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Instruments:
Laser
Scanning Confocal
Photo-activation
& Conversion
Spinning
Disc Confocal
Fluor. Emission
Fingerprinting
Nuance
Spectral Unmixing System
Total
Internal Reflection Fluor.
Axioskop Fluo. Microscope
Microinjector
Rotary Shadowing System
Ultramicrotomes
Trans. Electron Microscopes
Software:
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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Zeiss LSM 510 Laser Scanning Confocal Microscope
Important Features of Zeiss LSM 510
The laser scanning confocal microscope is designed for imaging with high
spatial resolution. It is not designed with temporal resolution in mind.
If you need to monitor rapid biological processes, you may want to consider
the spinning disc confocal system.
Z-sectioning
It is the unique feature of a confocal microscope to perform Z-section on
biospecimens. This is different from image deconvolution. While deconvolution
relies on mathematical algorithms to rid the image of out of focus intensity
data, confocal microscope relies on a small pinhole to block out of focus light.
By varying the distance between the pinhole and the specimen, users can generate
a Z-series that dissect through the specimen, as shown in the following example
of a clam egg, stained with three different fluorophores.
Z-sectioning from top to bottom of a clam egg. Video clip courtesy of
Robert Goldman's Lab
Co-localization of proteins
The LSM 510 can perform simultaneous multi-color imaging, allowing users to
detect the distribution of various fluorescently-tagged proteins. The most
commonly used colors are red, green and Cy5. It is also possible to perform
live cell imaging using cyan(CFP)- and yellow (YFP) fluorescent proteins.
Fluorescent Resonant Energy Transfer (FRET)
The availability of excitation and detection channels for CFP-YFP means
that users can perform FRET experiment to study dyanmic protein-protein
interaction in live cells. Ratio-imaging of the resultant images can be
performed on Zeiss LSM 510 software and also MetaMorph. Please discuss
FRET experiments in details with Teng-Leong Chew. FRET analyses require
many internal controls, and a lot of planning.
Photo-bleaching (FRAP and FLIP)
Zeiss LSM 510 provides user-defined, tunable photo-bleaching capability.
This allows users to perform experiments such fluorescent recovery after
photo-bleaching (FRAP) and fluorescent loss in photo-bleaching. These
fluorescent techniques are extremely powerful in study protein trafficking
in and out of various subcellular compartments. Please feel free to contact
us if you are thinking about studying these moecular events.
Fluorescent Intensity Measurement
Users can measure fluorecent intensity using LSM510 at any given region of
interest, as shown. It
is important to note that comparing fluorescent intensity from one image to
another requires internal normalization standards. Please consult the
facility staff for suggestions.
Before you begin:
First time users are encouraged to contact the director of the Cell
Imaging Facility (Teng-Leong Chew, 312-503-2841) to discuss the use of
confocal microscopes and fluorescent imaging in general. It is not always
necessary to use confocal microscope for your fluorescent imaging
purposes. We now also have two different confocal systems to be used for
different experiments. The laser lines of both confocal systems are
different, and they may not have the correct excitation wavelengths
available for the chosen fluorophores. It is crucial that you find out
these information in advance.
Our LSM 510 is equipped with the following lasers:
458 nm, 477nm, 488nm, 514nm, 543nm, 633nm (This scope does not have UV
excitation for DAPI, please refer to another LSM 510 META site here)
For an extensive list of fluorophore excitation and emission wavelengths,
please click here.
Other Documents/Software for Downloading
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