Name: Implementazione della microscopia di riflessione delle interferenze per limaging ad alta velocità e senza etichetta dei microtubuli (Video) | JoVE Journal
Uploaded: 2019-08-08T11:30:00.000+00:00
Duration: 9 min 45 s
Description: Scopri la microscopia di riflessione delle interferenze per l'imaging ad alta velocità e senza etichetta dei microtubuli. Questa tecnica semplice ed economica si integra facilmente con i microscopi a fluorescenza standard.

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The Authors thank Anna Luchniak and Yin-wei Kuo for critical reading and comments on the protocol.

1. Microscope modification and objective lens
<ol>
	<li>Insert a 50/50 mirror into the filter wheel of the fluorescent microscope using an appropriate filter cube (Figure 1). Handle the 50/50 mirror with care as often they have anti-reflection coating. 
	NOTE: We used a 50/50 mirror in an empty filter cube of the microscope. The 50/50 mirror is inserted where the dichroic mirror is located.</li>
	<li>Use a high magnification/high NA oil objective. 
	NOTE: In this protocol, we used a 100x/1.3 NA objective.</li>
</ol>
2. Chamber preparation to adhere microtubules to the surface
<ol>
	<li>Clean microscope slides and 22 mm x 22 mm coverslips (for upright microscope) or 22 mm x 22 mm and 18 mm x 18 mm coverslips (for inverted microscopes). Modify the surface as needed. For example, clean the coverslips using an alkaline detergent (see Table of Materials) followed by 100% ethanol with distilled water washes in between and after11.</li>
	<li>Cut 3 mm wide strips of plastic paraffin film (see Table of Materials) using a razor blade and another microscope slide as an edge.</li>
	<li>Place two plastic paraffin film strips 3 mm apart on a clean 22 x 22mm cover slip. Then place an 18 mm x 18 mm coverslip to form a channel. If using an upright microscope, place the strips on top of a clean microscope slide and then place a coverslip on top.</li>
	<li>Place the coverslip (or slide) on a heat block at 100 °C for 10-30 s for the paraffin film to form a sealed channel.</li>
	<li>Flow in 50 µg/mL of antibody (in Brinkley Buffer 1980 (BRB80)) by perfusion using a pipette. Incubate for 10 min. 
	NOTE: We use anti-rhodamine antibodies to bind rhodamine-labeled microtubule seeds to the surface. Alternatively, avidin can be used to bind biotin-labelled microtubule seeds or to biotinylated gold particles. To simply look at microtubules in the absence of tubulin in solution (i.e., a non-dynamic assay), an anti-tubulin antibody can be used.</li>
	<li>Wash five times with BRB80 buffer. It is recommended to filter the solutions using a 0.22 µm filter.</li>
	<li>Flow in 1% poloxamer 407 (a triblock copolymer) in BRB80 to block the surface against non-specific binding. Incubate for 10 min.</li>
	<li>Wash five times with BRB80 buffer.</li>
	<li>The sample is ready to be placed on the microscope. To prevent the sample from drying out, add two droplets of BRB80 buffer at the ends of the channel and replenish when needed.</li>
</ol>
3. Microscope alignment
<ol>
	<li>Place the sample on the microscope stage. Turn on the epi-illumination light source.</li>
	<li>Focus on the sample surface. You will observe multiple surfaces as the objective is moved up and down due to back reflection of light from optics within the optical path. A good method to find the sample surface is to focus on the paraffin film edge. Once the surface is found, set the field of view to the center of the chamber.</li>
	<li>Center the field diaphragm in the field of view by closing it half way and using the adjustment screws. Once aligned, reopen the diaphragm. 
	NOTE: Screw adjustment only needs to be done sporadically, perhaps every 3-6 months or when troubleshooting.</li>
	<li>Slide in the Bertrand lens to view the exit pupil (back focal plane) of the objective.</li>
	<li>Close the aperture diaphragm (AD) beyond the edges of the exit pupil of the objective.</li>
	<li>Use the adjustment screws to center the AD with the exit pupil. Double check by opening the AD and matching its edges with those of the exit pupil. 
	NOTE: This adjustment only needs to be done sporadically.</li>
	<li>Set the aperture diaphragm to about 2/3 of the NA of the objective. See section 7 for detailed procedures for optimizing the aperture diaphragm.</li>
</ol>
4. Imaging stabilized microtubules or 40 nm gold particle
NOTE: Stabilized microtubules and gold nanoparticles serve as good control samples. It is recommended to image surface attached microtubules or gold nanoparticles as a first step to assess IRM performance and help in setting the optimal aperture diaphragm opening (section 7).
<ol>
	<li>Set the exposure time of the camera to 10 ms and adjust the illumination to nearly saturate the camera dynamic range.</li>
	<li>Flow in 10 µL of guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCCP)- or taxol-stabilized microtubules in BRB804,12,13 (or 40 nm gold particles) by perfusion using a pipette to a fresh sample and monitor binding by imaging the surface. Once 10-20 microtubules are bound within the field of view, wash the sample 2x with BRB80. 
	NOTE: With a well aligned microscope, the microtubules should be visible without background subtraction.</li>
	<li>Acquire 10 images by setting a time lapse with 1 second delay period for 10 seconds (at 10 ms exposure).</li>
	<li>Acquire a background image. To do so, move the stage using the stage controller or computer software along the channel long axis while acquiring 100 images with no delay (i.e. streaming close to 100 fps @ 10 ms exposure). 
	NOTE: The background is the median of the 100 images. By taking the median, the illumination profile and other stationary features like dirt on the optics are preserved while everything else is filtered out. There should be no tilt on the sample as this will lead to change in the axial position as the stage is moved and ultimately degrade the background image. If the tilt cannot be avoided, then an alternative method is to acquire averaged background images before flowing the seeds.</li>
