title: Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy.
abstract: 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.
authors.affiliation: Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco 94143, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: Olveczky
authors.forename: N
authors.initials: N
authors.lastname: Periasamy
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D016475:3T3 Cells; D000818:Animals; D016880:Anisotropy; D002232:Carbocyanines / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004285:Dogs; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D007668:Kidney; D007834:Lasers; D008433:Mathematics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D012031:Refractometry
chemical_list: D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein