Name: Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM) (Video) | JoVE Journal
Uploaded: 2009-08-05T19:00:00.000+00:00
Duration: 19 min 16 s
Description: Learn fluorescent speckle microscopy to study F-actin dynamics in living cells using microinjection and specialized imaging chambers. This technique enables quantitative analysis of cytoskeletal protein kinetics.

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The development of qFSM is funded by the NIH grant U01 GM06230.

Section 1: Obtaining your fluorescently labeled actin for FSM
Materials required: purified actin, fluorophore (Alexa, X-rhodamine recommended). G-buffer, ultracentrifuge
<ol>
<li>A key component to acquiring good FSM movies requires the proper labeling of actin (or other cytoskeletal proteins) with the fluorophore of your choice. We recommend using fluorophores of Alexa (488, 568 wavelengths) and X-rhodamine succinimidyl ester derivatives that target exposed lysine residues on the surface of actin. </li>
<li>When choosing your fluorophore and its appropriate wavelength, ensure that your microscope setup has the appropriate filters (excitation and emission) and illuminators (mercury arc lamp and/or lasers) to accommodate your fluorescent tag. We recommend selecting wavelengths in the orange to red wavelengths (560-630 nm) to minimize the effects of auto-fluorescence and photo-damage and to ensure its compatibility with GFP-conjugates. </li>
<li>Pure actin can be extracted from muscle tissue belonging to Rat or Chicken, but requires a lengthy extraction procedure from muscle acetone powder. Muscle acetone powder can be purchased from Invitrogen. The labeling procedure is not discussed in this protocol, but can be found here[1] </li>
<li>Alternatively, you can purchase labeled actin proteins from several vendors including Cytoskeleton Inc., and Invitrogen. Custom labeling services are also available through Invitrogen. Purified, non-labeled actin (both muscle and nonmuscle actin) can be purchased from Cytoskeleton Inc.</li>
<li>Whether you decide to purchase or prepare your own labeled actin, you want to ensure that the labeling ratio is somewhere around 0.3 to 0.7 dyes per monomer of actin. Too high a labeling ratio may effect the turnover kinetics (and binding to associated proteins) of the actin filament, while too low a labeling ratio will lead to poor looking speckles (low signal to noise) and may cause microinjection problems (see below). This is a critical factor for getting good speckle images.</li>
<li>Prior to long term storage, the labeled actin solution should be clarified by spinning at 75,000 - 80,000xg for 20 minutes at 4 degrees Celcius. Labeled actin should be stored in G-buffer solution and kept -80 degrees Celcius. Preparing 2 uL aliquots is highly recommended to reduce the detrimental effects of repeated freezing-thawing (leads to insoluble aggregates). Refrain from exposing the labeled actin solution to direct light (will photobleaching of fluorophores). We recommend using opaque microcentrifuge tubes for long term storage.</li>
</ol>
Section 2: Preparation of Cells and Microinjection of labeled actin
Materials required: microinjection needles, microsyringe, microloaders, prewarmed cell media, injection buffer, bucket of ice, microscope (phase ring, phase contrast objective 40X, transjector, needle puller, micromanipulator)
<ol>
<li>Many cell types are amenable to FSM, but getting good speckle images is directly dependent on the success of the microinjection procedure. When selecting cells for FSM, cells should be large ( &gt; 50 um in diameter), should spread when plated, and be naturally adherent to substrates (or plastic tissue culture dishes). It also helps if the cell&rsquo;s cytosol occupies an area greater than its nucleus. The aforementioned criteria ensure that cells are amenable to microinjection. Successful candidate cell lines include (but not limited to) PtK1[2,3], newt lung cells[4], and Aplysia neurons[3]. </li>
<li>Cells should be plated on square glass coverslips (22 x 22 mm; no. 1-1/2) that have been acid-washed. Standard substrate coating can be applied to the coverslip and will not affect microinjection.</li>
