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Coverslip Secrets

Coverslips typically come in 4 thicknesses: #0 = approximately 0.10 mm thick #1 = approximately 0.12- 0.15 mm thick #1-1/2 = approximately 0.17 mm thick #2 = approximately 0.2mm thick. For the best image, you want the right coverslip: 170 microns. However, in the case where you want additional depth penetration into your sample, you might use a thinner coverslip, and trade optical quality for increased depth. This is because each objective has a maximum working distance at which it can focus. The thinner the glass, the deeper you can ultimately focus.

For example, our 40X/1.30 Plan Neofluar can penetrate 120 microns into your sample if you use the #1.5 (170 microns thick), but you could go 180 microns with a #1 (which is 90-110 microns thick), or 230 microns using a #0 (50-60 microns thick). Image quality will degrade at all depths with these thinner glasses, but you'll be able to image deeper into the sample.

Since these estimates assume an optically clear sample with no refraction, practical distances will be somewhat less than that, although you could approach these distances by employing clearing steps (with e.g. methyl salicylate) and a mounting medium with the proper index of refraction. Also note that the thickness values are nominal, and can vary by large amounts, but the general message is still the same.

All of the foregoing assumes that you've already taken care of the most common problem we encounter: Do not allow excessive distance from your coverslip to your sample. This can not be stressed too highly. Some people worry about crushing their cells. In practice, it's usually the opposite problem; if the cells have a 100 micron layer of medium between them and the coverslip, image quality will suffer greatly, unless you pay a lot of attention to the optical quality of the medium. The ideal situation is to grow the cells on the coverslip that you will be imaging through, reducing the distance to zero, and giving us the clearest possible image. This is why we frequently recommend Mat-Tek dishes.

Various Mounting Media and Anti-fade Reagents:
(Not a complete listing. This is a collection of suggestions from many users. Please contact me with your suggestions, corrections, and additions.)

Mounting Media:

The sample's surroundings will affect the image to a great degree. For optimal quality, you would ideally like your sample to be immersed in a material with the same index of refraction as our immersion oil (1.518). If the material is able to solidify, you will avoid problems with movement of the sample (or the coverslip) relative to the slide. And for unstable fluorophores, which tend to photobleach quickly, many of the commercial mounting media contain anti-fading reagents in them.

Note that your sample's characteristics will determine whether you want an aqueous medium or an organic one.

Anti-fade additives:

Many people prefer to add specific anti-fade reagents, such as the following, to their mounting media (often made from scratch). Although the mechanism of the photobleaching has not been completely elucidated, it has been found that you can reduce the effect by using free-radical scavengers, minimizing the amount of oxygen in the sample, etc.

Keep in mind that every fluorophore has an optimal pH range, and buffer your sample accordingly.


Where do I start?

Much information for new users can be found in an entire section of this web site dedicated just to you.
Head on over to the New User Information section.


How do I sign up to use a microscope?

ALL CISR microscopes are available ONLY to those who have been trained in their use by a member of the RESOURCE STAFF. After training, you will be able to sign up and use the microscopes as needed. Detailed instructions on microscope use can be found on most Equipment pages.


How much will this cost?

CISR SERVICE PRICES [as of September 1st, 2018]

SERVICE
*RATE / UNIT
MICROSCOPE USE
LSM 510 Confocals, scope time$35.00 / hour
Confocal, scope time$45.00 / hour
Widefield, scope time$35.00 / hour
Super Res, scope time$25.00 / hour
Multiphoton Electrophys, scope time$20.00 / hour
TEM, scope time$100.00 / hour
ESEM, scope time$50.00 / hour
 
Nikon Center of Excellence
Nikon Spinning Disk, scope time$25.00 / hour
Nikon SIM Super Resolution, scope time$25.00 / hour
Nikon STORM Super Resolution, scope time$25.00 / hour
 
EM PROCESSING
embed (Spurr's)$50.00 / capsule
thick section (routine)$50.00 / block
thick section (non-routine) > 1 hour process$60.00 / hour
thin section (routine)$100.00 / block
thin section (non-routine) > 2 hour process$60.00 / hour
negative stain, material only$6.00 / grid
Immunolabel, material only$20.00 / kit
ESEM prep, material only$10.00 / stub
TEM grid box$5.25 / each
Staff Review - thin section EM$60.00 / grid
 
Technical Help
Imaging for hire, training, protracted help$60.00 / hour


*Payment for service is processed through the VU CORES system using center numbers supplied by each user prior to receiving service. An account application is required for all new users. The CISR is supported, in part, by 5 NIH-funded Research Center Grants. Accordingly, members of the following Centers may be eligible to receive assistance to pay for service with a "scholarship" number: VICC, DRTC, DDRC, MMPC, and VVRC. Please contact your Center administration for details.


