Announcing a significant new opportunity for microscopic imaging at Washington University!
Dr. Heuser’s “deep-etch” electron microscopy laboratory is expanding to include a fee-for-service facility dedicated to advancing other investigators’ imaging projects.
If you would like to retain our services, please review this page, then download the DEEM Job Request Form (Word format), fill it out, and bring it to your initial consultation or fax to the included number.
Services to be provided by the laboratory include the following
1. Quick-freezing of all sorts of biological samples, (from whole organs and tissues, to individual cells and cell-cultures, to individual macromolecular complexes).
This alternative to traditional chemical fixation for electron microscopy is advantageous or even mandatory when samples are osmotically fragile, or when they are composed of materials that do not react properly with chemical fixatives, like complex sugars and lipids. It is also mandatory when it becomes important to capture fleeting biological or biochemical events, such as cellular movements or transient changes in macromolecular architecture associated with enzymatic or ‘motor’ activities.
The Heuser lab is still the only facility in the world that has optimal procedures and machines for accomplishing ‘near-perfect’ freezing. Alternatives that are in widespread use, like “high-pressure’ freezing with the expensive Balzers or Leica machines sold commercially, are widely acknowledged to be capricious, inconsistent, and even wholly inapplicable for many of the sorts of medical/biological samples currently under study, like monolayer cell cultures or whole tissues.
2. Preparation of quick-frozen samples for electron microscopy. The Heuser lab has developed a whole range of different techniques for ‘prepping’ samples after they have been quick-frozen, depending on the sorts of views that are required for the particular project. These are outlined briefly here.
2a. Freeze-fracture EM. The most straightforward way to view a frozen sample in the EM is to simply cleave it open under a vacuum and deposit a metal ‘replica’ on its surface, then view this replica in the electron microscope.
This is ideal for preserving the exact state of the sample as it was frozen and for avoiding any and all structural artifacts that might arise from subsequent procedures (below). This it is ideal for observing in vitro emulsions, suspensions, and extracellular polymers. It is also ideal for displaying ultrastructural details in membrane architecture, since membranes cleave preferentially after optimal quick-freezing.
2b. “Deep-etch” EM. This is an extension of procedure (1) above, whereby a freeze-cleaved sample is permitted to begin to freeze-dry in the vacuum for a minute or two, before metal replication.
This brings into view cell surfaces and organelle surfaces, as well as the formed elements in the cytoplasm (macromolecules in suspension and the cytoskeleton, itself.) Producing such images in dramatic 3-D views has long been the ‘bread and butter” of the Heuser lab, as it was pioneered by them over 30 years ago. Despite the fact that this procedure is so rarely used (since only Heuser and a handful of his progeny around the world are technically equipped to do so), it has yielded a wealth of information about pathogenic bacteria, pathogenic protists, yeast and other fungi, as well as all aspects of vertebrate cell structure and function....all because it creates the unique opportunity to image the true surfaces of biological membranes and thus, all the protein polymers that coat the inside and outside of living membranes.
2c. “Freeze-dry” EM. This is a simple shortcut that doesn’t even involve the freeze-cleavage used in procedures (1) and (2) above, but simply involves mounting any quick-frozen sample in a vacuum chamber and completely freeze-drying it (by raising its temperature to -100 degrees C for ~15min).
This brings into bold relief the entire upper surfaces of whole cells grown in monolayer culture, or bacteria and yeast grown in suspension, or tissues with free serosal surfaces, or any other biological or organic material that has a free surface.
It can also be used to obtain highly informative overall views of all sorts of subcellular components (their substructure, their organization. their purity, etc), so long as they can tolerate the added step of being adsorbed to a glass or mica surface, after which they are quick-frozen like any other sample. (The quality of freezing accomplished by the Heuser lab is not compromised by mounting a biological sample on a solid substrate, and as a result, their freeze-drying preserves the samples’ ultrastructure much more faithfully than any other approach.)
- This is the ‘quality’ alternative to traditional “negative staining” of subcellular samples for electron microscopy.
- This has also become, most recently, a highly desirable alternative method for viewing “immunogold”-decorated cryosections in the electron microscope.
This widely used and highly informative procedure, the true link between light and electron microscopy, is already performed by several EM labs that operate as service facilities at Wash U. However, not until the Heuser lab joined forces with these other labs and began to freeze-dry and replicate their cryosections, did it become possible to see clearly within these sections the biological membranes, their protein coats, and the cytoskeletal elements that associate with them.
2d. “Freeze-substitution” followed by traditional plastic thin-sectioning EM. This is the procedure that yields relatively ‘classical’ thin-section views of biological cells and tissues, but distinctly ‘non-classical’ in the sense that they display vastly improved preservation and are more ‘lifelike’ in their portrayal of all aspects of cell ultrastructure. (This is because “freeze-substitution” accomplishes chemical fixation and dehydration while the biological sample is still frozen, and thereby does not ‘lose ground’ from the high-quality preservation achieved during its initial quick-freezing.)