<li>The correct selection of microinjection needles is dependent on the size of the tip opening. Typically, hole sizes can very in size from 0.5 um to as large as 3 um. However, we recommend that injection of actin requires an optimal opening size between 0.5 to 1 um. If clogging problems are encountered consistently, larger hole sizes can accommodate for the delivery of monomeric as well as unwanted polymerized actin filaments. Microinjection needles can be purchased through several vendors including Eppendorf systems which make femtotip microinjection needles. Alternatively if a commercial needle puller is available, glass microinjection tubes can purchased from Sutter, and pulled to specification. For beginners, we recommend having 10-20 pulled needles on hand before starting the injection procedure.</li>
<li>Ideal &lsquo;injection concentration&rsquo; (final concentration of actin loaded in your needle) of actin is between 0.5 to 1.0 ug/ul based on a 40% labeling ratio of dye to actin monomer. Lower labeling ratio of your actin can be compensated by injecting at higher concentrations (1-3 ug/ul). However, the clogging frequency of your needle will be increased. This can be partially alleviated by using needles with larger openings. Stock concentration of actin can be diluted by adding ice-cold injection buffer. Ice-cold ddH2O can also be used to dilute for quick injections. At this point all working solutions should be kept on ice. </li>
<li>Load 0.5 to 1.0 uL of your actin solution using a microsyringe (narrow-gauge &ndash; Hamilton), or alternatively by using a microloader pipette tip. </li>
<li>Carefully dispense the actin solution, by gently releasing the solution. Bubbles should be avoided, but can be easily removed by taking up excess liquid surrounding the bubble. </li>
<li>Ensure that the actin solution is resting completely at the needle tip. Excess solution anywhere along the capillary of the needle can disrupt the injection flow. </li>
<li>Now, carefully attach the needle, avoiding contact with the needle tip, to the injector holder. Position the holder so that the needle makes a 35-50 degree angle to the base of the microscope stage. Lower angles are preferred.</li>
<li>Lower the tip of the needle until it barely breaks the surface of the media bathing your cells. </li>
<li>Now position the needle tip (without lowering) so that it rests directly above the center of the objective. If the needle tip is aligned with the objective, you should see a bright halo through the eyepiece. This is the end of the needle tip. Slowly moving the needle side to side (using the manipulator) may help better locate the halo. </li>
<li>Adjust the z-focus on the microscope until your cells are in clear view. Now, slowly lower the tip until the halo gradually constricts until finally converging into the needle tip &ndash; you will need to control the focus (z-position) simultaneously. An additional feature to look for is the shaft of the needle, which will cast linear shadows. Lowering the needle will gradually define the needle shaft. When done correctly the end of the needle and your cells should both be in clear view. Slight adjustment of the phase ring may be required to improve contrast. </li>
<li>When navigating, ensure the needle tip is well above your cells to prevent breaking the needle tip. </li>
<li>When selecting cells to inject, cells with free edges should be facing the needle tip. This will ensure declining slope of the cell (from thickest point, where the nucleus resides, to the thinnest point the leading edge) will meet the needle tip, rather than running parallel to it. </li>
<li>The needle pressure should be set at 0.3 to 0.8 psi. The constant pressure applied will result in a steady flow of the actin solution. </li>
<li>After deciding on a cell to inject, position your needle so the tip is next to the nucleus (perinuclear region, thickest part of the cell where the nucleus is not directly below). </li>
<li>Slowly lower, and readjust the position of the needle until the tip is depressing the membrane, now incrementally lower until the needle tip has pierced the membrane. If the needle has fully pierced the membrane, the cell will appear brighter, as soon as you see this difference raise the needle until it is no longer touching the cell. The actual piercing and injecting process will take 0.5 to 2 seconds. </li>
<li>When done correctly, the cell shape will appear unchanged, whereas too much injection will result in quick retraction of the cell edge and in some cases you will actually see the cell explode. </li>
<li>If you do not see changes in the cell when microinjecting (does not get brighter), the needle tip may be closed. You can test this by pressing the &lsquo;clean&rsquo; button if available on your transjector. If open, this will significantly increase the constant pressure flow which can be seen through the eyepieces &ndash; it will appear as ripples and nearby debris can be seen dispersing. </li>