	<li>For processing and analyzing the images, go to step 6.</li>
</ol>
5. Imaging microtubule dynamics
<ol>
	<li>For microtubule dynamics using brain tubulin, set the sample heater temperature to 37 °C.</li>
	<li>Flow in 10 µL of GMPCCP-stabilized microtubule seeds in BRB8011 by perfusion using a pipette to a fresh sample and monitor them binding to the surface by imaging the surface live (i.e., while streaming). Once 10-20 seeds are bound with field of view, wash the sample using 2x channel volume with BRB80 (BRB80 should be prewarmed to 37 °C or at least at room temperature). 
	NOTE: With a well aligned microscope, the seeds should be visible without background subtraction.</li>
	<li>Flow in the polymerization mix (7.5 µM unlabeled tubulin + 1 mM guanosine triphosphate (GTP) + 1 mM Dithiothreitol (DTT) in BRB80 buffer). For measuring microtubule growth, set a time-lapse using the acquisition software to acquire an image every 5 seconds (0.2 frames per second (fps)) for 15 minutes.</li>
	<li>To enhance the contrast, acquire 10 images (instead of one) at each time point and average them. For microtubule shrinkage, acquire images at 100 fps by setting the time delay to 0 and an exposure time of 10ms (higher fps is possible with smaller regions of interest depending on the camera used).</li>
	<li>Acquire a background image as in step 4.4.</li>
</ol>
6. Image processing and analysis
NOTE: For analysis, this protocol uses Fiji14 but the reader is free to use any software she/he find suitable.
<ol>
	<li>Open saved background images.</li>
	<li>Calculate the median image (i.e. background) using Image &gt; Stack &gt; Z project &gt; Median.</li>
	<li>Open microtubule dynamics movie as a stack (same for non-dynamic microtubules) using File &gt; Open.</li>
	<li>Subtract the background image from the stack using Process &gt; Image calculator. Make sure to check the “32bit (float) result” option.</li>
	<li>For dynamic microtubules, using the line tool draw a line along the microtubule of interest and add it to the region of interest manager by pressing “t”. Repeat for all microtubules of interest.</li>
	<li>For dynamic microtubules, run the Multi-Kymo macro (Supplementary File 1). The macro will generate a video and a kymograph for every microtubule in the ROI manager. Every microtubule will get a unique identifier.</li>
	<li>For dynamic microtubules, run the Kymo-Analysis macro (Supplementary File 2) and follow its steps to measure the growth rates and shrinkage rates of the microtubules.</li>
</ol>
7. Aperture diaphragm size
NOTE: An important factor for acquiring high contrast images of microtubules using IRM is setting the illumination numerical aperture (INA) correctly10,15. The INA can be changed by changing the size of the incoming illumination beam at the objective’s exit pupil which is controlled by the size of the AD (the AD is located at a conjugate plane with the exit pupil (back focal plane) of the objective, Figure 1): 
<img alt="Static equilibrium; equation INA = D_AD/(2*f_objective), for D_AD &lt; D_ep; formula analysis." src="/files/ftp_upload/59520/59520eq1.jpg" data-altupdatedat="1788282455000" data-alt="Equation"> 
where DAD is the diameter of the aperture diaphragm, fobjective is the focal length of the objective and Dep is the objective’s exit pupil diameter. Typically, the AD is left fully open for fluorescence imaging, so the INA equals the objective’s NA. In a fluorescence microscope, the AD scale does not indicate its diameter, thus the INA can’t be calculated. It’s possible to calibrate the AD size with the help of an objective. Yet, it’s not necessary since the AD size would be fixed to the size that produces the highest contrast.
<ol>
	<li>Prepare a sample of fluorescently labelled stabilized microtubules stuck to the surface (steps 4.1-4.2). 
	NOTE: We used tetramethylrhodamine labeled microtubules16 (Ex: 550 nm, Em: 580 nm).</li>
	<li>Bring microtubules into focus using the microscope focusing knob while fluorescently imaging them (if microtubules are not labelled, image them with IRM15 or DIC5).</li>
	<li>Set camera exposure to 10 ms using the camera software. 
	NOTE: This exposure is arbitrary and an exposure of 100 ms would also work.</li>
	<li>Close down the AD to its smallest opening. Adjust the illumination to nearly saturate the camera’s dynamic range or to the maximum possible. 
	NOTE: As a guide, use the look up table typically provided by the acquisition software.</li>
	<li>Acquire 10 images (by streaming or taking an image every second) of a field of view containing 10+ microtubules.</li>
	<li>Acquire a background image as in step 4.4.</li>
	<li>Change the size of the AD and repeat steps 7.5-7.6 for the whole AD opening range (from closed to the exit pupil size, Figure 2). Every time the AD size is changed, adjust the illumination intensity to match that of step 7.4.</li>
	<li>For every field of view acquired, subtract the corresponding background using process &gt; image calculator and choosing “subtract” from the drop-down menu. Make sure the “32bit (float) result” option is checked. Then average the resulting images using image &gt; stack &gt; Z project &gt; average.</li>
	<li>Measure the signal-to-background noise ratio (SBR) of the microtubules defined as the average intensity of the microtubule signal (intensity of the microtubule minus the intensity of the background) divided by the standard deviation of the background (Figure 3).</li>
	<li>Determine the optimal AD size (i.e. optimal INA) by calculating the average SBR of the microtubules for every opening size and set the AD size to the one producing the highest SBR (Figure 2). It is possible that there is a range of AD sizes that produce comparable contrast15.</li>
</ol>

