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 an....

Protocol

1. Seeding and Incubation of Cells

1. 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.
2. Incubate cells with a membrane marker, e.g. 6-dodecanoyl-2-dimethylamino naphthalene (laurdan) applied for 60 min at a concentration of 8 μM (in culture medium)⁴, or a cytoplasm marker, e.g. calcein acetomethlyester applied for 40 min at a concentration of 5 μM³

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

List of materials used in this article

Name	Company	Catalog Number	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