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Principle and working of a confocal microscope
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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