<ol><li>Widlund, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+purification+of+assembly-competent+tubulin+from+diverse+eukaryotic+sources%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">One-step purification of assembly-competent tubulin from diverse eukaryotic sources.</a> Molecular Biology of the Cell. 23 (22), 4393-4401 (2012).</li>
<li>Walker, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+instability+of+individual+microtubules+analyzed+by+video+light+microscopy%3A+rate+constants+and+transition+frequencies%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.</a> The Journal of Cell Biology. 107 (4), 1437-1448 (1988).</li>
<li>Hotani, H., Horio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+microtubules+visualized+by+darkfield+microscopy%3A+Treadmilling+and+dynamic+instability%5BTitle%5D)+AND+%22Cell+Motility+and+the+Cytoskeleton%22%5BJournal%5D)">Dynamics of microtubules visualized by darkfield microscopy: Treadmilling and dynamic instability.</a> Cell Motility and the Cytoskeleton. 10 (1-2), 229-236 (1988).</li>
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<li>Andrecka, J., Ortega Arroyo, J., Lewis, K., Cross, R. A., Kukura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+Imaging+of+Microtubules+with+Sub-nm+Precision+Using+Interferometric+Scattering+Microscopy%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy.</a> Biophysical Journal. 110 (1), 214-217 (2016).</li>
<li>Koch, M. D., Rohrbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+Imaging+and+Bending+Analysis+of+Microtubules+by+ROCS+Microscopy+and+Optical+Trapping%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping.</a> Biophysical Journal. 114 (1), 168-177 (2018).</li>
<li>Kandel, M. E., Teng, K. W., Selvin, P. R., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-Free+Imaging+of+Single+Microtubule+Dynamics+Using+Spatial+Light+Interference+Microscopy%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy.</a> ACS Nano. 11 (1), 647-655 (2017).</li>
<li>Curtis, A. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Mechanism+of+Adhesion+of+Cells+to+Glass%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">The Mechanism of Adhesion of Cells to Glass.</a> The Journal of Cell Biology. 20 (2), 199-215 (1964).</li>
<li>Weber, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%5B2%5D+Reflection+interference+contrast+microscopy%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">[2] Reflection interference contrast microscopy.</a> Methods in Enzymology. 361, 34-47 (2003).</li>
<li>Gell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+13+-+Microtubule+Dynamics+Reconstituted+In+Vitro+and+Imaged+by+Single-Molecule+Fluorescence+Microscopy%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Chapter 13 - Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy.</a> Methods in Cell Biology. 95, 221-245 (2010).</li>
<li>Nitzsche, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+14+-+Studying+Kinesin+Motors+by+Optical+3D-Nanometry+in+Gliding+Motility+Assays%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Chapter 14 - Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays.</a> Methods in Cell Biology. 95, 247-271 (2010).</li>
<li>Schiff, P. B., Fant, J., Horwitz, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promotion+of+microtubule+assembly+in+vitro+by+taxol%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Promotion of microtubule assembly in vitro by taxol.</a> Nature. 277 (5698), 665-667 (1979).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Mahamdeh, M., Simmert, S., Luchniak, A., Schäffer, E., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+high-speed+wide-field+imaging+of+single+microtubules+using+interference+reflection+microscopy%5BTitle%5D)+AND+%22Journal+of+Microscopy%22%5BJournal%5D)">Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.</a> Journal of Microscopy. 272 (1), 60-66 (2018).</li>
<li>Hyman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%5B39%5D+Preparation+of+modified+tubulins%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">[39] Preparation of modified tubulins.</a> Methods in Enzymology. 196, 478-485 (1991).</li>
<li>Ruhnow, F., Zwicker, D., Diez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+Single+Particles+and+Elongated+Filaments+with+Nanometer+Precision%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Tracking Single Particles and Elongated Filaments with Nanometer Precision.</a> Biophysical Journal. 100 (11), 2820-2828 (2011).</li>
<li>Mahamdeh, M., Schäffer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+tweezers+with+millikelvin+precision+of+temperature-controlled+objectives+and+base-pair+resolution%5BTitle%5D)+AND+%22Optics+Express%22%5BJournal%5D)">Optical tweezers with millikelvin precision of temperature-controlled objectives and base-pair resolution.</a> Optics Express. 17 (19), 17190-17199 (2009).</li>
<li>Kim, K., Saleh, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilizing+method+for+reflection+interference+contrast+microscopy%5BTitle%5D)+AND+%22Applied+Optics%22%5BJournal%5D)">Stabilizing method for reflection interference contrast microscopy.</a> Applied Optics. 47 (12), 2070-2075 (2008).</li>
<li>Ortega Arroyo, J., Cole, D., Kukura, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interferometric+scattering+microscopy+and+its+combination+with+single-molecule+fluorescence+imaging%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.</a> Nature Protocols. 11 (4), 617-633 (2016).</li>
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</ol>

