The BPHO Core provides training and equipment for both light and electron microscopy, sample processing, imaging and analysis. In addition to the Director, Dr. Daniela Boassa, the BPHO is staffed by two full-time imaging specialists, Dr. Elsie Quansah and Mr. Sammy Weiser Novak, who have expertise in light and electron microscopy, as well as data analysis.
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:
Light Microscopes
- Zeiss LSM 880 with Airyscan
- There are three LSM 880s with Airyscan in the Core (referred to as 880-1, 880-2, 880-3). They are equipped with the following laser lines: 405, 458, 488, 514, 561, 594, and 633 nm. They have 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. These microscopes boast the Airyscan FAST module for fast, enhanced resolution (~1.7x higher than standard confocal microscopes). Motorized X, Y stage and Z galvo focusing stage. The acquisition software is Zen Black (Zen 2.3).
880-1 (inverted) –
Objectives: 10x/0.45, 20x/0.8, 40x/1.0 W, 40x/1.0 Oil , 63x/1.4 Oil –
Equipped with a Definite Focus module and incubation for extended time-lapse imaging of living cells. Incubation chamber allows for regulation of temperature and CO2.
880-2 (inverted)–
Objectives: 10x/0.3, 20x/0.8, 40x/1.0 W, 63x/1.4 Oil, 63x/1.46 Oil –
Equipped with a Definite Focus module and incubation for extended time-lapse imaging of living cells. Incubation chamber allows for regulation of temperature and CO2.
880-3 (upright) –
Objectives: 10x/0.45, 20x/0.8, 20x/1.0 W, 63x/1.4 Oil – Not set up for live cell imaging.
- Zeiss LSM 710 Confocal Microscope
- This inverted microscope is equipped with the following laser lines: 405, 458, 488, 514, 561, 594, and 633 nm. 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. Equipped with a Definite Focus module. The acquisition software is Zen Black (ZEN 2.3).
Objectives: 10x/0.45, 20x/0.8, 20x/1.0 W, 63x/1.4 Oil
- Zeiss Spinning Disk Confocal Microscope
- This inverted microscope is equipped with the following laser lines: 405, 488, 561, and 635 nm, 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. The acquisition software is Zen Blue (ZEN 2.3).
Objectives: 5x/0.25, 10x/0.45, 20x/0.8, 40x/1.3 Oil, 63x/1.4, Oil, 100x/1.4 Oil
- Olympus FluoView FV3000 Confocal Microscope
- Inverted microscope with laser lines: 405, 445, 488, 514, 561, 594, 640, 730 nm. The wavelength detection capabilities to the NIR region of up to 890 nm through a suite of carefully engineered NIR, NIR-sensitive GaAs detectors enable up to 6 channels for multiplexed imaging from violet to NIR (400 nm – 890 nm), resonant scanner for time-lapse imaging. Long working distance silicone objectives enable detailed microscopic imaging at depth. Equipped with Tokai Hit incubation chamber to maintain optimal temperature, CO2 and humidity for live cell imaging. TruFocus Z-drift compensation system which maintains focus during live cell imaging despite changes in temperature or added reagents. The acquisition software is FV31S-SW.
Objectives: 4x/0.16, 20x/0.8, 30x/1.05 Sil, 40x/1.25 Sil, 60x/1.5 Oil, 100x/1.35 Sil.
- This microscope is equipped with 405, 445, 488, 515, 561, and 638 nm laser lines. It is ideal for large 3D or 4D volume imaging. It is a standalone sealed box system with temperature and CO2 control. It also has two PCO.Edge sCMOS cameras. The acquisition software is Zen Black (ZEN 2014 SP1 ).
Illumination objectives: two 5x/0.1 and two 10x/0.2
Detection objectives: 5x/0.16, 10x/0.5, and 20x/1.0 for both CLARITY and water-immersion.
- Olympus VS-120 Virtual Slide 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 Brightfield (histology). The acquisition software is VS-ASW.
