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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.