The authors have no conflicts of interest to disclose.

This protocol demonstrated the successful use of IRM for imaging and measurement of the microtubule dynamics. Care should be given to correctly set the illumination numerical aperture as it has the strongest impact on image contrast. Also, using high numerical aperture (NA) objectives is important for getting high resolution/high contrast images, as higher NA objective have higher light collecting power compared to low NA objectives. The cleaner the surface and solutions used the lower the noise as dirt ends up attaching to the surface and adding (over the course of the experiment) speckle like noise to the images. Acquisition of a background image is important as well as it removes illumination inhomogeneities, static noise and surface irregularities.
A recommended modification is to introduce a long pass filter (&#62;600 nm) in the illumination path. The spectrum of white light sources typically contains wavelengths in the UV which can damage microtubules. In addition, using long wave length for IRM comes in handy when combining IRM with fluorescence (e.g., when studying the effect of microtubule associated proteins (MAPs) on microtubule dynamics). Be aware that when imaging for expended period of times, sample drift (especially along the optical axis) decreases image contrast due to the deviation of the image plane from the background plane. Modern microscopes are often equipped with stabilization mechanisms (e.g., perfect focus (Nikon), Definite focus.2 (Zeiss), IX3-ZDC2 (Olympus)). An alternative solution is to thermally stabilize the setup either passively or actively18 or by correcting for drift19,20,21. Finally, microtubule contrast can be increased by reducing the size of field diaphragm (a 70% opening is good choice as it is a balance between increasing contrast and field of view size)15.
While IRM is suitable for imaging microtubules it is not sensitive enough to detect single proteins. For such application, iSCAT is a more suitable technique. Similarly, fluorescence and iSCAT are better suited if tracking precision of less than 10 nm is needed. For IRM, the measured length tracking precision is ~20 nm as shown in Figure 7.
Use of IRM in surface assays can go beyond microtubules; for example, molecular motors can be labelled with gold nanoparticles and tracked as they interact with microtubules. In addition a more advanced form of IRM known as reflective interference contrast microscopy (RICM)22 can, in principle, be used to further enhance microtubules contrast and obtain higher tracking precision.

