Multifocal multiphoton microscopy

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Multifocal multiphoton microscopy is a microscopy technique for generating 3D images, which uses a laser beam, separated by an array of microlenses into a number of beamlets, focused on the sample.[1] The multiple signals are imaged onto a CCD camera in the same way as in a conventional microscope. The image rate is determined by the camera frame rate, depending on the readout rate and the number of pixels and may range well above 30 images/s.[2]

By exploiting specific properties of pulsed-mode multiphoton excitation the conflict between the density of the foci, i.e. the degree of parallelization, and the axial sectioning has been resolved.[3] The laser pulses of neighboring foci are temporally separated by at least one pulse duration, so that interference is avoided. This method is referred to as time-multiplexing (TMX). Moreover, with a high degree of time multiplicity, the interfocal distance can be reduced to such an extent that lateral scanning becomes obsolete. In this case axial scanning is sufficient to record a 3D-image.


  1. ^ Straub, Martin; Hell, Stefan W (December 1998). "Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging". Bioimaging. 6 (4): 177–185. doi:10.1002/1361-6374(199812)6:4<177::AID-BIO3>3.0.CO;2-R. 
  2. ^
  3. ^ Bewersdorf, J; Pick, R; Hell, SW (1998). "Multifocal multiphoton microscopy.". Optics Letters. 23 (9): 655–7. doi:10.1364/ol.23.000655. PMID 18087301. 


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