Again, a number of expensive commercial devices are available today to carry out this procedure, but none of them work as well or as dependably as the homemade devices used in the Heuser lab.
(Once a sample submitted to the Heuser lab is optimally quick-frozen, freeze-substituted, and embedded in plastic in preparation for thin sectioning, it is the responsibility of the investigator involved to find another service lab at Wash U. to actually get the thin sections cut.)
Besides being more ‘lifelike’ in their portrayal of all aspects of cell ultrastructure, thin sections of quick-frozen and freeze-substituted cells display many features not seen at all after traditional EM preparation, like extracellular proteoglycan coats, intracellular lipid storage-deposits, etc., etc. They are considered “de rigueur” by many of the leading EM laboratories around the world today, but no other lab can achieve the quality of freezing that is available through the Heuser lab at Wash U., hence none achieve such quality results.
2e. Molecular imaging or so-called “single-particle” analysis. Deep etch EM is carried to its maximum resolution of fine molecular detail (4nm) by briefly adsorbing a dilute suspension of isolated & purified macromolecules to thin flakes of freshly-cleaved mica, and then quick-freezing, deep etching, and replicating these mica flakes.
This yields highly informative, 3-D images of macromolecular architecture in any biological molecule that is 40-50kDa or larger. It is the perfect in vitro complement to direct imaging of macromolecules ‘in situ’ in quick-frozen cells, as carried out in the ‘classical’ deep-etch EM procedure outlined in (2) above.
Ancillary advanced LM-imaging techniques available through the Heuser lab
EM projects that involve high-resolution EM imaging of whole cells typically benefit from correlative ‘livecell’ LM-imaging of cell dynamics. The Heuser lab can offer several different ‘livecell’ light microscopes for performing these studies, in conjunction with whatever outside EM projects are undertaken.
These include one of the latest ‘total internal reflection fluorescence’ (TIRF) microscopes, as well as one of the last of the fine BioRad confocal microscopes, plus the unique Yokagawa spinning-disk confocal microscope, especially suitable for imaging of ultra-sensitive living cells.
Also available are unique “Antiflex” optics, not available anywhere else at Wash. U., for imaging living cells by the completely benign technique of ‘interference-reflection microscopy’ (IRM). This powerful technique for imaging dynamics near the bottoms of cells has largely been forgotten, but turns out to be the perfect complement to modern-day TIRF-imaging.
Details of procedure for initiating a project with the Heuser ‘deep-etch’ EM laboratory
- Contact John Heuser at <firstname.lastname@example.org> or 314-362-5105 to set up a time for initial consultation and planning of your project. Note that Dr. Heuser will be available to ‘hold your hand’ at every step of your EM project, from the initial step of planning which procedure would be best for your samples and your experimental designs, to the final step of interpreting the 3-D electron micrographs that are made from your samples. (The electron microscopic images will initially be provided to you in a high-resolution (~30KB) “anaglyph” 3-D format suitable for viewing in Photoshop on either a Mac or a PC. Once in your hands, they can readily be converted to ‘flat’, 2-D images in B&W, for mounting in PowerPoint or Keynote presentations.)
- Prepare your samples, as planned together, and deliver them
to the Heuser lab at the time scheduled for them to be frozen.
- Users of the facility are welcome to “look over our shoulders” as their samples are being worked up, or as they are being examined in the electron microscope. This can be done by the PI’s themselves or by the relevant postdocs or students involved. (Past experience has shown, however, that most users of the facility find this too tedious; under certain circumstances we may be able to offer limited opportunities for personal instruction and “hands-on” experience with the workups or with the EM analysis, itself.)
- ‘Turnaround time’ from initial submission of the samples until some feedback from the Heuser lab is provided should generally be around one week, though certain forms of workup (such as freeze-drying) take less than a day, allowing for limited adjustments in protocol as a project is underway.
FY 2007 RATE SCHEDULE:
Deep-Etch Electron Microscopy (DEEM)
$500 per sample (Flat rate)
Up to 10 preliminary samples may need to be processed for each DEEM sample that is fully worked up. $500 minimum charge applies even if none of the preliminary samples are deemed worthwhile for further workup.
Rate includes up to 20 images per sample (generated initially on EM film, but provided in high-res digital and “anaglyph” 3-D versions).
$900 per set, plus technician-time for the initial quick-freezing @ $60 per hour (a “set” to consist of up to ten samples carried through complete freeze-substitution and plastic embedding)
$60 per hour
Live cell light microscopy
$102 per hour (includes technician time plus microscope time)
$60 per hour (including production and processing of more than 20 images per sample, if that becomes necessary for quantitation, etc.)