Label-free imaging of microtubules is of interest as it circumvents the need for fluorescent labeling of tubulin to generate contrast in the images. Fluorescent labeling has several drawbacks: it not feasible if the protein concentration is low1 and photobleaching and photodamage limit the observation time. Several techniques have been used to image label-free microtubules, including video-enhanced differential interference contrast microscopy (DIC) and darkfield microscopy2,3,4,5. Recently, interferometric scattering microscopy (iSCAT)6, rotating-coherent-scattering microscopy (ROCS)7 and spatial light interference microscopy (SLIM)8, have also been used. All these techniques are capable of imaging microtubules and have proved to be valuable for studying microtubule dynamics. However, each have their own limitations. In DIC the contrast depends on the angle between the microtubule and the Nomarski prism axis. &#160;In darkfield, the microtubule signal is degraded by scattered light from impurities or surfaces defects. Though iSCAT exhibits extraordinary sensitivity (down to single proteins) and ROCS can image microtubules deeper into the sample, both methods are technically demanding, requiring laser scanners.
This protocol demonstrates how interference reflection microscopy (IRM)9, 10 can be set up as an alternative technique for label-free imaging of microtubules. IRM is easy to implement as it requires only the addition of an inexpensive 50/50 mirror to a standard fluorescent microscope. When used in conjunction with the software described here, IRM produces high contrast microtubule images, can image large fields of view at high speed, requires a onetime alignment, and can easily be combined with other techniques such as fluorescence imaging.

<table><tbody><tr><td>Microscope</td><td>Nikon</td><td>Ti-Eclipse</td><td>An inverted microscope used for perfoming the expriments</td></tr><tr><td>50/50 beam splitter</td><td>Chroma</td><td>21000</td><td>When buying make sure to choose the splitter dimensions that fit the cube used in the microscope</td></tr><tr><td>NIKON PLAN FLUOR 100X/0.5-1.3 Iris objective</td><td>Nikon</td><td>MRH02902</td><td>Imaging objective. This objective has a NA adjusting iris that was opened to NA 1.3</td></tr><tr><td>Mucasol universal detergent</td><td>Sigma-aAldrich</td><td>Z637181-2L</td><td>Used for cleaning coverslips and slides</td></tr><tr><td>plastic paraffin film (commerical name Parafilm M)</td><td>Sigma-aAldrich</td><td>P7793</td><td>Used for constructing flow channels</td></tr><tr><td>Anti-TAMRA antibody</td><td>Invitrogen</td><td>A-6397</td><td>Used to bind TAMRA labeled molecules (e.g. microtubules) to the sample surface. RRID (AB_2536196)</td></tr><tr><td>Poloxamer 407 (commercial name Pluronic F-127)</td><td>Sigma-aAldrich</td><td></td><td>Used for blocking the channel surface to prevent nonspecific binding</td></tr><tr><td>40 nm gold nanoparticles</td><td>Sigma-aAldrich</td><td>753637</td><td>Used as a control sample</td></tr><tr><td>20 nm gold nanoparticles</td><td>Sigma-aAldrich</td><td>753610</td><td>Used as a control sample</td></tr><tr><td>Zyla 4.2 Camera</td><td>Andor</td><td>Zyla 4.2</td><td>2048x2048 pixles (6.5&amp;micro;m pixel size) with quantum efficiency of 72% and 16bit dynamic range</td></tr><tr><td>Feista tracking software</td><td></td><td></td><td>https://www.bcube-dresden.de/fiesta/wiki/FIESTA</td></tr><tr><td>Stabilized microtubles</td><td></td><td></td><td>prepared in house (see references in text)</td></tr></tbody></table>

implementation of interference reflection microscopy for label-free, high-speed imaging of microtubules

