Fluorescent layer and fluorescent micropattern. A 200-nm-thin layer of super-yellow polymer spin-coated on a glass coverslip was used to characterize the profile and depth resolution of the TF beam (see the Supplementary Materials). It was also used to generate a fluorescent micropattern that was then imaged through scattering media. The negative of a pattern of interest was encoded on the SLM, and the thin fluorescent layer was placed at the focal plane of the microscope without any scattering layer. The laser power was set to the maximum, and the negative pattern was photobleached on the fluorescent film. Therefore, the only portion of the FOV that remained fluorescent was exactly the desired micropattern.

Fluorescent beads. Green fluorescent polymer microspheres (G400, Duke Scientific) with a diameter of 0.39 μm were used to test the performance of the imaging system. A very small amount of beads was deposited on a glass coverslip and placed on top of the scattering samples to image them through the turbid media.

HEK cells. HEK293T/17-GFP cells were used to demonstrate the capability of the microscope in imaging real biological samples through scattering. HEK293T/17 cell line obtained from American Type Culture Collection was cultured in Dulbecco’s modified Eagle’s medium GlutaMAX-I supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and was transfected using TransIT-LT1 transfection reagent with the Vesicular Stomatitis Virus glycoprotein (VSV-G) pseudotyped lentivirus vector and the packaging plasmid psPAX2 to deliver the plasmid pLenti-GFP-Puro. Regarding the sample preparation, the HEK293T/17-GFP cells were replated on World Precision Instruments FluoroDish poly-d-Lysine–coated cell culture dishes at a low density to achieve ideal imaging conditions. The day after plating, before imaging, the HEK293T/17-GFP cells were fixed in a phosphate-buffered saline 4% paraformaldehyde solution. The scattering samples were attached directly on the bottom of the dishes containing the cells.

Scattering phantom. Polystyrene beads of 1 μm in diameter were used as scatterers to simulate a turbid sample. The beads were purchased in a 1% concentration solution in water (Microbead NIST Traceable Particle Size Standard, 1.00 μm; Polysciences). The solution was thoroughly stirred in a vortex mixer and then mixed with a 1% solution of agarose in water (preheated above melting point). Agarose and beads were mixed in the vortex mixer again and placed into sample wells of variable height. The wells consisted of a 100-μm glass slide with multiple 90-μm vinyl spacers stacked on top of each other. An additional coverslip was placed on top to seal the well. The concentration of polystyrene beads in the sample was chosen to roughly match the scattering coefficient of real biological tissue (48). Using an on-line Mie scattering calculator (49), we determined the reduced scattering coefficient of our phantom to approximately be Embedded Image, corresponding to a mean free path of about Embedded Image, where g is the anisotropy factor (g ~ 0.9 for most biological tissue at the wavelength considered in this investigation). Different scattering phantoms were used in some experiments in the Supplementary Materials as appropriately specified.

Rat brain tissue. Similar to our previous work (50), rat brain tissue was obtained from adult Sprague Dawley rats, in accordance with the UK Animals (Scientific Procedures) Act 1986. It was fixed and then sectioned into slices at thicknesses of 200 and 400 μm. The mean free path of the rat brain tissue was estimated by measuring the ratio of the incident laser intensity I0 with the intensity of the ballistic photons IB and by applying an exponential law IB = I0 exp(− L/ls), where L is the thickness of the brain tissue (18). The obtained value of ls = 55 ± 9 μm is consistent with other measurements reported in the literature for rat (51) and mouse brains (52).

Colon tissue. Thin fragments of unfixed normal human colon tissue were used as scattering samples. They were stored in a freezer at −80°C and mounted between two coverslips. Their thickness ranged from 200 to 250 μm, and their reduced scattering coefficient was approximately 12 cm−1 (53, 54), giving a mean free path of ls ≈ 85 μm. The colon tissue sample used in this study was obtained from the Tayside Tissue Bank, Ninewells Hospital and Medical School, Dundee (tissue request no. TR000289) with appropriate ethical permission.

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