Technical Tips
Excitation
and emission wavelengths of various fluorophores
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Immunofluorescence
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:
(a) the proper fixative
(b) the proper temperature
(c) the proper blocking agent
(d) 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.
David Piston's paper
Coverslip Preparation
Ted Salmon lab at UNC-Chapel Hill has detailed
protocol to pre-treat coverslips before use.
Link
Mounting
Media
There are many mounting media available in the
market. Users are advised to avoid using VectorShield as this mounting
medium does not solidify and generate a lot of problems during imaging.
There are also several home-made recipes that work very well for fluorescent
microscopy. One of these is gelvatol, which can be easily made in large
quantity and it stores very well for a long time.
Gelvatol
Recipe
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. Please refer to the fluorescent
emission fingerprinting link for further details. To perform proper
linear unmixing of these various contaminants in addition to the desired
fluorophore emission, users must supply the META LSM 510 system 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.
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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.
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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.
Temperature control for live cell imaging
The Zeiss LSM510 META confocal microscope is also equipped with a Zeiss 37-2 digital stage warmer. The stage warmer can hold both 60mm and 35mm cell culture dishes. The protocol for calibrating and fine-tuning the temperature controller can be downloaded here.
For labs seeking to perform more sophisticated and dedicated live cell imaging experiments, we recommend the FCS-2 perfusion chambers from Bioptechs, Inc or the stage-top CO2 incubator from Tokai HIT