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

As mentioned above, with a well aligned microscope, microtubules should be visible without background subtraction (Figure 4A). Subtracting the background (Figure 4B) enhances the contrast of microtubule (Figure 4C). To further enhance the contrast, averaging or Fourier filtering or a combination of both can be used (Figure 4D,F,E). The line scans in Figure 4G shows the incremental improvement of image quality. Notice the reduction of background noise with each processing step.
Examples of kymographs of microtubule dynamics generated from time-lapse movies are shown in Figure 5. The videos were acquired at two frame rates: 0.2 fps (slow) and 100 fps (fast). The former is suitable for measuring growth rates while the latter is more suitable for measuring shrinkage rate that is an order of magnitude faster than the growth rate.
For the case where gold nanoparticles are used for setting up the microscope, an example image is shown in Figure 6. Gold nanoparticles were passively attached to the surface. While 40 nm particles are recommended, it is also possible to image 20 nm particles, yet at a lower contrast.
<img alt="Microtubule extension setup in fluorescence microscopy with optical system diagram and assembly steps." class="xfigimg" src="/files/ftp_upload/59520/59520fig1.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1. Schematic representation of IRM. (A) Epi-illumination from the light source passes through the aperture diaphragm before reaching the 50/50 mirror. The aperture diaphragm sets the beam width thus the illumination NA. The 50/50 mirror partially reflects the light up to the objective to illuminate the sample. Light reflected from the sample is collected and then projected onto the camera chip (by the tube lens) where it interferes to generate the image. Image contrast is the result of the interference between the light reflected from the glass/water interface (I1) and the light reflected from the water/microtubule interface (I2). Depending on the microtubule/surface distance (h), the optical path difference between I1 and I2 will result in a constructive (bright signal) or destructive (dark signal) or anything in between. For example, if light with a wavelength of 600 nm is used for imaging, the contrast will switch between dark and bright when the microtubule height changes by about 100 nm. The asterisk indicate conjugate planes  (modified from15). (B) Example of the 50/50 mirror installation. A suitable filter cube was opened and the mirror was inserted where a dichroic mirror usually sits. The mirror was oriented as per manufacturer instructions. Then the cube was inserted in the filter wheel which was inserted back to the microscope (not shown). During the installation, gloves were used, and the mirror was only held by the edges. <a href="https://www.jove.com/files/ftp_upload/59520/59520fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy signal analysis; signal-to-background ratio chart; image processing stages; data analysis." class="xfigimg" src="/files/ftp_upload/59520/59520fig2.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2. Optimal Aperture diaphragm setting. (A) Same field of view was imaged at different aperture diaphragm openings without background subtraction. Visually, the contrast increased as the size of the aperture diaphragm increased till it reached a plateau and started to degrade afterwards. This was confirmed by (B) SBR measurements of background subtracted images. Error bars are standard deviation.  Scale bars are 500 µm (AD) and 3 µm (microtubules). <a href="https://www.jove.com/files/ftp_upload/59520/59520fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Signal-to-Background Ratio (SBR) analysis diagram; SBR=Signal/StDv(Background) with graph details." class="xfigimg" src="/files/ftp_upload/59520/59520fig3.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3. Measuring signal-to-background noise ratio. Microtubules were isolated in regions of interest. Each region of interest was thresholded to separate the microtubule from the background. The average microtubule signal was obtained from a line scan across the microtubule. The scan line width was set to equal the microtubule length. This way, every point on the scan is an average of the signals of all pixels along the microtubule axis that are parallel to that point. The background noise is the standard deviation of all the pixels below threshold cut off.  <a href="https://www.jove.com/files/ftp_upload/59520/59520fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Image processing diagram with FFT filter comparison, background removal, and data averaging results." class="xfigimg" src="/files/ftp_upload/59520/59520fig4.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
Figure 4. Image processing. After acquiring raw images (A), the background (B) was subtracted (C) to enhance the microtubule contrast. To further improve the contrast the images were either averaged (D) or Fourier filtered (E) or both (F). The line scans (G), whose location is indicated by the dashed red line in (A) are color matched to the various images in (A) to (F). The numbers at the lower corner are average SBRs measured for the whole field of view. The scale bar is 5 µm (modified from15) . <a href="https://www.jove.com/files/ftp_upload/59520/59520fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy image showing dynamic material deformation; data represents stress-strain cycles." class="xfigimg" src="/files/ftp_upload/59520/59520fig5.jpg" data-altupdatedat="1788282455000" data-alt="Figure 5"> 
Figure 5. Examples of kymographs. (A) Kymograph examples of microtubule dynamics generated from time-lapse movies acquired at 0.2 fps. (B) Kymograph depicting an example of a shrinkage event generated from a movie acquired at 100 fps. Dashed lines mark the seeds. <a href="https://www.jove.com/files/ftp_upload/59520/59520fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Nanoparticle analysis via microscopy; 20 nm vs 40 nm particle comparison in electron micrograph." class="xfigimg" src="/files/ftp_upload/59520/59520fig6.jpg" data-altupdatedat="1788282455000" data-alt="Figure 6"> 
Figure 6. Example of gold nanoparticles imaged with IRM. Gold nanoparticles of sizes 20 and 40 nm were passively attached to the surface. 10 images were acquired. After background subtraction, the images were averaged to enhance contrast. <a href="https://www.jove.com/files/ftp_upload/59520/59520fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Graph of length (nm) vs time (s) showing fluctuations; statistical process control chart." class="xfigimg" src="/files/ftp_upload/59520/59520fig7.jpg" data-altupdatedat="1788282455000" data-alt="Figure 7"> 
Figure 7. Microtubule length Tracking precision in IRM images. Stabilized microtubules (i.e., fixed lengths) were imaged 200x at 100 fps then averaged to 10 fps to enhance contrast. Next, the microtubules’ lengths were measured using Fiesta17 tracking software. For every microtubule the mean length and standard deviation were calculated as shown in the figure (dashed line represents the mean and solid red lines represents the standard deviation, length = 3971 ± 20 nm. The overall tracking precision was the average of the standard deviation of all tracked  microtubules (n = 6 microtubules x 20 data points = 120 data points). <a href="https://www.jove.com/files/ftp_upload/59520/59520fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1. <a href="https://www.jove.com/files/ftp_upload/59520/Multi_Kymo.ijm" target="_blank">Please click here to download this file.</a>
Supplementary File 2. <a href="https://www.jove.com/files/ftp_upload/59520/Kymo_Analysis.ijm" target="_blank">Please click here to download this file.</a>

