The authors thank the Land Baden-W&uuml;rttemberg and the Europ&auml;ische Union -Europ&auml;ischer Fonds f&uuml;r die regionale Entwicklung - for funding ZAFH-PHOTONn, the Bundesministerium f&uuml;r Bildung und Forschung (BMBF) for funding research grant no. 1792C08 as well as the Baden-W&uuml;rttemberg-Stiftung GmbH for financing the project "Aurami".

1. Seeding and Incubation of Cells
<ol>
	<li>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 &deg;C and 5% CO2.</li>
	<li>Incubate cells with a membrane marker, e.g. 6-dodecanoyl-2-dimethylamino naphthalene (laurdan) applied for 60 min at a concentration of 8 ...

<ol>
	<li>Gingell, D., Todd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+reflection+microscopy:+a+quantitative+theory+of+image+interpretation+and+its+application+to+cell-substratum+separation+measurement.">Interference reflection microscopy: a quantitative theory of image interpretation and its application to cell-substratum separation measurement.</a> Biophys. J. 26, 507-526 (1979).</li><li>Reichert, W. M., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+(TIRF)+microscopy+(I)+Modelling+cell+contact+region+fluorescence.">Total internal reflection fluorescence (TIRF) microscopy (I) Modelling cell contact region fluorescence.</a> J. Cell Sci. 96, 219-230 (1990).</li><li>Stock, K., Sailer, R., Strauss, W. S. L., Lyttek, M., Steiner, R., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+total+internal+reflection+fluorescence+microscopy+(VA-TIRFM):+realization+and+application+of+a+compact+illumination+device.">Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device.</a> J. Microsc. 211, 19-29 (2003).</li><li>Wagner, M., Weber, P., Strauss, W. S. L., Lassalle, H. -. P., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotomography+of+cell+surfaces+with+evanescent+fields.">Nanotomography of cell surfaces with evanescent fields.</a> Adv. Opt. Technol. 2008, 254317 (2008).</li><li>Lassalle, H. -. P., Baumann, H., Strauss, W. S. L., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+topology+upon+ALA-PDT+using+variable-angle+total+internal+reflection+fluorescence+microscopy+(VA-TIRFM).">Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).</a> J. Environ. Pathol. Toxicol Oncol. 26, 83-88 (2007).</li><li>Coates, C. G., Denvir, D. J., McHale, N. G., Thornbury, K. D., Hollywood, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+low-light+microscopy+with+back-illuminated+electron+multiplying+charge-coupled+device:+enhanced+sensitivity,+speed+and+resolution.">Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed and resolution.</a> J. Biomed. Opt. 9, 1244-1252 (2004).</li><li>Gustafsson, M. G. L., Shao, L., Carlton, P. M., Wang, C. J. R., Golubovskaya, I. N., Cande, W. Z., Agard, D. A., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination.">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.</a> Biophys. J. 94, 4957-4970 (2008).</li><li>Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., Hess, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91, 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Meth. 3, 793-796 (2006).</li><li>Willig, K. I., Harke, B., Medda, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+with+continuous+wave+beams.">STED microscopy with continuous wave beams.</a> Nat. Meth. 4, 915-918 (2007).</li><li>Schneckenburger, H., Wagner, M., Weber, P., Bruns, T., Richter, V., Strauss, W. S. L., Wittig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Dimensional+Fluorescence+Microscopy+of+Living+Cells.">Multi-Dimensional Fluorescence Microscopy of Living Cells.</a> J. Biophotonics. 3, 143-149 (2011).</li><li>Dougherty, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosensitizers:+therapy+and+detection+of+malignant+tumours.">Photosensitizers: therapy and detection of malignant tumours.</a> Photochem. Photobiol. 45, 879-889 (1987).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence.">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> J. Cell Biol. 89, 141-145 (1981).</li><li>&#214;lveczky, B. P., Periasamy, N., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+fluorophore+distribution+in+three+dimensions+by+quantitstive+multiple+angle+total+internal+reflection+fluorescence+microscopy.">Mapping fluorophore distribution in three dimensions by quantitstive multiple angle total internal reflection fluorescence microscopy.</a> Biophys. J. 73, 2836-2847 (1997).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+vry+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with vry high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+microscopy:+technical+innovations+and+novel+applications.">Total internal reflection microscopy: technical innovations and novel applications.</a> Curr. Opin. Biotechnol. 16, 13-18 (2005).</li></ol>

No conflicts of interest declared.

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 50&shy;100 W/cm2 is needed for single molecule methods...

When a light beam propagating through a medium of refractive index n1 meets an interface to a second medium of refractive index n2 &lt; n1, total internal reflection occurs at all angles of incidence &Theta;, which are greater than the critical angle &Theta;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) = I0 e-z/d(&Theta;). I(z) corresponds to the intensity of the electromagnetic field and d(&Theta;) to the penetration depth at wavelength &lambda;, as given by 
 
d(&Theta;) = (&lambda;/4&pi;) (n12 sin2&Theta; - n22)-1/2
As reported in the literature1,2, I0 corresponds to the intensity of incident light Ie multiplied with the transmission factor T(&Theta;) 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(&Theta;) = 4 cos2 &Theta; / [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 &eta; of the relevant dye as well as with the solid angle of detection &Omega;. As reported previously3, this intensity can be described by 
 
IF (&Theta;) = A T(&Theta;)&ograve;c(z) e-z/d(&Theta;) dz
if all factors which are independent from the angle of incidence &Theta; 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 &ge; &Delta;, e.g. within the cytoplasm of cells having a distance &Delta; from the interface. In this case, the integral has to be calculated from z= &Delta; to z= &infin; resulting in 
 
IF = A c T(&Theta;) d(&Theta;) e-&Delta;/d(&Theta;)
- or between z = &Delta; - t/2 and z = &Delta; + t/2, i.e. within a thin layer of thickness t, e.g. a cell membrane located at a distance &Delta; from the interface. In this case, the fluorescence intensity can be calculated as 
 
IF = A c T(&Theta;) t e-&Delta;/d(&Theta;), 
 
if t is small in comparison with &Delta;.
From Equations (4) and (5) cell-substrate distances &Delta; can be calculated, if images of fluorescence intensity IF are recorded for variable angles &Theta;, 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 &le; 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.

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

nanotopology of cell adhesion upon 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.

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

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

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

Nanotopology of Cell Adhesion upon 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 ...

nanotopology-cell-adhesion-upon-variable-angle-total-internal

Research

JoVE Journal

Bioengineering

10.1K visualizações.  Institut für Angewandte Forschung. Topologia de adesão celular num substrato é medida com uma precisão nanométrica de ângulo variável por microscopia de fluorescência de reflexão interna total (TIRFM-VA).