<li>If the needle tip is closed, you can choose to insert a new microinjection needle or attempt to break the tip of the needle by gently lowering the needle until the tip of the needle presses against the glass coverslip, thus causing the very end of the tip to break. The breakage area should be no more than a 1/5th of the area of the needle point (the portion of the needle that starts to converge to a point). </li>
<li>Continue with the injection, spending no more than 20 &ndash; 45 minutes for each microinjection session (will depend on cell type). As a general rule, shorter sessions are recommended to minimize stress on cells.</li>
<li>After finishing the microinjection, change the cell media in the dish by adding fresh prewarmed media. Then allow cells to recover for 30 minutes in an incubator before imaging. This will also allow for the actin to integrate into the existing cytoskeleton network. </li>
</ol>
Section 3: Assembling the Imaging Chamber
Materials Required: coverglass, double sided tape, q-tips, forceps, valap, kim wipe, oxyrase, imaging media, razor blade, p200 pipette, pure ethanol
<ol>
<li>Begin by prewarming the imaging media and thawing a 15 uL aliquot of Oxyrase. Make sure to protect the photosensitive oxyrase from light. The addition of oxyrase will greatly reduce the effects of photobleaching. </li>
<li>Prewarm the valap, by setting the hot plate temperature to low. Do not overheat/burn the valap for it will lose its efficacy as a sealant. </li>
<li>Wipe down a coverglass with a kimwipe doused with pure ethanol to clean and remove small debris. </li>
<li>Stack two equal sized lengths (2.5 cm long) of double-sided tape on top of each other, using a clean flat surface as a platform. Press out any trapped air bubbles to create a completely flat plane.</li>
<li>Using a razor blade, cut two straight strips along the long axis, so that each strip measures 0.5 cm in width. </li>
<li>Using forceps, place each strip against the long edge of your coverglass so that the two strips of tape creates a gap in the center of your coverglass. Press gently on the tape to make a nice seal with the glass. The strips of tape will act as spacers between the coverglass and the coverslip.</li>
<li>Make sure the valap has completely liquefied before you begin the next step. </li>
<li>Retrieve your prewarmed imaging media, adding 15 ul of oxyrase to every 500 uL of media.</li>
<li>Fold kimwipes into triangles to create 3 hard edges. This will be used to create a dry surface for the tape to stick on. </li>
<li>Retrieve your cells from the incubator. Using forceps, grab a corner of the coverslip. Quickly place the coverslip on a kimwipe (cell side is facing up, not touching the tissue), and use the folded kimwipe to wipe two opposing sides of the coverslip to create a semi-dry surface (on the surface where your cells rest). </li>
<li>Using forceps, grab a corner the coverslip and place it on your coverglass so that the dry surfaces are aligned to the two strips of tape. Notice now you have two closed sides and two open slits. Slowly pipette 200 uL of your imaging-media-oxyrase solution close to one opening. Importantly, no air bubbles should be introduced. The solution should be sucked up into the newly assembled chamber by capillary forces, thus fully immersing your cells in solution. </li>
<li>Using a Q-tip dab the four corners of your coverslip with melted valap to quickly affix and stabilize the coverslip. You&rsquo;ll notice that the valap dries instantly (within seconds at room temperature). </li>
<li>Now beginning with the open sides (non-tape side), lay a thin strip of valap by using a Q-tip and mimicking a brushing stroke. Repeat for the closed sides. Slowly build up the coat of valap, thus reinforcing the seal by repeating the coating steps. The valap should be confined to the edges, leaving the center of the coverslip clear. </li>
<li>Gently clean the center portion of the coverslip by using a Q-tip with ddH2O to remove any residual media, being mindful not to crush the cells. Repeat cleaning step with pure ethanol. </li>
<li>You will now have a fully enclosed imaging chamber optimized for speckle imaging. Having a fully sealed enclosure will prevent your fluorophores from quickly photobleaching.</li>
</ol>
Section 4: Imaging cells
Materials required: inverted widefield microscope, mecury lamp, appropriate filters, cooled CCD camera with 6.7 micron pixels, High numerical aperture, oil-immersion plan-Apochromatic objectives (60X to 100X, phase or DIC). Vibration table. Imaging software.
