PART I: 
DESCRIPTION OF SERVICES

Location:
A-103, A-105, and A-107

Telephone:
212-746-6146 

Staff:
Leona Cohen-Gould, M.S., Director (Rm. A-105)
Donald A. Fischman, M.D., Faculty Advisor

Facility Description
The facility provides transmission and scanning electron microscopy services, including specimen preparation, embedding, thin and ultrathin sectioning and staining (TEM) and critical point drying, sputter and/or carbon coating (SEM). The work can be done on an assisted or partially unassisted basis. Specialized techniques, such as immunolocalization may be arranged.

Equipment:
The facility has a JOEL 100CX-II transmission electron microscope equipped with a ASID unit enabling scanning EM, ultramicrotomes, a paraffin microtome, cryostat, 2 vacuum evaporators, critical point dryer and sputter coater, a low temperature UV chamber for polymerization of acrylic resins for immuno EM, and an automated tissue processor for paraffin embedding. 
 

ORGANIZATION AND SERVICES PROVIDED:

1. Consultation: Consultation on experimental design and approach is provided by Ms. Cohen-Gould.
2. Transmission Electron Microscopy: Full sample processing from fixation through embedding, sectioning and microscopy is available. The investigator may, after instruction, perform the embedding and microscopy unassisted. All ultramicrotomy is performed by the director. Film is processed in the facility and the investigator receives the set of negatives generated by his/her time at the microscope. Negative staining of cell fractions, viruses, etc. is also available. 
3. Scanning Electron Microscopy: Fixation, dehydration, critical point drying and sputter coating of small samples can be accomplished. Microscopy is conducted with the assistance of Ms. Cohen-Gould.
4. Microtomy: Sectioning for electron microscopy is done by the director. There is a second ultramicrotome available for those already versed in ultramicrotomy. Equipment is also available for the investigators' use to obtain paraffin and frozen sections for light microscopy. Ultrathin frozen sections for immuno EM are also available (see 5).
5. Ultrathin Frozen Sectioning technique for immunolocalization at the EM level utilizes the Facility's new RMC MT7000 ultramicrotome which is equipped with the CRX cryo chamber. Appropriate samples would be cell pellets (from culture) or small tissue samples. Convincing immunostaining, with appropriate controls in the form of photographs or microscope slides that can be examined in the epifluorescence microscope, is a pre-requisite for acceptance of material. The investigator will be responsible for providing the primary antibody or antibodies, and for having characterized the conditions for successful immunofluorescent labelling (fixation conditions, primary antibody concentration, etc.). The Core Facility will keep stocks of "standard" secondary antibodies (anti-mouse, anti-rabbit and protein A) tagged with colloidal gold for visualization of labelling at the EM level. The actual immunolabelling of the EM samples after sectioning can be performed by the investigator, working in the EM Facility under the supervision of Ms. Cohen-Gould. This would qualify as the "unassisted" rate which covers the cost of materials used. If the investigator wishes Ms. Cohen-Gould to carry out the immunolabelling in addition to the sample preparation and sectioning, he/she will be billed at the "assisted" rate. This is a time- and labor-intensive procedure: sectioning of a single sample may require 30-90 minutes exclusive of set-up and take-down time. Time per sample will depend on such things as orientation requirements and the number of grids needed. Samples will not be accepted unless the proposed procedure has been fully discussed, planned and scheduled with Ms. Cohen-Gould. 
6. Paraffin Embedding for Light Microscopy. The TissueTek VIP 150 automated tissue processing unit and the accompanying embedding console are now available for paraffin embedding for light microscopy applications. The tissue processing unit has up to 9 programs to allow for processing of a variety of tissues under controlled, reproducible conditions. It automatically steps your samples from fixative, through dehydration and xylene, into liquefied paraffin. Up to 150 sample cassettes can be handled at one time. Processing of 1 sample uses the same amount of time & reagents as 150, therefore "batching" your samples is encouraged. The user places his/her samples into individual cassettes, which are, in turn, placed in a carrier basket in the unit's reservoir. The lid is closed, a program selected and activated. The user returns at the end of the cycle to retrieve his/her samples and take them to the next station for embedding. The embedding console has a reservoir of warm, liquefied paraffin in which to hold your samples while you use the illuminated platform to embed individual samples in the reusable metal molds. The molds are then placed on the cooling platform to solidify the wax. Once cooled, samples are released from the metal molds and are ready for further handling.

The equipment will be maintained by Ms. Cohen-Gould. The facility will supply all reagents and the disposable cassettes, the reusable cassette lids and an adequate supply of reusable metal molds. Individuals who plan on processing large numbers of samples, or who do not want to unmold their blocks immediately must purchase their own molds. Users may also wish to purchase their own supply of cassettes so that they can color-code their samples. Ordering information can be obtained from Ms. Cohen-Gould.

Users will need to reserve the unit ahead of time. Once a cycle has begun, it must run to completion (Minimum processing time is about 3 hours with a 40 min. cleaning cycle after each use.). There will be a log book into which must be entered the user's name, number of samples processed and program used. With attention to scheduling and consideration of others, several runs/day are possible. Individuals will need to handle their own samples, from fixation to final sections. Because of both time constraints and the specific orientation requirements of many samples, this will not be offered as a drop-off service. 

7. Training: People who plan to use the facility on a regular basis are taught how to use the electron microscope to examine their samples and obtain electron micrographs. Instruction in sample preparation (embedding) for TEM and preparation of tissue for, and generation of, frozen sections for light microscopy is also available.

PART II: FEES FOR SERVICE

* Users may pay $3000.00/yr for unlimited, unassisted use of the electron microscope.
**3 grids per condition, 4 conditions tested as a standard. Additional samples will increase the hourly fee. 

PART III: USE OF THE ELECTRON MICROSCOPY CORE FACILITY

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