|
Test Yourself
Question:
How does confocal laser scanning microscopy (CLSM) work?
Answer:
The theory behind confocal laser scanning
microscopy (CLSM) comprises a variety of existing technologies;
it is a melding of fluorescence microscopy, computers and lasers.
This very sophisticated microscope has the ability to penetrate
deep beneath the surface of a tissue and to produce much higher
resolution images than possible with conventional fluorescence
microscopy. With the conventional fluorescence microscope, images
can be obscured by lack of focus and excess fluorescent light from
the specimen both above and below the plane of focus. The CLSM
has advanced optics which eliminate all the information outside
of a narrow plane of focus, thus yielding a well-defined image.
Material must be autofluorescent or be stained with fluorescent
dyes in order to be viewed with the LSCM. The use of a laser allows
optical slices to be produced. These optical slices or 'sections'
can be taken at specific focal depths, in unsectioned material,
and then stored in the accompanying computer as .pic or .bmp files.
These 'sections' can then be re-combined to form a 3-D image of
your specimen, or digitally enhanced using different graphics software.
Many results can be obtained using very simple preparations of
unfixed materials that display autofluorescence or become fluorescent
after staining with general fluorochromes. Labelling with fluorescent
antibodies can provide molecular localization, and can facilitate
in situ hybridization or ion ratio imaging.
U of G Facility
The confocal microscope facility provides services for all interested
users both on and off campus. This new facility was installed in
March of 2002 and was made possible by a multi-applicant equipment
grant from the Natural Sciences and Engineering Research Council
of Canada (NSERC). The instrument is a Leica TCS SP2, and has access
to the high speed network to allow easy downloading and manipulation
of images in remote sites. Specifications
Specifications
Filter free SP head: a spectrophotometer for each detector channel
(design your own filters, maximize sensitivity, minimize crosstalk,
record emission spectra).
Microscope: Upright microscope, Leica DMR
Lenses: 10x, 20x, 40x (Oil, Ph), 63x (Oil, DIC), 100x (Oil, DIC)
Laser System:
Ar (50mW): 458nm, 476nm, 488nm, 514nm
Green HeNe (1.2mW): 543nm
Red HeNe (10mW): 633nm
Two detection channels for fluorescence
Transmitted light detection
Leica confocal software (Version 2.5.1227a) for 2-D and 3-D image, ROI scanning,
and time course imaging.
Simulator for processing images:
Leica confocal software
Adobe Photoshop Version 6.0
Office 2000
CD Writer
Networked in CBSSRV workgroup.
Dr. Michaela Struder-Kypke
Co-ordinator CLSM
Department of Botany
University of Guelph
Guelph, ON N1G 2W1
Tel: 519-824-4120, Ext. 52737
E-mail: confocal@uoguelph.ca
TOP
| 