Objectives: 2x/0.08, 4x/0.16, 10x/0.4, 20x/0.75, and 40x/0.95.
- Zeiss Axioscan 7 Slide Scanner Microscope (referred to as Newton)
- Automated scanning of up to 100 slides in a single run. Both fluorescence and brightfield imaging possible. This system is equipped with Colibri 7 LED light source with excitation at 385, 430, 475, 555, 590, 630, 735 nm. Brightfield imaging with Axiocam 705 color camera and Fluorescent imaging with Axiocam 712 camera. It has a 10 position filter wheel consisting of single-band filter sets for specific imaging: FS96 for DAPI, FS38 for AF488, FS43 for AF555, FS64 for AF594, FS50 for AF647 and multi-band filter sets for fast imaging: FS90- DAPI, AF488, AF555, AF647, and FS110- DAPI, AF488, AF594, AF750. The acquisition software is Zen Blue (ZEN 3.9).
Objectives: 5x/0.25, 10x/0.45, 20x /0.5, 20x/0.8, 40x/0.95.
- Zeiss Axioscan 7 Slide Scanner Microscope (referred to as Einstein)
- Automated scanning of up to 100 slides in a single run. Both fluorescence and brightfield imaging possible. This system is equipped with Colibri 7 LED light source with excitation at 385, 430, 475, 555, 590, 630, 735 nm. Brightfield imaging with Axiocam 705 color camera and Fluorescent imaging with Orca Flash camera. It has a 6 position filter wheel consisting of single-band filter set for specific imaging: FS90 for AF488, AF555, AF647, FS64 for AF594 and multi-band filter sets for fast imaging: FS110 for fast imaging of DAPI, AF488, AF594, AF750. The acquisition software is Zen Blue (ZEN 3.10).
Objectives: 5x/0.25, 10x/0.45, 20x/0.8, 40x/0.9
- 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. The acquisition software is Zen Black (Zen 2.3 SP1).
Objectives: 10x/0.3, 63x/1.4, 100x/1.46, 100x/1.57
- Oxford Nanoimager (ONI)
- This inverted microscope is configured for TIRF, epifluorescence, HILO illumination, and PALM/STORM super resolution imaging. It is equipped with the following laser lines: 405nm, 488, 561, and 640. Two simultaneous imaging bands and laser filter sets. 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.
Objective: 100x/1.45
- 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 into adhesive caps of eppendorf tubes. Further analysis on the chosen sub-regions or isolated cells can be processed for DNA, RNA and protein analysis. The acquisition software is PALMRobo V4.5
Objectives: 5x/0.25, 10x/0.5, 40x/0.6
Electron Microscopes
- Carl Zeiss Libra 120kV PLUS Energy Filtered Transmission Electron Microscope (TEM) equipped with a 2kx2k fiber-optically coupled YAG CCD camera capable of montage imaging at high sensitivity and in-column energy filter which yields High-Contrast Imaging, 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.
- Carl Zeiss SIGMA Variable Pressure Field Emission Gun Scanning Electron Microscope (SEM) 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.
- 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 segmented.
Sample preparation instrumentation and services
The BPHO core has a dedicated sample preparation space within the facility, including processing for electron microscopy. The sample preparation instrumentation includes:
- Pelco BioWave Pro Microwave
- Leica UC7/FC7 ultramicrotome
- Leica vibratome
- Leica cryostat
- Leica EM ICE High-Pressure Freezer
- Leica AFS Automatic Freeze Substitution
- Leica EM CPD300 Automated Critical Point Dryer
- Leica EM SCD500 Sputter Coater/Evaporator
- Leica EM GP Automatic Plunge Freezer
- Vitrobot system
- Clipping station
- Solarus II Plasma Cleaner
- Logos X-CLARITY
Please inquire for further information: bpho@salk.edu.
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
- Nikon Microscopy
- http://www.microscopyu.com/
- Microlist – A resource for light microscopists
- https://www.microlist.org/
