Name: Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM) (Video) | JoVE Journal
Uploaded: 2012-10-02T12:00:00.000+00:00
Duration: 9 min 14 s
Description: Learn to measure cell adhesion topology with nanometer precision using variable-angle total internal reflection fluorescence microscopy. This method compares adhesion between cancerous and non-cancerous cell lines.

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5 minutes

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 °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 μM (in culture medium)4, or a cytoplasm marker, e.g. calcein acetomethlyester applied for 40 min at a concentration of 5 μM3, 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 Θc=61.3° for a glass-water transition as a fixed value. Θc can be visualized upon variation of Θ when a fluorescent dye (e.g. 1 mM flavin mononucleotide, FMN) is dissolved in water. The position of the mirror at Θ=Θc 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×/0.75 or 63×/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 (Θ≥Θc with Θc corresponding to 64.5° for a cell-glass interface). An angular range of 66°-74° with steps of 0.5° or 1° 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 Θ, 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(Θ) and the transmission factor T(Θ) are calculated automatically.</li>
	<li>Select images used for evaluation of cell-substrate distances Δ 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 μM; 60 min) are depicted for angles of incidence of 66.0°, 69.0° and 73.0° 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="Microscope optical setup; diagram of light path with mirrors, lens, prism for excitation studies." src="/files/ftp_upload/4133/4133fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. Illumination device for VA-TIRFM in an upright microscope. An adjustable mirror driven by a step motor permits an angular resolution of ±0.25°.
<img alt="Static equilibrium graph ln(I₀/T(ω)) vs. 1/d(θ) nm⁻¹ with linear fit; data analysis." src="/files/ftp_upload/4133/4133fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 Figure 2. Evaluation profile for one pixel (schematic). When ln[IF/T(Θ)] is plotted as a function of 1/d(Θ), the slope represents the cell-substrate distance Δ. This slope is commonly determined with a precision of about ±10%. One value - resulting from inhomogeneous illumination - has been marked and excluded from evaluation.
<img alt="Microscopic imaging analysis showing spectral fitting data in grayscale and color-coded diagrams." src="/files/ftp_upload/4133/4133fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
 Figure 3. TIRFM images of U251-MG glioblastoma cells with activated TP53 suppressor gene upon incubation with laurdan (8 μM; 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: ≥420 nm; image size: 210 μm × 210 μm. <a href="https://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Spatial distribution diagram; liquid saturation measurement in porous media, identified by color scale." src="/files/ftp_upload/4133/4133fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
 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: ≥420 nm; image size: 210 μm × 210 μm. <a href="https://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">Click here to view larger figure</a>.

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<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/pubmed?term=((Optimizing+low-light+microscopy+with+back-illuminated+electron+multiplying+charge-coupled+device%3A+enhanced+sensitivity%2C+speed+and+resolution%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">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>
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<li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+%28STORM%29%5BTitle%5D)+AND+%22Nat.+Meth%22%5BJournal%5D)">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/pubmed?term=((STED+microscopy+with+continuous+wave+beams%5BTitle%5D)+AND+%22Nat.+Meth%22%5BJournal%5D)">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/pubmed?term=((Multi-Dimensional+Fluorescence+Microscopy+of+Living+Cells%5BTitle%5D)+AND+%22J.+Biophotonics%22%5BJournal%5D)">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/pubmed?term=((Photosensitizers%3A+therapy+and+detection+of+malignant+tumours%5BTitle%5D)+AND+%22Photochem.+Photobiol%22%5BJournal%5D)">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/pubmed?term=((Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> J. Cell Biol. 89, 141-145 (1981).</li>
<li>Ölveczky, B. P., Periasamy, N., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+fluorophore+distribution+in+three+dimensions+by+quantitstive+multiple+angle+total+internal+reflection+fluorescence+microscopy%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">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/pubmed?term=((Selective+imaging+of+surface+fluorescence+with+vry+high+aperture+microscope+objectives%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Selective imaging of surface fluorescence with vry high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li>
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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><tbody><tr><td>Incubator</td><td>Nunc</td><td>Nunc Cellstar&lt;br/&gt; 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)

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.

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 ...

