Applications of Total Internal Reflection Fluorescence Microscopy for Studies of Chemical Phenomena at the Substrate-Liquid Interface

Abstract

Applications of TIRFM for quantitative measurements of cells are limited due to high background fluorescence which can result in a low S/N ratio and therefore contribute to inaccuracy of measurements. Time-resolved total internal reflection fluorescence microscopy (TR-TIRFM) was developed by temporally gating a CCD camera using a liquid crystal shutter to optically filtering the short-live fluorescence and recording only the long-live emission. This technique was then applied to examine the extent of cell-substrate contacts. Tb+3 chelates such as DTPA-PhenylAS-Tb+3 was synthesized and applied as a membrane staining agent but was observed to internalized into the cell nucleus. A modified chelate molecule was therefore synthesized using DOPE as a carrier molecule. DOPE-DTPA-pAS-Tb+3 has a similar emission lifetime (1.5 msec) and appeared to stain only the cell membrane. TR-TIRF was applied to examine adherent cells on polystyrene-coated substrate. TR-TIRF images showed cellular autofluorescence and polystyrene emissions were optically filtered out, while the long-lived emission intensity of Tb+3 chelate was recorded. These results conclude that TR-TIRFM, with the use of long-live emission label (Tb+3 and Eu+3 chelates), is suitable as an analytical tool for probing a large number of cellular and molecular events occurring in the cell membrane and on the cell surface where background fluorescence would usually be problematic. Detection of K+ transported across a cell membrane is a prerequisite in the development of devices for screening drugs targeting K+ ion channels. K+ sensing film was fabricated by encapsulating a squaraine dye (aza-crown-SQR) in a sol-gel matrix for detection of K+. Sol-gel films were prepared by the hydrolysis and condensation reactions of TEOS or TMOS, APTS and GOPS mixtures. Formation of a DPhPC bilayer on sol-gel films was achieved by the vesicle fusion method and had diffusion coefficients of 2.3 and 2.1x10-8 cm2s-1 as measured by FRAP on TEOS-APTS-GOPS and TMOS-APTS-GOPS film, respectively. The time-based fluorescence intensity data from the H+ blocking experiments showed the sol-gel-supported DPhPC bilayers are impermeable to H+, and the K+ blocking experiments showed K+ was passively transported across a DPhPC bilayer by valinomycin.