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.