The overall goal of this procedure is to measure a topology of cell adhesion on a substrate with nanometer precision. This is accomplished by first cultivating cells on transparent glass slides and incubating them with a fluorescent marker. Then the angular position of the microscope's adjustable mirror is calibrated and the position is stored in the mirror positioning program.The next step is to record the fluorescence images upon irradiation at about 10 different angles. The final step is to calculate cell substrate distances with a precision of a few nanometers. Ultimately, results of topology of cell adhesion on substrate are used to show differences of adhesion between various cell lines, for example, cancer cells and less malignant cells.The main advantage of this technique over existing methods like confocal microscopy, is that actual resolution is in the range of only a few nanometers. Though this method can provide insight into cell membranes. It can also be applied to other systems such as small vesicles or nanomaterials.Demonstrating the experiment will be our postdoc Petra, who will prepare the cell samples and our engineer, Michael Wagner, who will run the experiment To prepare cells for variable angle, total internal reflection. Fluorescence microscopy seed individual cells at a density of 100 cells per square millimeter on a glass slide in culture medium supplemented with 10%fetal calf, serum, and antibiotics. Grow cells for 72 hours in an incubator at 37 degrees Celsius and 5%carbon dioxide.Next, apply to the cells the fluorescent membrane, marker six D, decano two, dimethyl amino naphthalene, or lorane at a concentration of eight micromolar in culture, medium incubate for 60 minutes prior to fluorescence microscopy. Wash the cells with PBS pipette the buffer solution over the object slide to remove medium afterwards, wipe off the buffer solution from the backside of the object slide. An upright microscope such as a Zeiss Axio plan, equipped with a back illuminated and or exon E-M-C-C-D camera is used for variable angle.T-I-R-F-M replace its condenser by a custom made illumination device with a hemi cylindrical glass prism optically coupled to the object slide. The laser beam is incident from a collated mono mode fiber, which after imaging by the glass prism and further optical components, again results in a parallel beam on the surface of the sample. Take care that the electrical field vector is polarized, perpendicular to the plane of incidents.This position is marked on the output coupler of the fiber. Use an adjustable mirror located inside the condenser, which is imaged in the sample plane and illuminates the sample under a variable angle of incidents or object ray. The adjustable mirror may be driven by a step motor, which is controlled by software.Each position corresponds to a well-defined angle of incidents as determined by go metric proceeded measurements. To calibrate the angular position of the adjustable mirror, use a fluorescent dye such as one millimolar flavin nucleotide, dissolved in water. To visualize the critical angle upon variation of the angle of incidents, use the critical angle of 61.3 degrees for a glass water transition as a fixed value, the angular position of the mirror at which the angle of incidents equals the critical angle is stored in the mirror positioning program.Using the camera software, set the parameters of the E-M-C-C-D camera, including temperature gain, recording time, and shift speed. Use immersion oil to assure optical contact of the sample. With a glass prism, select an objective lens of appropriate magnification for the cells and moderate numeric aperture.To visualize the object in the microscope, use an appropriate long pass or band pass filter for fluorescence detection. Record fluorescence images of identical samples upon variation of the angle of illumination. The angles of incidents should be the same or greater than the critical angle.With the critical angle corresponding to 64.5 degrees for a cell glass interface, an angular range of 66 degrees to 74 degrees with steps of 0.5 degrees or one degree is recommended. To begin data analysis, use the lab view program to read the images recorded at various angles as well as the camera settings, the excitation wavelength, and the refractive indices. For glass, which is 1.52, and cells, which is 1.37, the settings are imported for proper evaluation of the data.For each set of parameters, the penetration depth and the transmission factor are calculated automatically For the success of the procedure. It is important that images are carefully recorded and selected for analysis. Select images used for evaluation of cell substrate distances according to the equation.For a membrane marker, individual images within homogenous illumination, such as shown in this example, may be excluded. Calculate images of cell substrate topology and select an appropriate color code for display during the evaluation. Profiles of individual pixels can be displayed as depicted in this figure.These profiles may be used as an indicator for the quality of the fit. Finally, store the evaluation protocol. This figure depicts total internal reflection images of U2 51 mg glioblastoma cells with an activated TP 53 tumor suppressor gene.Upon incubation with luan recorded at various angles of incidents, 66 degrees corresponding to a penetration depth of the evanescent electromagnetic field of about 140 nanometers, 69 degrees, corresponding to a penetration depth of 75 nanometers and 73 degrees corresponding to a penetration depth of 60 nanometers with decreasing penetration. Depth fluorescence decreases in intensity and originates from more superficial sites of the cells, such as focal contacts on their edges. Cell substrate distances are depicted in Panel D by a color code ranging from zero to 600 nanometers as seen here.Cell substrate distances vary greatly over the cells between only a few nanometers represented by white to yellow and 250 to 300 nanometers represented by dark red. This indicates focal contacts and larger cell substrate distances are in close vicinity. A similar variation in cell substrate distances is calculated for U2 51 mg glioblastoma cells with an activated tumor suppressor gene P 10.In contrast, cell substrate distances are rather constant for U2 51 mg control cells represented by yellow and wild type cells represented by orange to red After its development. This technique paved in the way for researchers in the field of cell substrate topology to explore tumor cells and the morphological changes upon laser therapy. After watching this video, you should have a good understanding of how to visualize surfaces with an actual resolution in the nanometer range at low light exposure, which is well tolerable to living organisms.

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

Research

JoVE Journal

Bioengineering

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.

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

This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Daniele, F., et. al TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis. J. Vis. Exp. (2015).This video demonstrates the imaging of human neuroblastoma cells using a total internal reflection fluorescence (TIRF) microscope to visualize fluorophore-tagged synaptic vesicles. The cells are first focused in epifluorescence mode. Adjustments are then made to achieve total internal reflection,...

Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

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.

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

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&amp;B) analysis.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

Learn to measure cell adhesion topology with nanometer precision using variable-angle total internal reflection fluorescence microscopy. This method compares adhesion between cancerous and non-cancerous cell lines.

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

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