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Instruments:
Laser Scanning
Confocal
Photo-activation
& Conversion
Spinning Disc
Confocal
Fluor. Emission
Fingerprinting
Total Internal
Reflection Fluor.
Axioskop Fluo. Microscope
Microinjector
Rotary Shadowing System
Ultramicrotomes
Trans. Electron
Microscopes
Softwares:
MetaMorph 6.0
Volocity
2.0
Zeiss LSM 510
Software
Zeiss Image Examiner
Resources:
Publications
Links
Technical Tips
References and
Books
Web Design: Teng-Leong Chew
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Technical Tips
Excitation and emission wavelengths of various fluorophores
PDF
Interactive Java site
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.
A quick way to search for antibody against your gene products is to search
the iGene database.
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.
Pictures courtesy of Dr. Peter Gann's Lab.
NU Department of Preventive Medicine
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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.
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 chamber from Bioptechs,
Inc.
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