Confocal Microscopy Principles

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Crosstalk problems Most of the time there is some overlapping between fluorophores emission spectra Example of FITC and TRITC Using 488 nm and 543 nm lines : 22% overlap If the fluorescence signals are not taken sequentially : some of the green fluorescence is assigned to the red channel
Crosstalk problems To avoid bleed-through of one fluorescence in another channel, multitrack configurations allow sequential acquisition of lines (or frames) by very fast switching of the laser lines by means of AOTF Minimize crosstalk between channels More accurate quantification in co-localization experiments
Page 1 and 2: Confocal Microscopy Dr. Serge Arnau
Page 3 and 4: Light source in focus Sample (objec
Page 5 and 6: Light source Confocal microscope pr
Page 7 and 8: How does a fluorescence microscope
Page 9 and 10: Advantage of fluorescence confocal
Page 11 and 12: LASER Light Amplification by Stimul
Page 13 and 14: Beam scanning Majority of laser sca
Page 15 and 16: Photomultiplier Tubes (PMT) The spe
Page 17 and 18: Scans averaging reduces noise Avera
Page 19 and 20: Airy disk and Resolution The radius
Page 21 and 22: Pinhole and Resolution Confocal pin
Page 23 and 24: Pinhole and Resolution Confocal PSF
Page 25 and 26: Z Optical sectionning Y X 5 4 3 2 1
Page 27 and 28: Sampling in confocal microscopy 260
Page 29 and 30: Sampling in confocal microscopy The
Page 31: Ideal emission separation Red emiss
Page 35 and 36: Spectral separation Fluorescence se
Page 37 and 38: FRAP experiments FRAP-recording for
Page 39 and 40: Other beam scanning techniques Mult
Page 41 and 42: Nipkow disk confocal microscope fac
Page 43 and 44: Other beam scanning techniques Slit

Confocal Microscopy Principles

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