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Department of Biosciences

Confocal Microscopy

The  Molecular Imaging facility is equipped with a Zeiss LSM510 Meta Confocal Microscope and a Biorad Radiance 2000 and has the software packages for image analysis, processing and 3D image reconstruction.

Confocal microscopy permits one to optically section a  sample (such as a cell that has been stained with contrasting fluorescent dyes) with superior resolution by using a pinhole to reject light that originates outside of the chosen area. By collecting a series of such images through the depth of a sample, the user may assemble a highly accurate three-dimensional reconstruction of the entire sample. The Zeiss LSM 510 confocal microscope is equipped with Blue diode, AR, HE/Ne and diode laser sources. Detailed description of this microscope can be found here. Trained users may reserve time on either of the instruments using our on line booking system ( password required).

Co-localization of proteins. The LSM 510 can perform simultaneous multi-color imaging, allowing users to detect the distribution of various fluorescent-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 dynamic protein-protein interaction in live cells. Ratio-imaging of the resultant images can be performed on Zeiss LSM 510 software and also MetaMorph. 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 sub cellular compartments.

Fluorescent Intensity Measurement Users can measure fluorescent 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.

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.

Booking System