** Services provided to non-Vanderbilt University investigators requires a completed, current "Research Core Services Agreement." Services are limited to non-clinical research samples. Commercial customer prices will be adjusted to equal 1.6 times the service rates in the table above.

When are the microscopes available?

All CISR microscopes are in secured rooms, most requiring a VU ID badge for entry. CISR light microscopes are available for use 24/7. TEM and SEM are available Monday through Friday 9:00 a.m. – 6:00 p.m. with use after hours or on weekends limited to users with extensive experience who have undergone additional training.

Please remember that ALL scopes should be reserved before use through the online calendar reservation system.


Will you help me plan my experiment?

For new experiments or for inexperienced researchers it is best to consult with CISR staff to insure that specimen preparation is optimized and to ensure you have a through understanding of microscope use. A brief meeting and/or training sesstion between the Resource staff and the researchers laboratory to iron out details can be arranged by contacting anyone on our Staff page. This meeting can help determine feasibility, provide suggestions for optimal use of the resource, and, if appropriate, alert the resource staff to unique details of specimen preparation for your project.


How do I prepare my samples for light microscopy?

That depends on the sample, the fluorophore, and the information desired, but here are a couple of examples: a tissue staining protocol, and a cell staining protocol. Some notes on coverslips can be found here, and some information on anti-fade reagents and mounting media is located here.


How do I prepare my samples for EM?

Samples must be fixed prior to submission. Please see the Tips for Fixation Procedures for EM question on this page. If you have samples for immunolabeling or any other specialized protocol, please discuss your project with EM staff prior to sample preparation.

Fixation is typically achieved with a 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer. Fixation should be initiated at room temperature. Tissue should be fixed in a solution volume of at least ten times the sample volume. Specimen vials pre-filled with fixative solution are available for users in the refrigerator of the Processing Lab located in room T-3208 of MCN.

Larger volumes of fixative and buffer are available for whole-animal perfusion fixation (recommended). Please contact a member of our staff to arrange for pick up.

Samples for SEM must also be fixed. Typically these samples will be processed in the critical point dryer by EM staff and mounted for examination in the SEM.


Do you have any tips for fixation procedures for EM?

To maintain ultrastructure, specimens must be fixed rapidly after the oxygen supply has been cut off. The specifics of fixation (fixative, concentration, time, temperature) and the buffering medium will vary for specific experiments. However, in general, fixation will involve buffered aldehydes which cross link proteins, carbohydrates, and to a certain extent lipids. The fixatives will cross link similar molecules that surround the sample (body fluids, cell culture medium, etc. ) so the sample should be washed briefly in a buffered solution before fixation.

Aldehyde fixatives penetrate tissue slowly, so samples must be small (less than 0.5 mm on two sides). For large specimens, mincing the sample directly in fixative after washing works best. This can be done by placing a drop of fixative on dental wax or parafilm and placing the excised and washed sample in the drop of fixative and then mincing with fresh razor blades. The EM staff can show you how this is done.

Cold depolymerizes cytoskeletal elements, so whenever possible, buffer washes, fixatives, and sample should be at room temperature or warmer to initiate fixation. For temperature sensitive samples, such as enzyme cytochemistry, the samples can be briefly warmed placed in fixative and then, after a minute, re-cooled. To store fixed samples for more than a few hours, the specimen should be placed on ice or in the refrigerator after fixation. For storage of samples more than a few days before processing, the fixative should be replaced with buffer after 24 hours.


How do I submit my EM samples?

All samples must now go through the EM online laboratory information management system located here. Please contact a member of our staff if you experience any problems or need access to the system.

Please note that specimens for EM processing should be fixed prior to submission and kept refrigerated until submitted. EM fixative and wash buffer can be obtained from the refrigerator in the Processing Lab located at T-3208 MCN.


How do I save my data?

CISRstore is a very large, very fast disk storage system available to all registered Cell Imaging Shared Resource users, and is the core's preferred location for storing image files. Click Here to view instructions on accessing the CISRstore.


Where do I get relevant software?

The free LSM Image browser is here.

The free NIS-Elements Viewer is here.

The free Olympus Viewer is here.

The free ZEN browser is here.


How do I get help with advanced imaging techniques?

Answer: Just ask!

CISR staff members are available to assist you with FRET, FRAP, time lapse, image tiling, and many, many other specialized procedures. Extended help may be subject to additional charges, but most answers are free. We are always ready to help.