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The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

We find that stimulated Raman excitation of an atom by a two-photon pulse can be enhanced by orders of magnitude if the photons are simultaneously frequency correlated and spatially anticorrelated. That is, a correlated photon pair must have an inherent time delay between its constituent photons. This counterintuitive feature is a manifestation of the uncertainty principle, which yields that frequency-correlated photons cannot be time (spatially) correlated. This is opposite to two-photon absorption by a three-level atom, for which the enhancement occurs if photons in the pulse are frequency anticorrelated and spatially correlated, that is, photons in the pair simultaneously interact with the atom. Our findings can be useful for imaging and spectroscopy of biological samples which demand low illumination intensity.