Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).
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.
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 Θc=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) = I0 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 literature1,2, I0 corresponds to the intensity of incident light Ie 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
IF (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
IF = 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
IF = 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 IF are recorded for variable angles Θ, and if ln (IF / T d) (Eq. 4) or ln (IF / 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.
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 detection8-10 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…
The authors have nothing to disclose.
The authors thank the Land Baden-Württemberg and the Europäische Union -Europäischer Fonds für die regionale Entwicklung – for funding ZAFH-PHOTONn, the Bundesministerium für Bildung und Forschung (BMBF) for funding research grant no. 1792C08 as well as the Baden-Württemberg-Stiftung GmbH for financing the project “Aurami”.
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 |