy Loss Spectroscopy (EELS), which allows the elemental analysis of samples. In addition, the TEM is also equipped with a tilt specimen holder (+/- 75 degrees) for tomographic imaging, an in-column energy filter for electron loss and high-contrast imaging of biological samples, and cryogenically cooled stage for the native imaging of proteins and frozen, hydrated sections. The stage is also designed for single-axis tilting for performing cryo-tomography.

Scanning Electron Microscope

Carl Zeiss SIGMA Variable Pressure Field Emission Gun Scanning Electron Microscope (SEM) that is equipped with secondary electron (SE), backscattered electron (BSE), and variable pressure (VPSE) detectors, STEM detector, and cold-stage. System is also equipped with hardware/software for the ATLAS montage imaging module and the Shuttle and Find correlative microscopy navigation module to generate large tiled images and high-throughput workflow.

Serial Block Face Scanning Electron Microscope System

The Gatan 3View is a fully automated Serial Block Face Scanning Electron Microscope (SBFSEM) system. An ultramicrotome is mounted inside the vacuum chamber of the SIGMA SEM. The 3View automatically sections and images the exposed block face. The data sets generated can be combined to create z-stacks of a volume and later subsequently manually segmented.

Sample preparation instrumentation and services

The BPHO core has a dedicated EM sample preparation space within the facility to process samples for electron microscopy. The sample preparation instrumentation includes:

• Pelco BioWave Pro Microwave
• Leica UC7/FC7 ultramicrotome
• Leica EM PACT High-Pressure Freezer
• Leica AFS Automatic Freeze Substitution
• Leica EM GP Automatic Plunge Freezing
• Leica EM CPD300 Automated Critical Point Dryer
• Leica EM SCD500 Sputter Coater/Evaporator

The BPHO core provides processing of samples by expertly trained staff using established protocols, described below:

• Morphology – Samples are processed for conventional or cryo-TEM or SEM. Images produced yield either ultrastructural (TEM) or topographical (SEM) information.
• Immunohistochemistry – Standard pre- and post-embedding immunolabeling is performed on monolayers or tissue samples. Both methods provide subcellular localization of antigens.
• Volumetric – Three-dimensional data sets can either be obtained using electron tomography, cryo-electron tomography, Serial Block Face SEM (SBFSEM) or array tomography. This is ideal for revealing relationships that are not fully revealed from a single section.
• Correlative Light and Electron Microscopy – CLEM offers accessibility to antigens and visualization of whole cells, live or fixed, to further elucidate dynamic processes, protein localization, and cellular structure revealed by EM, bridging the two methodologies.
• High Pressure Freezing – High-pressure freezing cryo-immobilizes samples for the ultimate combination of preserved ultrastructure and increased access to antigens.
• Expansion Microscopy – Expansion of samples for superresolution fluorescence imaging using established protocols and reagents from the Boyden lab (MIT) and ExT Inc. (Cambridge).
• Tissue Clearing – X-CLARITY electrophoresis-based tissue clearing optimized for our CLARITY optics.

Data Analysis and Storage

• A storage server for short and long term data storage.
• A computational cluster to analyze and reconstruct the nanoscale architecture in three-dimensions.
• Three dual quad-processor workstations with equipped with the MATLAB, Metamorph, Imaris, Amira, Icarus and ImageJ/Fiji image analysis software packages, Microsoft Office Suite, and Adobe Creative Suite software packages. These workstations are also equipped with microscope software from Zeiss (ZEN Black 2012), Olympus, and Nikon.
• A high-end workstation equipped with same software listed above with the Zeiss Elyra Super-Resolution image-processing package.
• A second high-end workstation equipped with MATLAB, Imaris, ImageJ/Fiji, and the Gatan Digital Micrograph Suite image-processing package.

Microscopy Information links:

Carl Zeiss On-line Microscopy Campus

http://www.zeiss.com/campus

Olympus Microscopy Resource Center

http://www.olympusmicro.com/

Nikon Microscopy

http://www.microscopyu.com/

Microlist – A resource for light microscopists

https://www.microlist.org/