Imaging Services - BIDMC

Imagine Core B- BIDMC Services

 

 

Histology BIDMC: https://www.bidmc.org/research/core-facilities/histology-confocal-em/histology-core

Confocal/Immunostaining BIDMC: https://www.bidmc.org/research/core-facilities/histology-confocal-em/confocal-imaging-core

Electron Microscopy BIDMC: https://www.bidmc.org/research/core-facilities/histology-confocal-em/electron-microscopy-em-core

 

 

The HDDC Imaging Core B at the Beth Israel Deaconess Medical Center (BIDMC) consists of two interacting components:

The Histology core, provides service for paraffin embedding, sectioning, staining, and frozen sectioning and the Imaging/Microscopy core, provides shared equipment and service for confocal, widefield, photodocumentation, electron microscopy, and digital image processing. In addition, the microscopy core has an immunostaining service.

All work is done on a fee-for-service basis to recover costs for equipment upkeep, supplies, and technical assistance. The fee schedule for HDDC members (July 1, 2021-March 31, 2022) include priority access and service for all HDDC users with no overhead charges for users with labs outside of the BIDMC. Substantially discounted rates for HDDC users will return as a membership benefit on April 1, 2022.

 

 

FACILITIES, EQUIPMENT and PRICING

Facilities

The HDDC Core B at BIDMC is located on the 8th floor of the Charles A. Dana Research Building on the East Campus of the BIDMC (please note: this is NOT at the Dana Farber Cancer Center!!) and consists of more than 2,000 square feet of space. Maps and other information about access, pricing, and services can be found at http://www.bidmc.org/Research/CoreFacilities.aspx.

Equipment

Histology

1) Leica TP1020 Tissue Processor

This tissue processor is used to dehydrate and infiltrate tissues/cells with paraffin. The instrument has vacuum function and a fume control system. The carousel configuration has 12 stations; 10 for reagents and 2 for melted paraffin. Two loading baskets can be run each night with an 80 cassette capacity per basket. Infiltration time is separately programmable for each station. There is a delayed start function up to 9 days; this allows processing over the weekend to be ready on Monday morning. The machine also has the capacity to interrupt an automatic process for reloading or removing cassettes for special applications before the end of a run.

 

 

2) Leica EG1160 Embedding Center Dispenser and Hot Plate

This equipment is used to embed tissues/cells that are infiltrated with paraffin. The machine has a separately heated paraffin dispensing system. It additionally has a compressor-cooled cold spot for cooling blocks and a melted paraffin holding reservoir for the newly processed cassettes.

3) Leica XL Autostainer

This equipment is used to stain paraffin sections. It has the capability of programming numerous special stains or customer-specific modifications of the H&E stain. There are18 reagent stations, 5 wash stations, and an integrated forced hot air oven that significantly reduces slide drying time. The instrument has the capability of continuous loading - it can load up to 11 racks (with 30 slides per rack) while processing. The capacity is 150 slides per hour with a typical H&E protocol. The machine also allows for simultaneous staining of several different protocols - the user can select any compatible program for each rack. The instrument has self-contained fume control with a replaceable filter. The microprocessor controller can handle up to 15 programs with up to 25 steps each.

4) Leica IP S Automated Slide Printer, which is an ink-jet printing module that is used for high-throughput printing of labels for histology slides.

5) Leica CM 1850 Cryostat, which is used to prepare sections from frozen tissue samples for immunostaining and analysis by confocal microscopy or for immunoperoxidase methods.

6) Leica RT2125 Rotary Microtomes (3), which are used to cut paraffin sections.

 

 

Electron Microscopy

1) Ultracut E Ultramicrotomes (2), which are used to cut thick and ultrathin plastic sections.

2) Ultracut-S Ultramicrotome, which is used to cut thick and ultrathin sections from frozen tissue. Although we have this instrumentation, there is currently no expertise in the core for cryo ultramicroscopy. This instrument would have to be used independently by the HDDC member.

