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 &mu;M (in culture medium)4, or a cytoplasm marker, e.g. calcein acetomethlyester applied for 40 min at a concentration of 5 &mu;M3, or a membrane associated photosensitizer, e.g. protoporphyrin IX induced upon 4 hr incubation with its precursor 5-aminolevulinic acid (5-ALA; 1 mM)4,5.</li>
	<li>Wash the cells with culture medium or phosphate buffered saline (PBS) prior to fluorescence microscopy.</li>
</ol>
2. VA-TIRFM
<ol>
	<li>Use an upright microscope, e.g. Zeiss Axioplan, and replace its condenser by an illumination device, as described in Ref. 3 with a hemi-spherical or a hemi-cylindrical glass prism optically coupled to the object slide (see Figure 1).</li>
	<li>Use a parallel collimated laser beam which after imaging by the glass prism and further optical components (e.g. concave mirror) again results in a parallel beam on the surface of the sample (telecentric pupil ray). Take care that the electrical field vector is polarized perpendicular to the plane of incidence.</li>
	<li>Use an adjustable mirror located inside the condenser which is imaged in the sample plane and illuminates the sample under a variable angle of incidence (object ray). The adjustable mirror may be driven by a step motor, where each position corresponds to a well defined angle of incidence, as determined by goniometric measurements3.</li>
	<li>Calibrate the angular position of the adjustable mirror using the critical angle &Theta;c=61.3&deg; for a glass-water transition as a fixed value. &Theta;c can be visualized upon variation of &Theta; when a fluorescent dye (e.g. 1 mM flavin mononucleotide, FMN) is dissolved in water. The position of the mirror at &Theta;=&Theta;c is stored in the mirror positioning program.</li>
	<li>Set the parameters of the camera (back illuminated Andor Ixon EMCCD, DV887DC, ANDOR Technology, Belfast , U.K.6), including temperature, gain, recording time and shift speed.</li>
	<li>Use an objective lens of appropriate magnification and preferentially moderate numeric aperture (e.g. 40&times;/0.75 or 63&times;/0.90 water immersion) and localize the object slide in the microscope. Use immersion oil to assure optical contact of the sample with the glass prism. Record fluorescence images of identical samples upon variation of the angle of illumination (&Theta;&ge;&Theta;c with &Theta;c corresponding to 64.5&deg; for a cell-glass interface). An angular range of 66&deg;-74&deg; with steps of 0.5&deg; or 1&deg; is recommended. Use an appropriate long pass or band pass filter for fluorescence detection.</li>
</ol>
3. Data Analysis
<ol>
	<li>Read the images (e.g. in ASCII format) recorded at various angles &Theta;, as well as the camera settings (for background discrimination), the excitation wavelength and the refractive indices for glass (n1 = 1.52) and cells (n2 = 1.37) into the evaluation program (LabView; NanoCell). For each set of parameters the penetration depth d(&Theta;) and the transmission factor T(&Theta;) are calculated automatically.</li>
	<li>Select images used for evaluation of cell-substrate distances &Delta; according to Equation 5 (membrane marker) or Equation 4 (cytoplasm marker), Individual images with inhomogeneous illumination (revealing e,g. stripes) may be excluded.</li>
	<li>Calculate images of cell-substrate topology and select an appropriate color code for display. During evaluation profiles of individual pixels can be displayed, as depicted in Figure 2. These profiles may be used as an indicator for the quality of the fit.</li>
	<li>Store the evaluation protocol.</li>
</ol>
4. Representative Results
Representative experiments are carried out with genetically engineered U251-MG glioblastoma cells kindly supplied by Prof. Jan Mollenhauer, Dept. of Molecular Oncology, University of South Denmark, Odense. In two subclones of those U251 cells the tumour suppressor genes TP53 or PTEN are over-expressed using a Tet-On inducible expression system, such that these cells exhibit a reduced tumorigenic potential and may be regarded as less malignant. Highly malignant U251-MG cells without suppressor gene are used as controls, and U251-MG wild type cells serve for a further comparison. A laser diode emitting a quasi continuous series of picosecond pulses (LDH400 with driver PDL 800-B, Picoquant, Berlin, Germany; wavelength: 391 nm; pulse energy: 12 pJ; repetition rate: 40 MHz) is used as an excitation source.
In Figure 3 TIRFM images of U251-MG glioblastoma cells with an activated TP53 tumour suppressor gene and incubated with laurdan (8 &mu;M; 60 min) are depicted for angles of incidence of 66.0&deg;, 69.0&deg; and 73.0&deg; corresponding to penetration depths of the evanescent electromagnetic field of about 140 nm, 75 nm and 60 nm, respectively. With decreasing penetration depth fluorescence decreases in intensity (see scales) and originates from more superficial sites of the cells (e.g. focal contacts on their edges). Cell-substrate distances (visualized in Figure 3d by a color code ranging from 0-600 nm) strongly vary over the cells between only a few nanometers (white-yellow) and 250-300 nm (dark red), i.e. focal contacts and larger cell-substrate distances are in close vicinity. The same holds for U251-MG glioblastoma cells with an activated suppressor gene PTEN (Figure 4d), while cell-substrate distances are rather constant for U251-MG control and wild type cells with typical values of 80-100 nm (yellow; Figure 4b) and 150-200 nm (orange-red; Figure 4a).
<img alt="Figure 1" src="/files/ftp_upload/4133/4133fig1.jpg" /> 
 Figure 1. Illumination device for VA-TIRFM in an upright microscope. An adjustable mirror driven by a step motor permits an angular resolution of &plusmn;0.25&deg;.
<img alt="Figure 2" src="/files/ftp_upload/4133/4133fig2.jpg" /> 
 Figure 2. Evaluation profile for one pixel (schematic). When ln[IF/T(&Theta;)] is plotted as a function of 1/d(&Theta;), the slope represents the cell-substrate distance &Delta;. This slope is commonly determined with a precision of about &plusmn;10%. One value - resulting from inhomogeneous illumination - has been marked and excluded from evaluation.
<img alt="Figure 3" src="/files/ftp_upload/4133/4133fig3.jpg" /> 
 Figure 3. TIRFM images of U251-MG glioblastoma cells with activated TP53 suppressor gene upon incubation with laurdan (8 &mu;M; 60 min) recorded at various angles of illumination (a-c); cell-substrate distances in the range of 0-600 nm shown by a color code (d); excitation wavelength: 391 nm, detection: &ge;420 nm; image size: 210 &mu;m &times; 210 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4133/4133fig4.jpg" /> 
 Figure 4. Cell-substrate distances of U251-MG glioblatoma cells in the range of 0-600 nm shown by a color code; (a) wild type (b) controls; (c) cells with activated suppressor gene TP53; (d) cells with activated suppressor gene PTEN; excitation wavelength: 391 nm, detection: &ge;420 nm; image size: 210 &mu;m &times; 210 &mu;m. <a href="https://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">Click here to view larger figure</a>.

<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 次观看.  Institut für Angewandte Forschung. 细胞粘附在基板上的拓扑结构是由可变角度的总内部反射荧光显微镜（VA-TIRFM）与纳米精度测量。

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...