<ol>
<li>Prewarm the microscope stage to 37 degrees. Place your newly completed imaging chamber, and wait 10-15 minutes for the temperature to stabilize before imaging. </li>
<li>Start by focusing your cells in Bright-field, and then find your injected cells by toggling the correct fluorescence settings. Try to minimize the time of fluorescence exposure to reduce photobleaching.</li>
<li>Look for injected cells that have healthy curved leading edges. Over-injected cells (exploded cells) will be characterized by retracted and jagged edges, resembling numerous filopodia &ndash; avoid imaging these cells. </li>
<li>Acquisition settings will depend on the cell type, the quality of the labeled actin, and microscope components. However, exposure settings should not be greater than 2 seconds, and time interval between frames should be set between 5 and 10 seconds. Typical lengths for a time series (per imaged cell) can vary anywhere between 10 to 30 minutes, and is limited by photobleaching effects. Gain settings on the camera can be turned up to increase the signal to noise.</li>
<li>Ideal speckle characteristics should have each speckle spaced by ~2 speckle diameters to the next neighboring speckle. This will warrant optimal spatial and temporal sampling of the actin structure. For epithelial cells, the speckled actin network will appear homogeneous, with speckles evenly spread apart. Cell lines that feature dense stress fibers or lamellipodia will be distinguishable, but will have a dotted/speckled appearance. Exposure settings should be adjusted to get the best speckled images. </li>
<li>Over-microinjected cells will have densely packed speckles that do not appear to be discrete. Stress fibers and the lamellipodia will lose its dotted appearance.</li>
<li>Under-microinjected cells will appear very dim through the eyepiece, containing speckles that are far apart (&gt;10 speckles apart) and appearing randomly scattered. Stress fibers and the lamellipodia in these cells will be indistinguishable.</li>
<li>Key to obtaining &lsquo;good&rsquo; speckle movies is maintaining focus. This is particularly important for post-analysis involving speckle flow and turnover measurements. Thus the quality of the quantitative analysis will depend on locking in the focus for the duration of the time-lapse. This can be particularly challenging if the imaging plane is quite thin. Other factors will depend on the stability of the microscope system, particularly the stage housing and the imaging chamber. Contrast-based focusing can be used if image interval lengths are sufficient to accommodate this feature. Other options include purchasing a near-infrared based focusing system that provides reliable real-time focus adjustments. You can find these offerings from Nikon, Olympus and ASI. </li>
</ol>
Section 5: qFSM analysis
This section is not discussed in detail, but the information on the analysis software can be found here [5]. Software download requests can be made on our website (lccb.scripps.edu). Topics Covered:
<ul>
<li>Noise calibration</li>
<li>Speckle detection</li>
<li>Tracking using a hybrid approach of image correlation-based tracking of the coarse motion of speckle areas and single particle tracking of the detailed motion of individual speckles.</li>
<li>Computation of the polymer assembly and disassembly rates from the intensity fluctuations during speckle appearance and disappearance events. </li>
</ul>

<ol><li>Waterman-Storer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+speckle+microscopy+%28FSM%29+of+microtubules+and+actin+in+living+cells%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells.</a> Curr Protoc Cell Biol. Chapter 4, Unit 4.10-Unit 4.10 (2002).</li>
<li>Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+distinct+actin+networks+drive+the+protrusion+of+migrating+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Two distinct actin networks drive the protrusion of migrating cells.</a> Science. 305, 1782-1786 (2004).</li>
<li>Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rho-dependent+contractile+responses+in+the+neuronal+growth+cone+are+independent+of+classical+peripheral+retrograde+actin+flow%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.</a> Neuron. 40, 931-944 (2003).</li>
<li>Salmon, W. C., Adams, M. C., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual-wavelength+fluorescent+speckle+microscopy+reveals+coupling+of+microtubule+and+actin+movements+in+migrating+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.</a> J Cell Biol. 158, 31-37 (2002).</li>
<li>Danuser, G., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+fluorescent+speckle+microscopy+of+cytoskeleton+dynamics%5BTitle%5D)+AND+%22Annu+Rev+Biophys+Biomol+Struct%22%5BJournal%5D)">Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.</a> Annu Rev Biophys Biomol Struct. 35, 361-387 (2006).</li>
</ol>

The authors have nothing to disclose.

