Total internal reflection fluorescence (TIRF

Total internal reflection fluorescence (TIRF) microscopy I. Modelling cell contact region fluorescence

W. M. REICHERT* and G. A. TRUSKEY Department of Biomedical Engineering, and Center for Emerging Cardiovascular Technologies, Duke University, Durham, NC 27706, USA * Author for correspondence

Summary Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescent]}' labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associ-

ated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 run if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10 % as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1 %.

Introduction

magnetic theory for TIRF illumination of adherent cells for the case of a water-soluble fluorescent label in which the cell-substratum contact region was modeled as a dielectric lamellar structure (Gingell et al. 1987). Calculations of expected fluorescence intensity for several incident angles of the totally reflected beam were presented as a function of the water gap distance between the cell membrane and the substratum surface. TIRF is much more sensitive to gap thicknesses of the order of 0-70 nm than is the more widely used technique of interference reflection microscopy (Gingell et al. 1987; Bailey and Gingell, 1988). In the Gingell model, cell-substratum contact regions were treated optically as a four-phase system consisting of a glass substratum, a water-filled gap, a lipid membrane and the cell cytoplasm. The glass and cell were treated as semi-infinite media while the water and cell membranes were treated as thin dielectric films. Closed form expressions were derived for the distance dependence of the electric field amplitude in the watei1 gap regions for the four conditions under which the electric field in the water gap, cell membrane and cell cytoplasm was either continuous (propagating) or evanescent (decaying). Although the closed form expressions are exact solutions to the field equations, the expressions presented are difficult to apply in the determination of separation distances. An alternate approach, adopted in this paper, is to develop simpler expressions for the fluorescence that are

Total internal reflection fluorescence (TIRF) has become a standard technique for exciting spectroscopic phenomena at interfaces (Reichert, 1989). Briefly, the total internal reflection of visible light at a glass/solution interface produces a region of electromagnetic intensity (evanescent wave) that penetrates a few tenths of a micrometer into the lower refractive index medium and can excite spectral phenomena in a region confined to the solution side of the interface. The specific application of TIRF to the imaging of contacts of adherent cells to surfaces was developed by Axelrod et al. (1982). The ability to label specific regions of the cell has made TIRF an attractive technique for characterizing cell—substratum contacts. Recent applications of TIRF microscopy include the visualization of amoeba (Todd et al. 1988), fibroblast (Lanni et al. 1985), muscle (Gingell et al. 1986), erythrocyte (Axelrod et al. 1986), endothelial (Nakache et al. 1986) and leukemia (Weis et al. 1982) cells on glass or quartz substrata. Cell adhesions are classified in terms of close contacts (membrane within 30 nm of substratum surface) and focal contacts (substratum within 15 nm of substratum surface) (Burridge et al. 1988). An important factor in the quantitation of cell-surface separations is the development of a model that relates image intensity with the proximity of the membrane to the substratum surface. Gingell, Heavens and Mellor recently published the general electroJoumal of Cell Science 96, 219-230 (1990) Printed in Great Britain © The Company of Biologists Limited 1990

Key words: TIRF, cell contact, cell membrane.

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valid for the particular conditions of cell-glass contacts in an aqueous medium. Characteristic matrix theory (Hansen, 1968), in which the same matrix form is used whether the field in a given medium is either evanescent or continuous, was used to determine the expected TIRF emission excited using transverse electric (TE)-polarized incident radiation totally reflected at the glass-cell interface. Fluorescence excited from fluorescently labelled cell membranes and cells with cell-substratum water gaps infused with fluorescent solutions are considered. These calculations show that the lipid membrane may be ignored with an error of no greater than 2.5% and the cell-substratum contact may be modelled as a comparatively more simple three-phase system. Approximate analytical expressions are developed for these two TIRF excitation configurations that permit straightforward calculation of the cell-substratum water gap thickness from experiment.

homogeneous (c(z)=c), then for a given angle of incidence equation (1) becomes:

-'J*

T(z)6z,

where K^iAi/cosd^Qae. The above assumptions constitute an ideal case and actual experimental conditions may be subject to spatial variations in fluorescence emission resulting from fluorophore orientation with respect to the incident beam, fluorophore proximity with respect to the substratum interface, and molecular aggregation of fluorophore molecules. The importance of these phenomena to the quantitative interpretation of TIRF images is considered in the Discussion. For the ideal case, TIRF will occur when the incident angle exceeds the critical angle for total internal reflection (0c) defined by the higher refractive index of the incident medium (rei) and the lower refractive index of a semiinfinite homogeneous solution of fluorophores (n2): ec = sin-\n2/ni),

Theory Fluorescent emission excited during total internal reflection Consider a beam of light containing both transverse electric (TE, perpendicular, or s) and transverse magnetic (TM, parallel, or p) polarized electric vectors incident upon a dielectric interface at the angle 6x (Fig. 1). The fluorescence emission excited by the transmitted component of the incident beam between the limits Zi and z2 relative to the reflecting interface is given by the general expression (Suci and Reichert, 1988):

(3)

(4)

in which case the term T\z) reduces to the simple exponential: = T 12 (0)exp(-z/d 12 ),

(5)

where 7^(0) is the transmitted amplitude squared at the n\jni interface and d12 is the depth of penetration of the evanescent wave. Substitution of equations (4) and (5) into equation (3) followed by integration yields the expression for fluorescence excited by TIRF in homogeneous aqueous solutions between the limits zx and z2:

(6) F = #r 1 2 (O)d 1 2 [exp(- 2 l /d 1 2 )-exp(-z 2 /d 1 2 )]. (1) For solutions considered to behave optically as transparent media, the terms 7\2(0) and d12 for TE polarized where

0.10

E

v-0.00

0.00

0.0 0.5 1.0 1.5 2.0 Water gap thickness (Ai/lambda) Fig. 6. Normalized values offluorescenceexcited in the cell membrane (equation (23)) and in the water gap (equation (22)) plotted as a function of water gap thickness. Values calculated using a three-phase glass/water/cytoplasm model for an incident angle of 70 degrees and TE-polarized light.

(14)

Fm =

Since Kg and K, are assumed to be constant for a given incident angle, dx, the following primed expressions are used for further analysis:

= fJ Al o

z

(15) (16)

Equations (15) and (16) may be interpreted as excited fluorescence normalized to the incident intensity and the product of the fluorophore concentration and quantum efficiency. In the following calculations, Km is assumed to be equal to Kg, although in practice they may be quite different. Numerical integration of the data in Fig. 5 according to equation (15) (using Simpson's rule), and a direct matrix calculation of equation (16) yields values of normalized fluorescence excited from solutions in the water gap and from fluorescently labeled membranes. The result of these calculations are presented in Fig. 6 in which fluorescence intensities for both labeling techniques are plotted as a function of water gap thickness for an incident angle of 70 degrees and TE polarized light. The excitation of fluorophores in the water gap increases with water gap thickness, while the fluorescence from a dye-labeled membrane decreases with water gap thickness as the membrane moves farther from the glass surface. Both techniques reach an asymptotic intensity for a water gap thickness of the order of the wavelength of light, while possessing a similar sensitivity to changes in the distance of the cell membrane to the glass surface. Effective interfacial amplitude and depth of penetration Values for the decaying evanescent field (Fig. 5) and fluorescence intensities (Fig. 6) appear to fit the general shape of simple exponentially decaying functions for A2