Confocal Microscopy Unit
The Confocal Microscopy Unit (CMU) is part of the Microscopy Imaging Facility located within the University of Guelph Advanced Analysis Centre. The unit provides confocal microscopy services for departmental and non-departmental users, both on and off campus. Specifically, the facility houses three confocal laser scanning microscopes (CLSMs) including two Leica TCS SP2 CLSMs and a Leica TCS-SP5 Multiphoton CLSM.
The unit also provides computer workstations for CLSM image processing and a wide range of standard microscopy services, including standard transmitted light, epi-illumination, fluorescence, and DIC microscopy. Specific details on the three CLSMs and the epifluorescent microscope are provided below.
Background
The basic theory behind confocal laser scanning microscopy (CLSM) includes a variety of technologies such as fluorescence microscopy, computer systems, and lasers.
The CLSM is a sophisticated instrument with the ability to penetrate beneath the surface of a tissue and to acquire much higher resolution images than possible with a conventional fluorescence microscope. For instance, with the conventional fluorescence microscope, images can be blurred by excess fluorescent signal 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. With the multiphoton CLSM, even deeper penetration into the sample is possible. At the same time, activation of the molecules only takes place in the plane of focus. Therefore, bleaching of the surrounding tissue/sample does not happen. This makes this technique especially suitable for life imaging of thick samples.
Material must be either autofluorescent or stained with fluorescent dyes in order to be viewed with the CLSM. 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 stored in the computer. Subsequently, the 'sections' can be rendered into a 3D image of your specimen, quantified, or digitally enhanced using different graphics software.
Many results can be obtained using 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.
New technologies facilitate life cell imaging: FRAP (Fluorescent Recovery After Photobleaching) and FRET (Fluorescent [or Förster] Resonance Eergy Transfer) enable the study of mobility and interactions of cellular components, whereas FLIM (Fluorescent Lifetime Imaging Microscopy) provides information on the molecular microenvironment (lifetime and not just intensity) of fluorescent molecules.
Imaging
The Confocal Microscopy Unit provides basic methods for capturing and analyzing of microscopical images. However, it is also equipped for the processing of image data files. Images can be captured directly from microscopes using a video camera and saved as electronic files. We provide this service for a range of microscope techniques, including standard transmitted light, epi-illumination, fluorescence, and DIC.
Advantages are the elimination of film, the possibility to capture low light images (e. g., those frequently encountered in fluorescence microscopy) and to enhance picture quality (e. g., background, colour, contrast). Image capturing and image analysis is accomplished through the use of OpenLab and Volocity software. Linear measurements as well as densitometry, sorting or counting are possible.
Captured images can be manipulated in various ways using the different image analysis programs. Besides the enhancement of picture quality, the software enables the acquisition of vertical stacks or time series of images and the rendering of 3D-reconstructions or movies from the data files.
Specifications of Microscope Systems in the Confocal Microscopy Unit
CLSM-U: An upright Leica DM RE microscope connected to a Leica TCS SP2 system with 3 different visible light lasers, covering 6 excitation wavelengths. This instrument is located in the Science Complex, Room 1213. More information . . .
CLSM-I: An inverted Leica DM IRE2 microscope connected to a Leica TCS SP2 system with 2 different visible light lasers, covering 4 excitation wavelengths. This instrument is located in the Science Complex, Room 1212. More information . . .
CLSM-MP: An upright Leica DM 6000B microscope connected to a Leica TCS SP5 system with 8 visible laser excitations and additionally a Radius 405 nm laser and a Chameleon Ultra Infrared Laser (consisting of modulated infrared beam ranging from 690-1040 nm). The system also contains the Leica LAS AF Imaging software and FLIM, as well as allied software provided by the company Becker & Hickl. This instrument is located in the Science Complex, Room 1215. More information . . .
Epifluorescence Microscope: An upright Leica DM RA2 connected to a Hamamatsu digital camera and a Macintosh G4 computer with OpenLab™ and Volocity™ software. This instrument is located in the Science Complex, Room 1217. More information . . .
Fees
The Confocal Microscopy Unit is open to anyone interested in confocal microscopy or post-acquisition image analysis. Billing is on a pay-for-use basis, unless arranged otherwise.
User Information
The Zimbra Calendar system is used for booking time on any microscope or workstation in the Confocal Microscopy Unit. Usually, the calendar of the required instrument is available to the user after the second training session.
Users should come with prepared samples and ready to use the microscope, so that the time on the microscope is optimized.
Backup of the data on CDRs DVDs, USB memory sticks or external hard drives are the responsibility of the user. Temporary storage on the hard drive is provided but the hard drive is cleared and all files are deleted on a regular basis.
Internal billing will be done as a journal entry. A billing information sheet providing the trust fund number must be filled out, with accompanying authorization, before the facility can be used. These sheets are available from the manager at the demonstration session. External billing will be done by invoicing the user, institution or company following usage. Billing
is generally done on a monthly basis unless otherwise arranged.
Please contact the CMU manager, Dr. Michaela Strüder-Kypke, to arrange a training session if you are interested in using the facility.
