confocalmultiphoton

Confocal multiphoton microscopy is an advanced optical imaging technique that combines the principles of confocal microscopy with multiphoton excitation. In multiphoton microscopy, two or more low-energy photons are simultaneously absorbed by a fluorophore to achieve excitation, which typically requires a single higher-energy photon. This simultaneous absorption process is highly dependent on the photon density, meaning excitation occurs primarily at the focal point where the laser intensity is highest. This intrinsic optical sectioning capability dramatically reduces out-of-focus fluorescence, leading to clearer images with less photobleaching and phototoxicity in deeper tissue layers compared to single-photon excitation.

The "confocal" aspect of confocal multiphoton microscopy further enhances image quality by using a pinhole in the detection path. This pinhole blocks scattered or out-of-focus light, ensuring that only light originating from the focal plane reaches the detector. The combination of multiphoton excitation and confocal detection provides excellent spatial resolution and allows for deep penetration into scattering biological tissues. This makes it an invaluable tool for in vivo imaging of live cells and tissues, studying dynamic biological processes, and investigating structures deep within samples without the need for extensive sample preparation or staining.