carbocyanines and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

The decay of evanescent field intensity beyond a dielectric interface depends upon beam incident angle, enabling the 3-d distribution of fluorophores to be deduced from total internal reflection fluorescence microscopy (TIRFM) images obtained at multiple incident angles. Instrumentation was constructed for computer-automated multiple angle-TIRFM (MA-TIRFM) using a right angle F2 glass prism (n(r) 1.632) to create the dielectric interface. A laser beam (488 nm) was attenuated by an acoustooptic modulator and directed onto a specified spot on the prism surface. Beam incident angle was set using three microstepper motors controlling two rotatable mirrors and a rotatable optical flat. TIRFM images were acquired by a cooled CCD camera in approximately 0.5 degree steps for >15 incident angles starting from the critical angle. For cell studies, cells were grown directly on the glass prisms (without refractive index-matching fluid) and positioned in the optical path. Images of the samples were acquired at multiple angles, and corrected for angle-dependent evanescent field intensity using "reference" images acquired with a fluorophore solution replacing the sample. A theory was developed to compute fluorophore z-distribution by inverse Laplace transform of angle-resolved intensity functions. The theory included analysis of multiple layers of different refractive index for cell studies, and the anisotropic emission from fluorophores near a dielectric interface. Instrument performance was validated by mapping the thickness of a film of dihexyloxacarbocyanine in DMSO/water (n(r) 1.463) between the F2 glass prism and a plano-convex silica lens (458 mm radius, n(r) 1.463); the MA-TIRFM map accurately reproduced the lens spherical surface. MA-TIRFM was used to compare with nanometer z-resolution the geometry of cell-substrate contact for BCECF-labeled 3T3 fibroblasts versus MDCK epithelial cells. These studies establish MA-TIRFM for measurement of submicroscopic distances between fluorescent probes and cell membranes.

Topics: 3T3 Cells; Animals; Anisotropy; Carbocyanines; Cell Line; Cell Membrane; Dogs; Fluoresceins; Fluorescent Dyes; Kidney; Lasers; Mathematics; Mice; Microscopy, Fluorescence; Refractometry

A preparation of viable epithelial cells, suitable for transport studies, was prepared from rabbit distal colon. Enzymatic digestion of scraped mucosa liberated a population of intact colonic glands that were dissociated into cells by gentle agitation in culture medium. Metabolism of [14C]glucose was constant over 2 h and was inhibited 70% by 1 mM ouabain. Cells were loaded with the trapped, pH-sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxy methyl ester and leaked 0.1% of the dye per minute. Cell pH was 7.21 +/- 0.03 (SE) (n = 15) in pH 7.4 Ringer medium. Intracellular potassium concentration, as measured by a nigericin null-point technique, was 128 +/- 8 mM. Plasma membrane potential measured with the potential-sensitive fluorescent dye 3,3-dipropylthiadicarbocyanine iodide was -52 +/- 2 mV. Recovery of intracellular pH in acid-loaded cells occurred after exposure to sodium-containing medium and was inhibited by 5 x 10(-4) M amiloride. It is concluded that viable epithelial cells can be prepared from rabbit distal colon with relative simplicity and in high yield. These cells are suitable for measurement of intracellular pH and membrane potential and are thus a convenient model system for study of colonic cell physiology.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Separation; Cell Survival; Colon; Epithelial Cells; Fluoresceins; Fluorescent Dyes; Glucose; Glycolysis; Kinetics; Male; Membrane Potentials; Microscopy, Electron; Potassium; Rabbits