| 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 |