TempoRAl Focusing microscopy with single-pIXel detection. An illumination laser (Coherent Chameleon Ultra II) delivers 140-fs pulses with an 80-MHz repetition rate, up to 4-W average output power at a variable central wavelength between 680 and 1080 nm. The central wavelength of the laser was set to 800 nm for all the experiments performed. The illumination beam was expanded four times to cover the active area of a phase-only SLM (LCOS-SLM, Hamamatsu Photonics). The SLM was then imaged onto a blazed reflective grating (1200 g/mm) with a 4f (f = 400 mm) telescope to create wide-field TF illumination. The first diffraction order from the SLM was transmitted through an iris in the telescope, while all other orders were blocked. The beam was diffracted from the grating, and all wavelengths were collimated with an f = 400 mm lens relayed onto the back focal aperture of the illumination objective. Two different illumination objectives were used in this work. A water dipping objective (40× NA = 0.8; Nikon), which is enclosed in a custom-made chamber filled with water, generates a TF illumination plane with a size of 90 μm by 90 μm. The highest average laser power per unit area used in this configuration is 64 ± 5 μW/μm2. An air objective (20× NA = 0.75; Nikon) was used for additional studies presented in the Supplementary Materials, as accordingly specified. Backscattered fluorescent light propagated through the turbid media and was collected by an EMCCD camera run without amplification (iXonEM+ 885, Andor Technology) via the same illumination objective in epifluorescence configuration. To provide reference images for this paper, forwardly emitted photons from the sample were collected by a CCD camera (Clara, Andor Technology) in transmission via a long working distance air objective (100× NA = 0.7; Mitutoyo). Appropriate short-pass filters were used to reject the illumination laser at 800 nm and transmit fluorescence below 700 nm. In contrast to other single-pixel imaging approaches (25, 26), the EMCCD camera with 64 × 64 binning was used as a bucket detector instead of using a single-element detector such as a PMT or an avalanche photodiode. Using high binning helps reducing the effect of readout noise. All objectives, samples, and cameras were attached on the body of an inverted microscope (Eclipse Ti, Nikon) accordingly.

Point-scanning two-photon microscope. The 2PM shares the same setup as TRAFIX, except for the diffraction grating and a lens that are replaced with a mirror to obtain a focused beam on the focal plane of the illumination objective. A Nikon 20× NA = 0.75 air objective was used for all experiments. A variable iris was used to adjust the size of the focused spot. An X-Y-Z motorized stage (Nano-LP200, Mad City Labs) scanned the sample in a stepwise motion across the fixed focused beam covering the entire FOV. The same binned EMCCD camera run with no amplification (iXonEM+ 885, Andor Technology) collected the fluorescent light emitted by the sample.

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