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

The significance of this protocol is that it introduces a label-free imaging technique capable of acquiring high contrast images at high frame rates that is simpler and cheaper than other techniques like DIC and dark field. The main advantage of this technique is that it's easy to implement and it's easy to use. It's inexpensive and it can be combined with fluorescence microscopes easily.The method was developed for visualizing individual microtubules. It has the potential to be used for visualizing large protein complexes and nanoparticles. The main difficulty is the activation energy required to tinker with the filter cube to add the half silver mirror.My advice to someone who is trying the technique for the first time is just do it. To begin, insert a 50/50 mirror into the filter wheel of a fluorescent microscope using an appropriate filter cube. Handle the mirror with care as often they have an anti-reflection coding.Turn to a high magnification objective that also has a high numerical aperture. The one shown here is a 100x oil objective with a numerical aperture of 1.3. Next, use a razor blade and a microscope slide as a straight edge to cut three millimeter wide strips of plastic paraffin film.Place two of the plastic paraffin film strips three millimeters apart on a clean 22 by 22 millimeter cover slip, then place an 18 by 18 millimeter cover slip on top of the strips to form a channel. Transfer the cover slip to a heat block at 100 degrees celsius for 10 to 30 seconds for the paraffin film to form a sealed channel. Using a pipette, flow in 50 micrograms per milliliter of an anti-rhodamine antibody by perfusion and incubate the slide for 10 minutes.Following incubation, wash the channel five times using filtered BRB80, then flow in 1%Poloxamer 407 in the filtered BRB80 to block the surface against non-specific binding and incubate the slide for 10 minutes. Again, wash the channel five times using filtered BRB80. To prevent the sample from drying out, add two droplets of the filtered BRB80 at the ends of the channel and add more buffer as needed.Place the sample on the microscope stage and turn on the epi-illumination light source. Focus on the paraffin film edge to find the sample surface and then move the field to set the view to the center of the chamber. You will observe multiple surfaces as the objective is moved up and down due to back reflection of light from optics within the optical path.Next, center the field diaphragm in the field of view by closing it halfway and using the adjustment screws. Once the diaphragm is properly aligned, re-open it. Then, slide in the Bertrand lens to view the back focal plane, also known as the exit pupil of the objective.Close the aperture diaphragm beyond the edges of the exit pupil and use the adjustment screws to center the aperture diaphragm with respect to the exit pupil. Double check by opening the aperture diaphragm and matching its edges with those of the exit pupil. Then, set the aperture diaphragm to about two-thirds of the numerical aperture of the objective.To begin, set the exposure time of the camera to 10 milliseconds and adjust the illumination to nearly saturate the camera dynamic range. Next, use a pipette to flow in 10 microliters of GMPCPP-stabilized microtubules in 0.22 micrometer filtered BRB80. Monitor the microtubule binding on the imaging of the surface.Once 10 to 20 microtubules are bound within the field of view, wash the sample twice with the filtered BRB80. Acquire 10 images of the microtubules by setting up a time lapse with a 10 millisecond exposure and a one second delay period for a total of 10 seconds. Then, acquire background images by moving the stage using the stage controller along the channel's long axis while acquiring 100 images with no delay.To image microtubule dynamics using brain tubulin, start by setting the sample heater temperature to 37 degrees celsius. Using a pipette, flow in 10 microliters of GMPCPP-stabilized microtubule seeds and monitor them binding to the surface by imaging the surface live. Once 10 to 20 seeds are bound within the field of view, wash the sample using twice the channel volume of pre-warmed and filtered BRB80.Next, flow in 10 microliters of the polymerization mix. To measure microtubule growth, set up a time lapse using the acquisition software to acquire an image every five seconds for 15 minutes. Enhance the contrast by acquiring an averaged image of 10 at each time point.Acquire background images as shown in the previous section. Calculate the median by going to image, selecting stack, then Z project, then median. Subtract the corresponding background by going to process, going to image calculator, and choosing subtract from the dropdown menu.Make sure the 32-bit float result option is checked. For microtubule shrinkage, acquire images at 100 frames per second by setting the time delay to zero and keeping the exposure time at 10 milliseconds. An important factor for acquiring high contrast images with the interference induction microscopy is to properly set the numerical aperture of the illumination.This can be done by guiding the size of the illumination beam at the exit pupil of the objective, which is controlled by the aperture diaphragm size. Using a sample of florescently-labeled stabilized microtubules, bring the microtubules into focus using the microscope's focusing knob while fluorescently imaging them. Set camera exposure to 10 milliseconds and close down the aperture diaphragm to its smallest opening.Also, adjust the illumination to nearly saturate the camera's dynamic range or until the maximum is reached. Acquire 10 images by streaming a field of view containing 10 or more microtubules. Then acquire a background image.Change the size of the diaphragm and adjust the illumination intensity to match that which was previously determined. Acquire 10 new images and a new background. Repeat this process until the entire range of the diaphragm is completed.For every field of view acquired, subtract the corresponding background and average the resulting background subtracted images as shown previously. Then, calculate the average signal to background noise ratio of the microtubules for every opening size. Set the diaphragm size to the one producing the highest signal to background noise ratio.It is possible that there is a range of sizes that produce comparable contrast as shown here. With a well-aligned microscope, microtubules should be visible without background subtraction. Subtracting the background, however, does enhance the contrast of the microtubule.To further enhance the contrast, averaging, 4a filtering, or a combination of both can be used. The line scans shown here describe the incremental improvement of image quality. These describe a noticeable reduction of background noise with each processing step.Microtubule dynamics can be reported in kinographs, which are generated from time lapse movies. An example kinograph acquired at a frame rate of 0.2 frames per second is shown here. The dashed lines mark the seeds.When performing this procedure, it's important to work with high numerical aperture objectives. It's also important to align and set the size of the aperture diameter frame correctly. It is easy to combine IRM with fluorescence imaging to study, for example, microtubule binding proteins and how they modify microtubule dynamics.The technique reduces photo damage considerably and allows for higher frame rate acquisition, making it easier to study labeled biopolymers such as microtubules for extended periods of time with high temporal resolution and good tracking precision.