Several key factors are crucial for successfully obtaining speckle images, however good speckles begin and end with the quality of your fluorescently labeled actin. A high labeling ratio of dye to actin monomer, set around 0.4 to 0.7 will ensure that speckles will appear bright and discrete, while the labeled protein itself should be soluble and free of aggregates. Equally important is the microinjection procedure. To ensure normal cell homeostasis (and survival), cells need to be injected with a low flow pressure, int...

<table><tbody><tr><td colspan="4">&lt;p&gt;&lt;strong&gt;&lt;em&gt;Injection buffer (IB)&lt;/em&gt;&lt;/strong&gt;&lt;br/&gt; 50 mM potassium glutamate&lt;br/&gt; 0.5 M MgCl2 (&lt;em&gt;APPENDIX 2A&lt;/em&gt;)&lt;br/&gt; Store up to 2 years at &amp;#8722;20&amp;#x00B0; C&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;&lt;/em&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;G-buffer&lt;/em&gt;&lt;/strong&gt;&lt;br/&gt; 2 mM Tris&amp;#8901;Cl, pH 8.0 (&lt;em&gt;APPENDIX 2A&lt;/em&gt;)&lt;br/&gt; 0.2 mM CaCl2 (&lt;em&gt;APPENDIX 2A&lt;/em&gt;)&lt;br/&gt; Add just before use:&lt;br/&gt; 0.2 mM ATP (see recipe for 100 mM)&lt;br/&gt; 0.5 mM 2-mercapt&amp;#8211;thanol&lt;/p&gt;Store up to 1 day at 4&amp;#x00B0; C</td></tr></tbody></table>

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Fluorescent speckle microscopy is used to probe the dynamics of various cytoskeletal proteins in living cells. When used correctly, quantitative measurements of the velocity and turnover kinetics of cytoskeletal proteins can be made. We begin this procedure by micro injecting a small volume of fluorescently labeled acton into living cells.We then assemble the imaging chamber to reduce the effects of photobleaching. First two pieces of double-sided tape are placed on both sides of the micro slide where our cell cover slip will rest. Then imaging media is pipetted into the chamber by dispensing the media near one of the open slits.Lastly, all four sides are sealed with valet. Hi, my name is James Lim and the laboratory of Galden's Deucer in the Department of Cell Biology at the Scripps Research Institute. Today we're gonna be showing you a procedure on fluorescent speckle microscopy.We use this procedure to probe Acton Dynamics in migrating epithelial cells. So let's get started. A key component to acquiring good fluorescent speckle microscopy movies is the proper labeling of acton With the fluoro four of your choice, we recommend using fluoro fours of Alexa and X rodine cin, a middle Esther derivatives that target exposed lysine residues on the surface of Acton.It is important for the labeling ratio to be high around 0.3 to 0.7 dyes per monomer of acton to ensure that speckles will appear bright and discreet prior to long-term storage. Clarify the labeled Acton solution by centrifuging at 75, 000 to 80, 000 times G for 20 minutes at four degrees Celsius. Labeled Acton should be stored in G buffer solution at negative 80 degrees Celsius.Prepared two microliter aliquots to avoid repeated freeze thawing, which can lead to insoluble aggregates. To avoid photo bleaching of floral fours, do not expose the labeled actin solution to direct light for extended periods of time and use opaque micro centrifuge tubes for long-term storage plate. The cells for microinjection on acid washed square glass cover slips.Standard substrate coating can be applied to the cover slip and will not affect microinjection. Store the cells at 37 degrees while the actin is being prepared to select cells that are amenable to microinjection. For FSM, consider these criteria.The cells should be large, greater than 50 micrometers in diameter should spread flat one plated and be naturally adherent to substrates. It is also helpful if the site isol of the cell occupies an area greater than that of its nucleus. Some suitable cell lines include PTK one, newt lung cells and AIA neurons.In this demonstration, we will inject PTK one epithelial cells. The correct selection of microinjection needles depends on the size of the tip opening. Typically, hole sizes can vary from 0.5 micrometers to as large as three micrometers.We recommend an optimal opening size between 0.5 to one micrometer. For the injection of Acton Microinjection needles can either be purchased or made. If a commercial needle puller is available for beginners, we recommend having 10 to 20 pulled needles on hand before starting the injection procedure.The next step is to dilute the labeled acton with ice cold injection buffer. Keep all working solutions on ice. The ideal injection concentration, which