Confocal Microscopy

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CONFOCAL MICROSCOPY Yadya M. Chawla M.Sc. Biotechnology South Asian University

description

Principle and working of a confocal microscope

Transcript of Confocal Microscopy

CONFOCAL MICROSCOPY

CONFOCAL MICROSCOPY

Yadya M. ChawlaM.Sc. BiotechnologySouth Asian University

WHAT IS?CONFOCAL MICROSCOPYAlso known as confocal laser scanning microscope.Pioneered by Marvin Minskey in 1955 and patented as US PATENT 301467 in 1957.Optical imaging technique used to increase optical resolution of a micrograph.Allows optical sectioning of a specimen.Does not allow rays of light from out-of-focus points to pass through the detector.

WHY is it needed ?LIMITATIONS OF WIDE FIELD MICROSCOPY

Out of focus (blurred) imageLess contrastLess resolved No z- axis (depth cannot be ascertained)Cell destruction due to physical process of sectioning

HOW does it work?THE OPTICAL PRINCIPLE

It incorporates 2 ideasPoint-by-point illumination of specimenRejection of out-of-focus light

LASER beam illuminates a single part of the sample

Emitted fluorescent light passes through a pinhole that rejects out-of-focus light, producing a sharp image

Intensity of light from these in-focus areas is recorded by photomultiplier tube and image is stored in the computer.HOW does it work?THE INSTRUMENT

APPROACHES OF CM

WHERE IS IT USED?APPLICATIONSIncreased resolution3-D reconstructionOptical sectioning