Microscopy Core Services

 

Instruments within the Diabetes Center Microscopy Core include two electron microscopes, two confocal microscopes, a structured illumination optical sectioning microscope, a high throughput microscope, two widefield microscopes, three microtomes, a cryostat, a tissue processor, a histoembedder, a brightfield microscope and a suite of three computers available exclusively for post-acquisition image analysis. The microscope and image analysis stations are available for use, after training by any UCSF and, pending availability, non-UCSF laboratories. The other equipment is heavily used and available only to Diabetes Center-affiliated personnel.

All equipment, the training in their proper use and assistance in the use of the equipment is available to all campus members on a recharge basis. Portions of equipment use by members of members are reminded to acknowledge DERC Center grant (P30 DK063720) contributions with ‘This research was supported by grants x, y and facilities support from NIH P30 DK063720”.

 

The JEOL 1400 Transmission Electron Microscope

The JEOL 1400 Transmission Electron Microscope is configured for high contrast of biological samples. Attributes include:

  • Accelerating Voltage - 40-120 KV
  • Magnification: Standard - 200x to 1,200,000x / Low Mag - 50x to 1,000x
  • Sample Holder: Single grid, quick change, +/-30•
  • Digital/Graphic User Interface
  • On-line help, tutorials and guides
  • Gatan Orius CCD camera 4008 X 2672 pixels
  • SerialEM software for montages and tomography

 

Sample preparation or instruction and guidance in sample preparation is available.

 

The JEOL JCM-5000 Neoscope Scanning Electron Microscope

The JEOL JCM-5000 Neoscope scanning electron microscope is a versatile instrument:

  • Accelerating voltage is selectable at 5, 10 or 15 kV.
  • Detectors include secondary electrons or back-scattered electrons in low vacuum mode.
  • The X-Y sample stage can accommodate specimens up to 70mm in diameter and 50mm high. There is no external control of tilt and rotation at this time.
  • The digital image can be acquired at 1280 X 1024 pixels.
  • The digital user is interface is very easy to learn and use.

 

Sample preparation or instruction and guidance in sample preparation is available.

 

Leica SL Laser Scanning Confocal Microscope

The Leica SL laser scanning confocal system consists of an upright microscope with two detector channels and three lasers generating six lines for fluorescence excitation (458, 476, 488, 514, 543, 633 nm). Emissions are split using fixed dichroic mirrors. The system contains spectral scanning and three-dimensional acquisition/display capabilities. Emissions are split into a spectrum using a prism and are collected through two separate tunable emission slits/photomultiplier tubes. A parallel transmitted light image can be collected. Currently available objectives on the system are: 63x oil immersion (Plan-Apo, 1.4 NA), 40x oil immersion (Plan-Apo, 1.25 NA), and 10x multi-immersion (oil, water, glycerin) (Plan-Apo, 0.4 NA). The SL microscope is located in HSW-1142.

 

Leica SP5 Laser Scanning Confocal Microscope

The Leica SP5 laser scanning confocal system is used for advanced multi-channel fluorescence and live-cell applications. This system has five lasers generating nine lines for fluorescence excitation (405, 458, 476, 488, 496, 514, 543, 594, 633 nm). The system is equipped with an inverted microscope encased with an incubator for live-cell imaging. An acousto-optical beam splitter allows the investigator to rapidly switch collection parameters without the mechanical movement of dichroic mirrors. Emissions are split into a spectrum using a prism and are collected through five separate tunable emission slits/photomultiplier tubes. A parallel transmitted light image can be collected. The system also contains spectral scanning, three-dimensional acquisition/display and time-lapse capabilities. A programmable stage allows automated ‘return-to-site’ and multiple-field stitching. Currently available objectives on the system are: 63x water immersion (Plan-Apo, 1.2 NA), 63x oil immersion (Plan-Apo, 1.4 NA), 40x oil immersion (Plan-Apo, 1.25 NA), 20x multi-immersion (oil, water, glycerin)( Plan-Apo, 0.7 NA) and a 10x air objective (Plan-Fluotar, 0.3 NA). All objectives (except 10x) are capable of DIC imaging. The SP5 microscope is located in HSW-1142.

 

Molecular Devices IX-Micro High Throughput Microscope

The Molecular Devices IX-Micro high throughput microscope provides automated widefield imaging of live, fluorescent protein-labeled cells or fixed, stained cells in 96-well or 384-well format. Plate read times vary with number of fields collected per well, number of fluorescent channels collected per field and the image capture time per field but, as a rough estimate, a 384-well plate is collected in 18 minutes for one field/well and one fluorescent channel captured for 150 ms. The automated microscope also contains a fluidics handling capability which can allow the automated generation of time course data collection on living cells in follow-up studies.

Equally important is the data handling components of the system with includes MetaXpress4 to operate the microscope, a file server to store and collate images, three offline workstations for analysis of the data as it is collected, a data server into which that analytical data is stored and AcuityXpress4 software which organizes the image and analytical data and conducts comparative analyses to rapidly identify hits from the thousands of wells collected.