is the final concentration of acton loaded in the needle, is between 0.5 to one microgram per microliter.Based on a 40%labeling ratio of D to actin monomer load 0.5 to one microliter of the diluted actin solution. Using a micro syringe needle, carefully dispense the acton solution. Try to avoid bubbles.If there are bubbles, they can be easily removed by taking up excess liquids surrounding the bubble. Ensure that the Acton solution is resting completely at the needle tip. There should not be excess solution anywhere along the capillary of the needle because it can disrupt the injection flow.The loaded needle is now ready to be attached to the injection holder for microinjection. Retrieve your cells from the incubator and place the cover slip on the microscope stage. To begin the microinjection procedure, take the loaded needle to the microscope.Carefully attach the needle avoiding contact with the needle tip to the injector holder. Position the holder so that the needle makes a 35 to 50 degree angle to the base of the microscope stage. Lower the tip of the needle until it barely breaks the surface of the media bathing.Your cells now position the needle tip so that it rests directly above the center of the objective. If the needle tip is aligned with the objective, you should see a bright halo through the eyepiece. This is the end of the needle tip.Slowly moving the needle side to side may help better locate the halo. When navigating ensure the needle tip is well above your cells. To prevent breaking the needle tip, adjust the Z.Focus on the microscope until your cells are in clear view.Then slowly lower the needle tip while controlling the focus until the halo gradually constricts and finally converges into the needle tip. You can also look for the shaft of the needle, which will cast linear shadows. Lowering the needle will gradually define the needle shaft.Slight adjustments of the phase ring may be required to improve contrast both the end of the needle and your cells should now be in clear view. When selecting cells to inject cells with free edges should be facing the needle tip. This will ensure the declining slope of the cell from the thickest point where the nucleus resides to the thinnest point of the leading edge will meet the needle tip rather than run parallel to it.Set the needle pressure at 0.3 to 0.8 PSI. The constant pressure applied will result in a steady flow of the acton solution. When you have decided on a cell to inject, position the needle so that the tip is next to the cell's nucleus slowly, lower and readjust the position of the needle until the tip is depressing.The membrane incrementally lower the needle until its tip has pierced the membrane. If the needle has fully pierced the membrane, the cell will appear brighter. As soon as you see this change, raise the needle until it is no longer touching the cell.The actual piercing and injecting process will take 0.5 to two seconds. When done correctly, the cell shape will appear unchanged. To minimize stress on cells, spend no more than 20 to 45 minutes for each microinjection session.Sometimes you may not see changes in the cell when injecting and the cell does not get brighter. This indicates that the needle tip may be closed. You can test this by pressing the clean button if available on your trans injector.If the needle tip is open, this will significantly increase the constant pressure flow, which can be seen as ripples and the dispersion of nearby debris. If the needle tip is closed, you can either insert a new microinjection needle or attempt to break the tip of the needle by gently lowering the needle until the tip of the needle presses against the glass cover slip. Thus causing the very end of the tip to break.The breakage area should be no more than a one fifth of the area of the needle point the portion of the needle that starts to converge to a point when the microinjection is finished. Exchange the cell media in the dish with fresh prewarm media to remove any fluorescent residue. After changing the media, allow the cells to recover for 30 minutes in a 37 degree incubator before imaging.This will also allow for the actin to integrate into the existing cytoskeleton network while the cells are recovering in the incubator, begin assembling the imaging chamber. First prewarm the imaging media in a 37 degree water bath. Next thaw, A 15 microliter aliquot of oxa erase at room temperature.The addition of RA to the media later will greatly reduce the effects of photobleaching. Keeping the oxa erase wrapped in aluminum foil will ensure that the photo