DERC Microscopy and Imaging Core 1. Core description
The Microscopy and Imaging Core provides research groups with ready access to microscopy equipment and training in the use of that equipment. Currently, the Microscopy Core contains two confocal microscopes, two widefield fluorescence microscopes, a brightfield microscope, offline computer workstations for image analysis and equipment for sectioning and histochemical processing of tissues. Image collection is performed by individual investigators, who must be certified to use the equipment. Certification requires the successful passing of a training course provided by the Microscopy Core. Recharges offset the costs of equipment maintenance and training.
2. Services provided
Access.
The Microscopy Core provides access to specialized instrumentation and software for microscopy applications.
Microscopy Training.
Training classes in general microscopy and for the operation of the confocal microscopes are provided. Training is done once a month in group sessions as follows:
Part 1: Introduction to Microscopy.
All users are required to attend this 75 minute lecture which is held on the first Tuesday of a month at 1:00 pm in HSW-1057. Please see Contact Information to confirm the lecture as it can vary depending on the travel plans of the lecturer.
Part 2: Hands-On Training
Hands-on training should only be initiated when use of the microscopy by the investigator is imminent. Please note that all training is subjected to recharges, as listed in Section 4. Thus, initial training is free provided that the trainee uses the equipment. Those requesting training a second time do not receive a credit.
2a. Confocal Training-1. Immediately following the Introductory lecture, a time convenient to all attendees will be scheduled for a four hour group training on one of the confocal systems. For those who attended an Introductory lecture in an earlier month, please come at the end of the Introductory lecture (2:15 pm) to schedule your hands-on training with the others.
2b. Confocal Training-2. There are minor differences in the two confocal systems. After a user is comfortable working on the first system, trainees should schedule a 30 minute training session on the second confocal microscope. See 2a for scheduling information.
2c. Widefield Fluorescence Training, Brightfield Training, Histology Training. Due to heavy usage, there currently is limited capacity to accommodate new investigators from non-Diabetes Center-affiliated laboratories. Please attend the Introductory Lecture to familiarize yourself with the basics of microscopy that will assist your image collection. Then contact a laboratory colleague who is already a user.
Advice.
The Core is primarily community-driven. Please contact fellow experienced users for assistance in the development of your imaging protocols.
3. Equipment available.
The Microscopy and Imaging Core Laboratory consists of two fluorescence microscopy rooms, a brightfield microscope and an image analysis room and a histology room, all within the U.C.S.F. Diabetes Center. Instruments currently in the Core include two confocal microscopes, 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 two confocal microscopes are available for use, after training (Section 2) by non-Diabetes Center-affiliated laboratories. The other equipment is heavily used and available only to Diabetes Center-affiliated personnel.
The Leica SP2 laser scanning confocal microscope
The Leica SP2 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 three separate tunable emission slits/photomultiplier tubes. A parallel transmitted light image can be colleted. 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 SP2 microscope is located in HSW-1141.
The Leica SL laser scanning confocal microscope
The Leica SL laser scanning confocal system consists of an upright microscope with 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 colleted. Most routine confocal applications should be collected with this microscope. 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-1141.
The Zeiss upright Axioscope 2 Plus flourescence microscope
The Zeiss upright Axioscope 2 Plus fluorescence microscope uses fixed filter cubes to capture widefield blue, green and red wide-field images of standard fluorophores. Image acquisition is to on a CCD camera. Most routine widefield applications should be collected with this microscope. 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). The Axioscope 2 microscope is currently is located in HSW-1141.
The Olympus IX-70 inverted flourescence microscope
The Olympus IX-70 inverted 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). The IX-70 microscope currently is located in S-1230.
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 onto a color CCD camera. The AxioImager A1 brightfield microscope currently is located in HSW-1000.
