Strategies for sample preparation
Questions
- characteristics of your sample: living or fixed cells, tissue etc?
- labeling: fluorochromes, balanced brightness of labels, low crosstalk?
- magnification needed?
Choice of fluorochrome
Balance labeling intensity of the different channels. To minimize crosstalk between fluorescence channels in green / red fluorescence labeling, do not label too strong with the green fluorochrome, you may detect part of the signal in the red channel, especially when labeling with the red fluorochrome is only faint) Similar strategies can be applied for other fluorochrome combinations.
Cover slips
Use high quality cover slip (nr 1.5): Most lab suppliers offer only 0.15 mm (nr. 1) cover slips, which are not adequate for high resolution microscopy. Do not use them for high resolution (confocal etc.) microscopy.The objectives designed for the use with cover slips are generally designed for 0.17 mm (nr. 1.5) cover slips.
Think of cover slips being lens elements (actually they act as optical elements and are taken into account in objective optical design!)
Such cover slips are e.g. available in Germany from ASSISTENT (Germany, product # 1014, different sizes available), From ROTH, or from other companies.
Caution Nr.1: ASSISTENT assigned identical order numbers to cover slips of different thickness and different size! (but different order numbers to different containers). You cannot deduce thickness from order number!
Caution Nr.2: We were faced with the following issue: Many of the ASSISTENT #1014 cover slips we bought several years ago were denoted as 0.17 +- 0.01 mm, but probably were standard ones (at least in the packages we have)! There was a broad range in thickness (from 0.160 to 0.200 mm)!.
For studies where 0.170 mm thickness is a prerequisite, you can check and select cover slips using a micrometer measuring device.
Cover slips (selected thickness) from ROTH, Karlsruhe, Germany seem to match the requirements. But always check some when you open a new or unknown package!
Table cover slip number vs. thickness
| number | thickness |
| 0 | 0.08-0.12 mm |
| 1 | 0.13-0.17 mm |
| 1.5 | 0.16-0.19 mm |
| 2 | 0.19-0.23 mm |
| 3 | 0.28-0.32 mm |
Clean your cover slips: under certain conditions, some cover slips show some surface fluorescence generated by contaminating material. Clean your cover slips with analysis grade methanol, ethanol or acetone (any of these will probably work) and air-dry cover slips.
Mount cover slips correctly onto the slide: Mount samples / cover slips close to the center of the slide (to prevent tilting of the sample on the stage of an inverted microscope). Usually about 5-10 mm of the slide (at the left and right side) should be kept free (check the microscope stage slide holder). In addition, you may not be able to image samples too far off the center of the slide.
Embedding (mounting) media
Reduce bleaching. To minimize bleaching, you can enclose antibleach in embedding medium (see below). Caution: several combinations of antibleach/embedding medium and fluorochromes are not recommended!). Choose the appropriate embedding medium/antibleach combination! Since several years I mainly use Vectashield (Vector Labs.), which may be diluted with glycerol 1:1. DABCO (up to 2%) can also be added to this mix, but seems to interfere with the Hoechst dyes. Vectashield, on the other side, seems not to be compatible with Texas Red, but caused no problems with FITC, GFP, TRITC and the Cy dyes. Check any combination first!
Try to match the refractive index of the mounting medium and the lens immersion medium. The media mentioned above (refractive index of about 1.45) are suited better than buffer-based media (about 1.35, e.g. PBS alone with DABCO etc.) when using oil immersion lenses. For thick sample mounted in aqueous media (e.g. living cells), water immersion objective lenses are preferred (63x 1.2 N.A., to be used with cover slip).
Use liquid embedding media, if possible. Preparations mounted in liquid media (and your objects embedded within!) seem not to shrink in volume over time when sealed properly. Shrinking is at least less than in embedding media which harden by drying (like Mowiol, or ProLong from Molecular Probes). Samples embedded in liquid media like Vectashield or glycerol plus DABCO can be stored in the freezer (-20°C).
Mount your samples onto the cover slips, not onto the slides, if possible: The distance between the cover slip and the structure of interest should be as small as possible to minimize possible aberrations caused by a mismatch of refractive indices of objective immersion medium and embedding medium.
Use spacers between cover slips and slides, if possible - to keep both parallel to each other. Spacers can be made by cutting cover slips (use a diamond knife or marker).
Seal your samples properly (in case of liquid embedding media). Some embedding mixtures stay liquid, so the samples have to be sealed. Preparations embedded in Mowiol (hardening overnight) or Vectashield (liquid) stored at -20 C seem to be stable for weeks and even for months and can repeatedly be taken out of the freezer (be cautious with interpretations based on such old samples, but they still can be used for demonstration purposes).
Mixtures for sealing
Nail polish / nail hardener: To seal and to fix cover slips (separated from the slide by a thin layer of plastic cut off a freezer bag, or by other spacers), I use nail hardener, but sometimes I run into problems with its bad long-time contact with the slide surface. (Slides may be "contaminated" with embedding medium, or some mounting medium may penetrate slowly into or below the "hardened" glue).
VALAP: This is a 1:1:1 mixture of vaseline, lanolin and paraffin. It is prepared by mixing the components in a glass beaker or bottle on a heating plate. The mixture is applied by a small brush with a thin tip and hardens immediately. It can be reheated / reused many times. The mixture is preferred in cases where solvents of nail polish etc. may interfere (e.g. for life cell preparations).