3) Zeiss Stemi 508 Stereomicroscope

This diissecting microscope is used to prepare samples for electron microscopy, particularly for high pressure freezing applications. It is equipped with incident light and a cold-light source CL 6000 LED. The photoport has magnifications from 6x to 50x. There is an eyepiece camera available, if customers wish to photograph a sample.

4) JEOL JEM-1400 Transmission Electron Microscope

This microscope is used for the visualization of tissues by electron microscopy. This updated electron microscope is outfitted with both side (rapid acquisition with a wide viewing area) and bottom (high resolution) Gatan Orius 11 mexapixel ccd cameras, the latter being used to acquire images for electron tomography. The microscope is also outfitted with a 70-degree tilting goniometer, a Fischione dual axis specimen holder and a single tilt specimen holder for doing both single and dual-axis tomograms (can be rented by HDDC members).

5) Bal-Tec MED 020 Vacuum Evaporator, which is used to deposit a fine carbon film on formvar coated grids and to glow discharge the grids before ultrathin sections are placed on the grid.

The electron microscopy core also has a glass knife breaker, numerous diamond knives for both plastic and cryosectioning, a custom machine to pull thin films for making formvar coated grids, and ovens, rotators, and other small equipment needed to prepare samples.

 

Confocal Microscopy

1) Zeiss LSM880 Upright Confocal System

This laser scanning confocal system is outfitted with 6 laser lines (404, 458, 488, 514, 561, and 633), 3 PMT's, a motorized scanning stage and stepper motor, 10x (air), 20x (air), 40x (water corrected), 63x (oil), 100x (oil) objective lenses with DIC optics; Zen acquisition software with the tiling module, a high-capacity computer and 30-inch high resolution monitor.

2) Zeiss LSM880 Live-Cell Confocal System with Fast Airyscan

This inverted confocal system is used to visualize physiological processes in living cells. This instrument has a completely enclosed incubator system with an O2/CO2 regulation for long-term support of live cell experiments. The system is configured to acquire long time-lapse data, to do FRET, FRAP, and ratio imaging using calcium and intracellular pH dyes, and also serves as a secondary instrument for the acquisition of data from fixed tissue/fixed cell samples, when needed. A list of specific features are as follows: Axio Observer Z1 stand with joystick control; Zeiss environmental incubator; heating insert; Zeiss O2/CO2, and humidity control; assortment of live imaging chambers and inserts; Eppendorf InjectMan N1 2 with FemtoJet microinjection system; 10x (air), 20x (air), 40x (water corrected), 63x (oil), 100x (oil) objective lenses and DIC optics; 6 laser lines (405, 458, 488, 514, 561, and 633), 3 PMT's, a motorized scanning stage and stepper motor, Zeiss Zen Physiology software for FRET, time lapse acquisition, etc..computer, and a 32 inch high-resolution monitor.

Recently, the Airyscan detector with software capability to image in near super-resolution mode was added to the LSM880 Live Cell Confocal System. This equipment also facilitates use of spectral unmixing technology, used for multi-label applications being developed in the core. Further capability was added with the Fast module, which allows much faster acquisition via parallel excitation and detection.

 

 

Widefield fluorescence microscope with photodocumentation

1) Zeiss Axioimager M1 microscope with color and B/W CCD cameras

This upright widefield microscope is used for photodocumentation of stained paraffin sections and for fluorescence imaging of tissue sections. A sophisticated physiology software package accompanies this software to extend its processing capabilities. The specific features of this instrumentation are as follows: AxioCam MRm (B/W) and HRc (color) CCD cameras; X-Cite Mercury Vapor Short Arc Lamp; 5x (air), 10x (air), 20x (air), 40x (oil), 63x (oil), and 100x (oil) objective lenses; red, green, blue and far-red fluorescence filters; Zeiss AxioVision software including the Physiology package; additional off-line work station.

Immunostaining

  1. DAKO Autostainer Plus (Agilent)

Although many of our staining protocols and done by hand, new in the core (2021) is the ability to do large-volume repeated datasets using the DAKO Autostainer Plus. This instrument can process 48 slides/run and is automated. Work using this equipment has to be via service work contracted through the Confocal Imaging Core component of Core B.

