Generating the best image involves many factors, all dependent on what is ideal for imaging the marker of interest in its biological context. Here are some questions to ask yourself when choosing the right reagents and instruments.

Do I want to image tissue thicker than 10 µm? Confocal or Multiphoton Microscopy
Do I want to image more than 4 colors on a cell sample? Spectral Unmixing
Do I want to reconstruct the sample in 3D? Confocal or ApoTome
What level of resolution is desired/required? Deconvolution, Structured Illumination, STED or PALM
Do I want to demonstrate colocalization/binding or bioactivity in live cells? FRET or FLIM

1. Number of Targets

Using the spectral data of each fluorophore, you can make choices about optimal filter selection to minimize spectral spillover in experiments up to 4 colors for both confocal and widefield microscopy. Above 5 colors, a microscope employing spectral detection becomes useful to unmix the spectral spillover. If using antibodies for detection, problems can arise with the species-dependence of the primary and secondary antibody combination. Ideally, the use of directly labeled antibodies or haptens like biotin/streptavidin can help.

 

2. Fluorophore Combinations with Overlapping Spectra

One tip when using fluorophores where one spills into the other is to make sure the two antibodies are not imaged on markers that co-localize. For example, image one on a marker in the nucleus and the other at the cell surface, when possible. Also, make sure the fluorophore that is spilling over into the neighbor filter is on the less abundant antigen.

3. GFP or a fluorescent protein variant

Fluorescent proteins do not survive exposure to methanol or acetone. If the GFP signal was present prior to fixation but signal is lost upon fixation, check to see if the paraformaldehyde was reconstituted with the help of methanol. If the fixative can’t be changed to be organic solvent-free, anti-GFP antibodies can be employed to recover the GFP signal.

4. Instrument Choice

The instrument is made to be an ideal tool for the biological question, not the reverse. The better you understand the goal of the image, the better you can match the application to the instrument.

5. Sensitivity

Sensitivity is a balance between the signal strength and non-specific staining/autofluorescence/background. Biological autofluorescence will be endogenous in certain tissues, like brain, liver, lung, etc. The following can be applied to minimize background and improve signal strength:

  • An appropriately complex blocking step, for example serum instead of BSA or milk, prior to adding antibodies.
  • If streptavidin is used and the tissue will be fixed and permeabilized, an endogenous biotin-blocking kit can prevent the biotin found naturally in mitochondria from binding the streptavidin.
  • Use directly conjugated primary antibodies for sufficiently abundant antigens.
  • Use secondary antibodies or other amplification methods for lowly expressed antigens. Another option is to use biotin and streptavidin or other hapten-based amplification methods.
  • Use enzymatic amplification kits like tyramide signal amplification (TSA) kits for extremely lowly expressed antigens. As a cautionary note, amplification and secondary techniques will also likely increase your background.

6. Antifade

Finally, mounting media containing antifade is required for the maintenance of signal strength. All organic fluorophores photobleach, a process where reactive oxygen species created in the process of imaging attack the structure of the fluorophores, irreversibly neutralizing their ability to fluoresce. Using antifade is more difficult when the cells are imaged live, since any antifade scavenges oxygen from the media, thus suffocating the cells. This is why regenerating signal, like proteins expressing GFP, are desirable for long-term, live-cell imaging.