Technical Tips

Excitation and emission wavelengths of various fluorophores


Please note that some antibodies, cellular structures, and samples will respond very differently to different fixation methods. You can save yourself a lot of time if you first find out:

  • the proper fixative
  • the proper temperature
  • the proper blocking agent
  • the optimal antibody concentration

Try fixatives such as formaldehyde, paraformaldehyde, glutaraldehyde, ice-cold methanol, ice-cold acetone etc. Also be aware that certain structures, such as the microtubule network, are sensitive to temperature change. Immunofluorescence almost always requires higher antibody concentration than Western blots, and may require different blocking agent. Users must be mindful of these variables and perform appropriate experiments to survey the proper condition for their samples.

Magnification versus Numerical Aperture

If there is a single, most important basic knowledge about microscopy, it would be to know the difference between the magnification and numerical aperture of the objective lenses one is using. It is a very common misconception that the higher the magnification the better the resolution of the image one will acquire. Resolution is determined by the numerical aperture of the objective lens, and not by the magnification. To obtain a basic understanding of this concept, the facility recommends user to read David W. Piston's paper available as a PDF file below.

Optimizing pixel size selection, magnification and Z-step size

The choice of objective lens will affect the optimal pixel size and step size in the Z-axis when performing a confocal Z-stack. The document below lists the relationship between these various factors based on Nyquist calculation.

Coverslip Preparation

Ted Salmon lab at UNC-Chapel Hill has detailed protocol to pre-treat coverslips before use.

Mounting Media

There are many types of mounting media available in the market. Users are advised to avoid using VectorShield as this mounting medium does not solidify and generates a lot of problems during imaging. I addition to commercial options, there are also several home-made recipes that work very well for fluorescent microscopy.

Tissue sample

Users performing immuno-staining must be aware that autofluorescence is a very common problem in all tissue samples. Those who are familiar with chromogenic stain will be dismayed to find that even unstained tissue samples emits fluorescence across very wide spectrum, spanning multiple fluorescent channels. This inherent background autofluorescence cannot be easily removed with biochemical treatment, and must be spectrally dealt with before any accurate data can be used for image analysis. To perform proper linear unmixing of these various contaminants in addition to the desired fluorophore emission, users must supply the Nikon A1R a few control spectra, as listed below:

1. Pure background autofluorescence

Provide a tissue sample that was not stained by any fluorescent probe. This will generate a reference spectrum for the autofluorescence background. Be aware that every fixation protocol will generate distinct autofluorescence spectrum. If you switch fixation protocol, please supply a corresponding control.

2. Positive fluorescent signal

Provide a positive control containing pure fluorophore for each probe you will be using. The easiest way to make this control is mix a little of your undiluted fluorescent probe (such as a fluorescently labeled secondary antibody) with the mounting agent and mount the coverslip to a slide without any tissue/cell sample. Please note that this is different from a regular positive control wherein a sample with positive target is incubated with the probe. That is a positive control at the biochemical level (meaning you are testing if the antibody binds specifically) and not at the spectral level.

After the proper spectral information is fed into the system, an unmixing process can be performed, through which a high autofluorescent background from a tissue sample such as the one shown on the left can be converted into a background-free image as displayed on the right. Picture source: Laboratory of Dr. Peter Gann.


Many users, on the other hand, also expect us to spectrally clean up the non-specific binding pattern created by their antibodies. Please note that this is an impossible problem to correct by any optical system -- because it is a biochemical problem!

There are numerous things one can try:

  1. Confirm the species and isotype of the antibodies used. Do not use multiple primary antibodies from the same species in one sample.
  2. Pre-incubate antibodies with tissues that generate non-specific binding can sometimes reduce non-specific pattern.

Emission crosstalk

Many widely used fluorophores have extensively overlapping excitation and/or emission spectra. Overlapped excitation spectra means that the excitation light for one fluorophore can partially excite the other porbe used in the same sample. Overlapped emission spectra means that users can see the emitted light of one fluorophore in the channel used for the other fluorophore. One such example is the commonly used pair of cyan fluorescent protein (CFP and yellow fluorescent protein (YFP). The emission spectrum of CFP overlaps that YFP significantly, as shown below:

It is thus important for users to properly correct for the potential channel "bleed-through" if the nature of the experiment (such as imaging rapid biological process) precludes the use of emission fingerprinting, and when the relative intensities of CFP and YFP are critical part of the experimental data, as in the case of FRET experiments. There are multiple methods to perform intensity correction for CFP-YFP FRET. Please click here for a PDF file.