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• light energy cannot be lost kinetically because the rings are constrained
• some is lost as vibration but most is re-emitted at a longer wavelength
• forbidden triplet state (D in the Jablonski diagram) important for dSTORM
• emission spectrum is shifted to the red and is a mirror image of excitation
• the Stokes shift of excitation and emission mean the two can be separated
• excitation and emission filters and a dichroic mirror are used
• these can be mounted in a cube or separately in filter wheels
• some confocal systems use a method where light is spectrally separated
Alexa 647 spectra filter and dichroic mirror anatomy of a filter arrangement of Leica’s
spectra for Alexa 647 cube confocal detector system
Anatomy of a fluorescence
microscope
• confocal pinholes reduce blur by excluding out-of-focus light from the detector
• deconvolution computationally reassigns blur to the correct focal plane and
can be used on wide-field and confocal data