Microcopy - L5

• similar to one-photon

• each photon has one half of the energy, necessary to excite the molecule

• wavelength two times longer than one-photon excitation

• emitted photons have higher energy than exciting photons -> fluorescence has shorter wavelengths than excitation light

linear

• low intensity light

• change pathway of light, but not frequency

• mostly natural phenomenas: reflection, absorption, scattering

non-linear

• high intensity light

• change color of a beam (frequency)

• requires powerful lasers

• absorb two photons of same frequency

• transmission over virtual state in femtoseconds

• trasmitted throw non-symmetrical chrystal (tissue in biology)

• emission energy w is exactly twice the extinction energy wi: w = 2wi -> conserved

• unlike TPW: w < 2wi

• near infrared light -> greater penetration depth in tissue

• wide range of wavelengths

• short impulse

• long wavelength excitation light -> less effected by scattering -> higher penetration (up to 1 mm)

• low phototoxicity (infrared light) and photobleaching (short impuls only in focus)

• does not require pinhole -> easier to build