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Research

JoVE Journal

Bioengineering

In this video, we describe the interference reflection microscopy (IRM) technique to visualize microtubules growing on a coverslip surface in the presence of a suitable buffer. Upon illumination with incident light, the coverslip-buffer interface and buffer-microtubule interface reflect light, which combines to create an interference pattern, enabling the visualization of microtubules as high-contrast images against a bright background.

Interference Reflection Microscopy for Label-Free Visualization of Microtubule Dynamics

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform  2D dual color dSTORM  images of microtubules and intermediate filaments in fixed cells...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction 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

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

In this article, we present a protocol to detect microtubule-loaded oligodendrocytes in a model of tubulinopathy through a simple, innovative second harmonic generation microscopy approach.

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

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 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

This protocol describes a label-free optical diffraction tomography-based workflow for real-time imaging and quantitative analysis of macrophage morphodynamics. The method enables continuous tracking of single-cell morphology and migration, providing time-resolved measurements of projected area, perimeter, and average migration speed during long-term live-cell imaging.

Real-Time Label-Free Imaging and Quantitative Analysis of Macrophage Morphodynamics Using Optical Diffraction Tomography

Scopri la microscopia di riflessione delle interferenze per l'imaging ad alta velocità e senza etichetta dei microtubuli. Questa tecnica semplice ed economica si integra facilmente con i microscopi a fluorescenza standard.

Implementazione della microscopia di riflessione delle interferenze per limaging ad alta velocità e senza etichetta dei microtubuli (Video) | JoVE Journal

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

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