Currently available objectives on the system are: 60x air (Plan-Fluor, 0.85 NA), 40x air (Plan-Apo, 0.95 NA), 40x long-working distance air (Plan-Fluor, 0.60 NA), 20x air (S Fluor, 0.75 NA), 20x long-working distance air (Plan-Fluor, 0.45 NA), 10x air (S Fluor, 0.60 NA), 4x air (Plan-Apo, 0.20 NA). Filter sets are available for a wide variety of fluorophores ranging from DAPI to Alexa 647. Up to four different channels can be cleanly distinguished. The IX Micro high throughput microscope is currently is located in HSE-1690.

 

Zeiss Apotome/ Widefield Upright

Zeiss Axio Imager2 with Apotome microscope is an upright with an Axio Cam MrC5 color camera and a Roper/Photometrics CoolSnap HQ2 monochrome camera for fluorescence. Filter cubes installed are:

  • Filter Set 49 DAPI,EX G365
  • Filter Set 38 HE GFP, FITC
  • Filter Set 43 HE Cy3
  • Filter Set 64 HE mPlum
  • Filter Set 50 Cy5, EX BP640/30

The microscope can be used in widefield or Apotome structured illumination mode which provides reduced background and moderate optical sectioning capability. The system uses Axiovision 4.8 software. The installed lenses are: EC Plan-Neofluar 5x/0.16, EC Plan-Neofluar 10x/0.30, Plan-Apochromat 20x/0.8, EC Plan-Neofluar 40x/1.3 DIC. The stage is manually operated.

 

The Olympus IX-70 Inverted Fluorescence Microscope

The Olympus IX-70 inverted widefield fluorescence microscope uses computer-controlled excitation and emission filter wheels to collect multi-channel wide-field fluorescence images on a CCD camera. This system is optimized for quantitative fluorescence microscopy. A programmable stage allows automated ‘return-to-site’ and multiple-field stitching. Currently available objectives on the system are: 100x oil immersion (Plan-Apo, 1.4 NA), 60x air (Plan-Apo, 0.9 NA), 40x air (Plan-Apo, 0.95 NA), 20x air (Plan-Apo, 0.75 NA), 20x lwd air (Plan-Fluar, 0.45 NA), 10x air (Plan-S-Apo, 0.4 NA). 

 

The Zeiss Upright Axioscope 2 Plus Fluorescence Microscope

The Zeiss upright Axioscope 2 Plus widefield fluorescence microscope uses fixed filter cubes to capture widefield blue, green and red wide-field images of standard fluorophores. Image acquisition is via a CCD camera. Currently available objectives on the system are: 100x oil immersion (Plan-Apo, 1.4 NA), 40x air (Plan-NeoFluar, 0.75 NA), 20x air (Plan-NeoFluar, 0.5 NA), 10x air (Plan-NeoFluar, 0.3 NA), 5x air (Plan-NeoFluar, 0.16 NA), 2.5x air (Plan-NeoFluar, 0.075 NA). The Axioscope 2 microscope is currently is located in room RMB 921E.

 

The Zeiss AxioImager A1 Brightfield Microscope

The Zeiss AxioImager A1 brightfield microscope is the work-horse microscope for the collection of histochemically and immuno-histochemically stained samples. Currently available objectives on the system are: 40x air (Plan-Apo, 0.95 NA), 20x air (Plan-Apo, 0.75 NA), 10x air (Plan-Apo, 0.45 NA), 5x air (Plan-NeoFluar, 0.15 NA) 1.25x air (Plan-NeoFluar, 0.03 NA). Collection is via a color CCD camera. The AxioImager A1 brightfield microscope is temporarily located in HSW1230 while its HSW-1000 home is undergoing renovation.

 

Image Analysis Workstations

Data analysis and processing should be conducted post-image acquisition on one of three workstations provided by the Microscopy Core in HSW-1000 (temporarily in HSW1230 during 10th floor renovation). These workstations are linked together allowing users to sit at any one of the terminals and access data. Three other image processing workstations dedicated to the IX Micro high throughput microscope are located next to that microscope in HSE-1690.

The data analysis and processing workstations each contain Leica, Zeiss, and FIJI (ImageJ) software for viewing and analyzing of the confocal images, as well as Metamorph 7.6 and NX and Metafluor software for more advanced quantitative image analysis. The use of these Offline analysis packages allows the investigator to minimize microscope recharges and keeps the microscopes free for other users. Data captured on the Leica TCS SP5, Leica TCS SL, Zeiss Apotome, Zeiss fluorescence and Olympus IX-70 microscopes are saved directly onto a server accessed by the workstations.

 

Histology Room

Major histology equipment for the sectioning and processing of tissues for staining and imaging in a dedicated histology room (HSW-1026) complete with fumehoods. Equipment available includes: Leica RM 2153 and two 2155 Microtomes; Leica CM1900 Cryostat; Leica TP1050 Tissue processor; Histoembedder.

 

Direct Assistance

The Core Manager and/or Faculty Director are available, time-permitting and subject to recharge, to directly assist in the collection or analysis of your microscopy data.