Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Summary

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Abstract

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Introduction

When a light beam propagating through a medium of refractive index n₁ meets an interface to a second medium of refractive index n₂ < n₁, total internal reflection occurs at all angles of incidence Θ, which are greater than the critical angle Θ_c=arcsin(n₂/n₁). Despite being totally reflected the incident light beam evokes an evanescent electromagnetic field that penetrates into the second medium and decays exponentially with perpendicular distance z from the interface according to I(z) = I_{0 ^e-z/d(Θ)}. I(z) corresponds to the intensity of the electromagnetic field and d(Θ) to the penetration depth at wavelength λ, as given by

d(Θ) = (λ/4π) (n₁² sin²Θ – n₂²)^-1/2

As reported in the literature^1,2, I₀ corresponds to the intensity of incident light I_e multiplied with the transmission factor T(Θ) and the ratio n₂/n₁. If the electric field vector of this light beam is polarized perpendicular to the plane of incidence (i.e. the plane spanned by the incident and the reflected beam), this transmission factor is given by

T(Θ) = 4 cos² Θ / [1 – (n₂/nL₁)²]

For the detected fluorescence intensity in TIRFM measurements, light absorption has to be integrated over all layers of the sample and multiplied with the fluorescence quantum yield η of the relevant dye as well as with the solid angle of detection Ω. As reported previously³, this intensity can be described by

I_F (Θ) = A T(Θ)òc(z) e^-z/d(Θ) dz

if all factors which are independent from the angle of incidence Θ and the coordinate z are included within the experimental constant A. Equation 3 can be solved analytically, if the concentration c(z) of fluorophores is regarded to be almost constant

– either at all coordinates z ≥ Δ, e.g. within the cytoplasm of cells having a distance Δ from the interface. In this case, the integral has to be calculated from z= Δ to z= ∞ resulting in

I_F = A c T(Θ) d(Θ) e^-Δ/d(Θ)

– or between z = Δ – t/2 and z = Δ + t/2, i.e. within a thin layer of thickness t, e.g. a cell membrane located at a distance Δ from the interface. In this case, the fluorescence intensity can be calculated as

I_F = A c T(Θ) t e^-Δ/d(Θ),

if t is small in comparison with Δ.

From Equations (4) and (5) cell-substrate distances Δ can be calculated, if images of fluorescence intensity I_F are recorded for variable angles Θ, and if ln (I_F / T d) (Eq. 4) or ln (I_F / T) (Eq. 5) is evaluated as a function of 1/d for these angles. For the membrane marker laurdan used in this paper (s. below) the evaluation was performed according to Eq. 5.

As reported previously³, image acquisition and evaluation requires
– homogenous distribution of excitation light over the sample,
– validity of a 2-layer model (with the refractive indices n₁ and n₂ for the substrate and the cytoplasm, respectively). This holds, if the plasma membrane is very thin, and if the layer of the extracellular medium (between the cell and the substrate) is small compared with the wavelength of incident light.

In addition, a moderate numerical aperture (typically A ≤ 0.90) of the microscope objective lens is advantageous to avoid deviations of brightness due to anisotropy of radiation in the near field of the dielectric interface.

Protocol

1. Seeding and Incubation of Cells Seed individual cells, e.g. U373-MG or U-251-MG glioblastoma cells at a typical density of 100 cells/mm2 on a glass slide and grow them for 4872 hr in culture medium (e.g. RPMI 1640 supplemented with 10% fetal calf serum and antibiotics) using an incubator at 37 °C and 5% CO2. Incubate cells with a membrane marker, e.g. 6-dodecanoyl-2-dimethylamino naphthalene (laurdan) applied for 60 min at a concentration of 8 …

Discussion

A method is described for measuring cell-substrate distances with nanometer precision. Presently, methods of super-resolution microscopy, e.g. based on structured illumination⁷ or on single molecule detection^8-10 as well as stimulated emission depletion (STED) microscopy¹¹ are of considerable interest. None of these techniques, however, permits an axial resolution below 50 nm. In addition, rather high irradiance of 50­100 W/cm² is needed for single molecule methods…

Materials

Name	Company	Typ	Comments
Incubator	Nunc	Nunc Cellstar QM1300SVBA
Laminar flow	Holten Safe	S2010 1.2 EN GG 1LN
DMEM + 10%FCS + 1 % Penicillin / Streptomycin	BIOCHROM		Cultivation medium
Glass object slides	Marienfeld	Pure white glass	Special cleaning procedure used
6-dodecanoyl-2-dimethylamino naphthalene (laurdan)	Molecular Probes		Fluorescent marker (Stock solution: 2 mM in ethanol)
Microscope	Carl Zeiss	Axioplan 1
Laser diode	PicoQuant	LDH 400 with driver PDL 800-B	Wavelength: 391 nm
Single mode fiber system	Point Source	kineFlex	Used with collimating optics
EMCCD camera	Andor	DV887DC	Back illuminated camera