Services
The EMRL is both a full service facility that will help plan and perform electron imaging experiments for the research community at the University of Pennsylvania and regional research institutions as well as a resource facility open to trained, experienced researchers who need access to our state of the art facility to perform aspects of their own research. Below is a list of our full service offerings which is followed by a schedule of our current fees.
- Consultations are required before initiating a project with the facility. These are designed to acquire an initial literature base for the project, discuss the preceding work that has led to electron microscopy experiments, the feasibility of the desired approach, the expected outcomes, the financial costs of the experimentation, and define the experimental roles of the two parties to obtain desired results.
- Routine EM analysis is broken down into sub categories based on the desired preparation and ultra-structural detail our clients demand. This is not a menu, but a general overview of our approaches and experimental design may require various combinations or unique approaches not listed below.
- Thick sectioning: microtome sections exceeding 100nm thickness. These are appropriate for tomographic volume imaging and histo-pathological examination in light microscopes for correlative EM studies or identifying specific tissue structures that are the target of the EM examination.
- Thin sectioning: standard sectioning of blocks routinely around 60-100 nm thickness. Thinner sections provide greater surface area for antigenicity and structural detail due to less occlusion of epitopes or structures.
- Microwave processing is used for rapid processing of samples for EM (typical processing times are one week). Microwave energy can also be used to provide better infiltration of epoxy resin into difficult to embed material. It also is a preferred method for histo-pathological fixation for light microscopy.
- Pre-embedding immune-EM is labeling of samples similar to methods used for immune-fluorescent microscopy but is followed by standard plastic embedding for TEM.
- Post embedding immune-EM (single label and double labeling) labeling of thin sections of acrylic embedded tissue from high pressure frozen material is our typical first approach. However, multiple approaches are available including correlative light and EM methods and Tokuyasu methods.
- TEM and S/TEM imaging is charged by the hour and scheduled preferably online through the online scheduler. Three TEMs are available for imaging and each is best for certain applications so please contact us to discuss your needs. Training is absolutely required and provided for TEM use.
- SEM imaging is also charge by the hour in our facility with a Quanta 250F eSEM. This instrument is low vacuum and wet STEM capable for imaging unfixed biological material such as plant, bacterial, and yeast surfaces. The facility also has sputter coaters and critical point driers for specimen preparation for more traditional high vacuum SEM.
- Negative stain analysis is used to identify the molecular size and composition of biochemically pure viruses, proteins, nucleic acids, or liposomal complexes.
- Specimen fixation, dehydration, embedding, and sectioning are standard protocols within the lab and carry with each sample routine TEM time of one hour/sample. TEM time beyond this one hour limit will now need to be charged.
- Sectioning and staining of a duplicate block is available for return examination of previously processed samples. This service also carries with it one our TEM time/sample.
- High pressure freezing The HPF machine generates a pressurized stream of liquid nitrogen that rapidly freezes (<10 ns) samples of up to 300 µm thickness without forming large ice crystals. This allows for near instantaneous preservation of cellular events so transient events typically lost by standard chemical fixation procedures, can be preserved for imaging. It also produces a superior quality of preservation when low temperature dehydration and embedding are used to process the samples.
- Freeze substitution is performed at a controlled rate in a freeze substitution machine that ramps frozen material up from -100°C to +60°C in a controlled manner such that cells are fixed, dehydrated, and embedded at low temperatures to avoid ice crystal formation that can damage cellular structures.
- Cryosectioning is performed in a specialized microtome that can operate at liquid nitrogen temperatures. Cryosections are either directly imaged or are prepared for Tokuyasu immunolabeling. We are not strong advocates of cryo-sectioned material because the technique is fraught with quality and preservation issues. To use it is a last resort of difficult antigens.
- Plunge Freezing is the preferred method of forming vitreous ice around subcellular sized biological materials (<5 µm). It requires cooling ethane to liquid nitrogen temperatures and plunging samples absorbed and blotted to a vicinal layer into the liquid N2 cooled ethane slurry.
- Single particle image reconstruction is performed on homogeneous macromolecular complexes of molecular weights greater than 100,000 Daltons. Software algorithms are used to classify images into bins of similar views, compare these views to one another to determine the angular and translational relationships, and the views are then back projected to generate a three dimensional volume of the macromolecule.
- Tomography is performed on cellular or non-unique structure to generate three-dimensional volumes. The sample is tilted relative to the optical axis of the camera for a series of images. These tilt series images can then be aligned and back-projected to form a three-dimensional volume of this material. Additional averaging can be subsequently performed, or the volume segmented to generate models of specific sub-cellular or tissue layer structures.
- Montages can be generated of large areas at relatively high magnifications so that a large atlas of the tissue is created that can be explored at both low and high magnifications. Montaging and serial joining can be combined with tomography to generate volumes of areas that far exceed the TEM’s capabilities to image in a single section or image.
- Scripting can be requested for streamlining image processing.
- Computational assistance and access to EMRL computers is available upon request.
- Training is provided for all methods and techniques we provide at cost plus staff time. A commitment to training is required for any long term project initiated within the EMRL facility. Single particle EM is one such project where multiple training sessions may be required. If a long term commitment is not necessitated by a project the EMRL facility reserves the right to deny training to transient users. Training is required to access EMRL instrumentation.
- Electron dispersive microscopy: Inelastic events provide x-ray emissions that are characteristic of elemental content. The resolution of this technique is micron in scale and does not give high resolution. For high resolution compositional mapping, STEM energy loss spectroscopy is advised. Contrast imaging on a HAADF is available for this approach.
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SERVICE
- Hourly use EM T12 and JEOL 1010, $50/hr
- Routine analyses TEM, $350/sample
- Pre-embedding IEM, $350/sample
- Plunge freezing, $125/sample
- Thin sectioning only, $75
- Image construction tomography, $450/tomo (dual axis)
- Single particle data collection, $100/hr
- Single particle analysis - software, $100/hr
- High pressure freezing, $125/sample
- Freeze substitution fixation, $250/run
- Cryosectioning, Quote only
- Training, $100/hr
- Microwave tissue processing, $500/run
- Electron cryo-microscopy, $120/hr
- Electron dispersive x-ray spectroscopy, $150/hr
- Post-embedding IEM, $478/sample
- Each additional antibody, $120/sample
- Duplicate block for TEM analyses, $172/sample
- Duplicate block for post-embedding IEM, $225/sample
- Specimen processing only, $170/sample
- Thick section only (exsisting block), $50/sample
- Negative stain anaylses, $150/session
- SEM analyses, $250/sample
- Montage imaging, $100/montage
- TF-20 use, $125/hr (assisted), $75/hr (unassisted), $1500/24hr EPU run
- Quanta 250 SEM, $85/hr
- EMRL does not charge for consultation unless this requires extensive instruction which we bill as training