Computers-Image Processing

1) Two Dell Precision 3630 computer workstations.

These computer workstations contain an 8 core i9 processor; Windows 10; 64 GB RAM; a P4000 video card; 1TB NVMe SSD with a 4 TB data hard drive; DVD player; 32 inch high resolution monitor, and laser mouse.

Software-Image Processing

1) Image J is freeware from the NIH that is used by most labs, thus it will not be discussed further.

2) Imaris Software (Oxford Instruments)

This software allows for the rapid, interactive, high-resolution volume rendering of 3D and 4D data sets. This program will accept a range of file types from confocal, wide field, and electron microscopes. The user is able to detect objects, do very high-quality rendering, navigate through 3D/4D volumes, visualize and quantify colocalized regions, segment and analyze cells and their compartments, create images to export, take snapshots, and animate the data. This software is most effective when using fluorescence images.

4) SerialEM (the University of Colorado, Boulder)

This software is used to acquire tilt series for electron tomography, large image areas for 3-D reconstruction from serial sections, and images for reconstruction of macromolecules by single-particle methods. The main focus of the program is automated tilt series acquisition. For this, it uses an approach based on prediction of specimen position during the tilt series from the position at previous tilts. It does not count on the microscope or the specimen being particularly well-behaved, so unless a prediction appears reliable, it falls back to measuring and adjusting defocus and/or specimen position when necessary. With this method, it achieves high quality tilt series with both robustness and speed.

5) IMOD (the University of Colorado, Boulder)

This software is a set of image processing, modeling, and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. This program is free-ware, readily available to academic users. We typically use IMOD for assembling 3-D tomograms and use Volocity software for assembling 3-D stacks from the confocal system.

6) Chimera (UCSF)

This software is a highly extensible program for interactive visualization and analysis of molecular structures and related data, including density maps, supramolecular assemblies, sequence alignments, docking results, trajectories, and conformational ensembles. We use this program, in particular, to produce high-quality images and animations (movies). Chimera is free-ware for academic uses.

Fees for use of BIDMC Core B equipment/services 

 

 

Available through Core B: Harvard Center for Biological Imaging, Harvard University

https://hcbi.fas.harvard.edu/equipment

Below is a listing of equipment available for HDDC members to use at the HCBI. Please visit their website (above) for details and capability of the equipment. All work that HDDC members want to do at the HCBI must be arranged through Core B. Please contact Susan Hagen (shagen@bidmc.harvard.edu) for details.

  1. Zeiss Lightsheet.7 Microscope
  2. X-CLARITY Tissue Clearing System
  3. LifeCanvass SmartClear Pro Tissue Clearing System
  4. Zeiss ELYRA7 Super-resolution Microscope system
  5. Zeiss LSM980 Confocal System with Airyscan2
  6. Zeiss LSM880 Confocal System with FLIM
  7. Zeiss LSM980 NLO Multi-Photon Microscope
  8. Zeiss LSM700 Confocal System
  9. Zeiss LSM900 Confocal System
  10. Zeiss Axio Scan.Z1 (two systems are available in the HCBI)
  11. Zeiss AxioZoom V16 Zoom Microscope
  12. Celldiscoverer 7 Live/High Content Microscope
  13. PALM Laser MIcrodisection Microscope

 

Available through Core B: Harvard Center for Biological Imaging, Harvard University

https://cns1.rc.fas.harvard.edu/

Below is a listing of equipment to do FIB-SEM applications that are available for HDDC members to use at the CNS. Please visit their website (above) for details and capability of the equipment. All work that HDDC members want to do at the CNS must be arranged through Core B including the initial sample prep. Please contact Susan Hagen (shagen@bidmc.harvard.edu) for details.

  1. Zeiss CrossBeam FIB
  2. Various sputter coaters, saws, etc.. for final sample prep prior to FIB milling.