Post-Acquisition Data Analysis
Data analysis and processing should be conducted post-image acquisition on one of three workstations provided by the Microscopy Core in HSW-1000. These workstations are linked together allowing users to sit at any one of the terminals and access data. The data is also automatically backed-up by being stored as duplicates in two different locations.
The data analysis and processing workstations each contain Leica software for viewing and analyzing of the confocal images, as well as Metamorph 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 SP2, Leica TCS SL and Olympus IX-70 microscopes are saved directly onto the workstations.
Histology Equipment
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
4. Recharge Rates
The philosophy of the Core is to provide access to necessary instrumentation in a highly efficient and economical fashion. Thus, the Core is primarily User-initiated. Those requiring a fee-for-service level of support are recommended to contact other Cores.
The Core sustains equipment maintenance through recharges. Consolidating equipment use amongst a wide variety of investigators ensures optimal equipment use, thereby providing economy in equipment maintenance.
Recharge rates for the confocal and wide field systems are based upon maintenance and service contract costs. The current recharge rates are:
| Equipment | Training |
| | Recharge/ | Recharge/ |
| | Hour | Person |
| Leica SP2 | $34.71 | $150.58 |
| Leica SL | $23.21 | $133.33 |
| Zeiss widefield | $9.41 | $75.09 |
| Olympus widefield | $9.41 | $75.09 |
| Zeiss brightfield | $6.46 | $72.13 |
| Offline Workstation 1 | $2.35 | $68.02 |
| Offline Workstation 2 | $2.35 | $68.02 |
| Offline Workstation 3 | $2.35 | $68.02 |
| IXpress | $29.91 | $143.38 |
| IXpress Workstation | $2.35 | $68.02 |
| Assistance | $65.67 | |
5. Information on recharge arrangements
Please use the attached Microscopy Core Recharge Request Form to provide account and fund numbers to UCSF Core Administration Services when initiating use of the Microscopy Core. Investigators are required to update that information.
Please use the attached to provide account and fund numbers to UCSF Core Administration Services when initiating use of the Microscopy Core. Investigators are required to update that information.
Once the Recharge Information is submitted, investigators will be provided with login information to access the equipment. Logins are provided for laboratories, not for individuals within the laboratory. Use and recharge information will be returned to the PI and the Accounts Manager indicated on the form.
6. Hours of operation
The equipment will be available 24 hours a day to trained and authorized users. Access after 6 pm requires a key card and a code. User laboratories will need to have their key card activated in order to access the floor in the evening. All users of the equipment are required, without exception, to be trained and certified in Microscope use. Anybody who facilitates work by unauthorized people on the equipment shall have his/her authorization revoked.
7. Contact information
Fred Schaufele
8. Additional Information
DERC Intro Powerpoint introduction to the Core and to fluorescence microscopy
Training Synposis Very brief highlight of essential confocal control elements
Scheduling a Reservation Protocol for on-line reservation of Core Equipment by trained users
Confocal Start Up Reminder of generic start-up/shut-down procedures for our confocals
Oil Use Notice Important reminder of correct use of immersion oil on inverted microscopes
Olympus Operation Reminder of essentials of operating this widefield microscope
Zeiss Fluorescent Scope Policy Use policy on this heavily used microscope
9. Complementary UCSF Imaging Facilities & Resources
With the number and types of imaging equipment necessary to conduct modern biomedical research, it is beyond the domain of a single Core to deliver all equipment needs for all laboratories. Those following Campus Cores provide access to additional instruments, not available within the DERC Microscopy and Imaging Core, including the:
a) UCSF Biologic Imaging Development Center. The BIDC is closely affiliated with, and assists the introduction of new technologies to the DERC Microscopy and Imaging Core and other Cores on campus.
b) Laboratory for Cell Analysis of the UCSF Comprehensive Cancer Center.
c) Microscopy and Advanced Imaging Core of the UCSF Liver Center
d) UCSF Nikon Imaging Center
e) UCSF Small Molecular Discovery Center f) UCSF Center for Molecular and Functioning Imaging
