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Table 3 2PE vs. 1PE optical microscopy.

From: Multi-photon excitation microscopy

  2PE 1PE CONFOCAL
Excitation source Laser, IR, fs-ps pulsed, 80–100 MHz repetition rate, tunable 680–1050 nm Laser VIS/UV CW (365, 488, 514, 543, 568, 633, 647 nm)
Excitation/emission separation wide close
Detectors PMT (typical), CCD, APD PMT (typical), CCD, APD
Volume selectivity Intrinsic (fraction of femtoliter) Pinhole required
Image formation Beam scanning (or rotating disks) Beam scanning (or rotating disks)
Deep imaging > 500 μm (problems related to pulse shape modifications and scattering) Approx. 200 μm (problems related to shorter wavelength scattering)
Spatial resolution Less than confocal because of the focusing of IR radiation, compensated by the higher signal to noise ratio; pinhole increases resolution, good for high fluorescence Diffraction limited depending on pinhole aperture
Real time imaging Possible Possible
Signal to noise ratio High (especially in non descanned mode) Good
Fluorophores All available for conventional excitation plus new designed for 2PE Selected fluorophores depending on laser lines in use
Photobleaching Limited to the focal volume Whole double cone of excitation
Contrast mechanisms Fluorescence, high order harmonic generation, higher order n-photon excitation, autofluorescence Fluorescence, reflection, transmission, autofluorescence.
Commercially available Yes Yes
  1. Comparison of 2PE and 1PE confocal imaging systems.