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This lecture covers the limitations of conventional fluorescence microscopy, the principles of confocal microscopy, and the factors limiting imaging depth in confocal microscopy. It discusses the absorption and scattering of light in tissues, the challenges of imaging thick biological samples, and the use of near-infrared dyes for optical imaging. Additionally, it explores two-photon microscopy, the theory of two-photon absorption, and the relative two-photon excitation spectra for various fluorescent probes and proteins.