Figure 3.

Experimental validation using cell-embedded gels and solutions simulating tissue optical properties. (A) Agarose gels containing SP2/0 cells giving specified α were bathed in PBS containing calcein and rhod-dextran. Top: Dye fluorescence shown during microfiberoptic insertion into the overlying solutions and gels. Bottom: Brightfield photographs show different cell densities, giving α = 1 (gel not containing cells), 0.5, and 0.3. Bar: 50 μm. Right: Experimentally measured (from fluorescence ratios) versus calculated (from cell density and size) α (SE, n = 4, except n = 2 for α = 0.15). (B) Effects of illumination/detection geometry. Left: A coverglass supports a 400-μm-thick agarose gel equilibrated with calcein and rhod-dextran in PBS. Right: Fluorescence shown during insertion of the microfiberoptic through the gel down to the bottom surface. The example is representative of six separate experiments. (C) Effect of light scattering. PBS/dye solutions were supplemented with nonfat milk (up to 16%) to produce light scattering. Calcein and rhod-dextran fluorescence changed by <1% for <12% milk (equivalent scattering from brain slices 7–10% milk, SE, n = 6 for each point). (D) Effect of light absorbance. PBS/dye solutions were supplemented with India ink (0–0.5%) to produce light absorbance (optical density at 1 mm pathlength from 0 to 1.5 at 595 nm). Calcein and rhod-dextran fluorescence changed by <1% for 0.2% India ink solution (equivalent optical density from brain slices: 0.3; SE, n = 6 for each point).