If you are imaging cells or tissue with the intention of comparing changes in protein expression or colocalisation it is important to keep everything consistent. This is because big variations in fluorescent intensity can occur between objectives as well as between microscopes. Even if you are not making sensitive measurements it is still a good idea to choose a microscope for your project and stick with it – this way if there any changes through your project you can be sure they are not related to the microscope.
To keep things consistent remember to:
- Use the same microscope – different microscopes will have different bulbs and have usually been running for different amount of time – this means different light intensity and subsequently different exposure times.
- Use Colibri/LED illumination where possible – LEDs don’t change in intensity very much as they age. Colibri can cause increased crosstalk between channels so check for this.
- Use the same objective
- Use the same exposure time – use your brightest sample to set the exposure time, this will help you to avoid overexposure.
- Image your samples as close together as possible – if it is an important experiment try to do all your staining and imaging at the same time – as staining ages it can become less fluorescent so imagining some slides one week and other slides a few weeks later will give you confounding results. Similarly, as the bulb in the microscope ages it will become less intense or if it is replaced it may be much brighter than when you first set your exposure times.