sensitive compound is protected from light. Prewarm the VAP sealant on a hot plate that is set to a low temperature do not overheat or burn the vap, or it will lose its efficacy as a sealant.Wipe down a micro slide with a Kim wipe doused with pure ethanol to clean and remove small debris. Stack two equal sized lengths, 2.5 centimeters long of double-sided tape on top of each other using a clean flat surface such as another micro slide. As a platform, press out any trapped air bubbles to create a completely flat plane.Next, use a razor blade to cut two straight strips along the long axis of the tape so that each strip measures 0.5 centimeters in width. Using forceps, place each strip along the long edges of your micro slide so that the two strips of tape create a gap in the center of your micro slide. Press gently on the tape to make a nice seal with the glass.These strips of tape will act as spacers between the micro slide and the cover slip. Check on the valve to make sure it has completely liquified. Before you begin the next step, retrieve your prewarm imaging media.Add 15 microliters of oxy rays to every 500 microliters of media. Fold a chem wipe into a triangle to create three hard edges. This will be used to wipe the cover slip later.Retrieve the cells from the incubator using forceps. Pick up a corner of the cover slip quickly. Place the cover slip on a kim wipe with the cell side facing up and not touching the tissue, and use the folded Kim wipe to wipe two opposing sides of the cover slip to create a semi-dry surface.Then pick up a corner of the cover slip with forceps and place the cover slip on the micro slide so that the dry surfaces are aligned to the two strips of tape. Notice now you have two closed sides and two open slits. Slowly pipette 200 microliters of your imaging media RA solution close to one opening.It is important to not introduce any air bubbles. The solution should be sucked up into the newly assembled chamber by capillary forces, thus fully immersing your cells in solution. Using a cotton swab dab the four corners of your cover slip with melted vap to quickly affix and stabilize the cover slip.You'll notice that the VAP dries within seconds at room temperature. Then beginning with the closed sides, lay a thin strip of VAP by using the cotton swab and mimicking a brushing stroke. Repeat for the open sides.Slowly build up the coat of vap, reinforcing the seal by repeating the coating steps. The VAP should be confined to the edges leaving the center of the cover slip clear. After the edges of the cover slip are sealed, gently clean the center portion of the cover slip by using a Q-tip with DD H2O to remove any residual media.Be careful not to crush the cells. Repeat this cleaning step with pure ethanol. You now have a fully enclosed imaging chamber optimized for fluorescent speckle imaging.Having a fully sealed enclosure will prevent your floral fours from quickly photobleaching. The cells in the fully enclosed chamber are now ready for imaging. We use an inverted wide field microscope with a mercury lamp, epi illuminator, appropriate filters and oil immersion plan.Apochromatic objectives images are captured with a cooled CCD camera with 6.7 micron pixels and an image acquisition software. Prewarm the microscope stage to 37 degrees. Place the imaging chamber and wait 10 to 15 minutes for the temperature to stabilize before imaging.Once the temperature has stabilized, focus your cells in brightfield and then find your injected cells by toggling the correct fluorescent settings. Try to minimize the time of fluorescence exposure to reduce photobleaching. Look for injected cells that have healthy, curved leading edges.Over injected cells will be characterized by retracted and jagged edges resembling numerous phylo podia. Do not image these cells. Acquisition settings will depend on the cell type, the quality of the labeled acton and microscope components.However, exposure settings should not be greater than two seconds and time interval between frames should be set between five and 10 seconds. Gain settings on the camera can be turned up to increase the signal to noise. Ideal speckle characteristics should have each speckle spaced by approximately two speckle diameters to the next neighboring speckle.For epithelial cells, the speckled actin network will appear homogeneous. With speckles evenly spread apart. Exposure settings should be adjusted to get the best speckled images.The key to obtaining good speckle movies is maintaining focus. This is particularly important for post-analysis involving speckle flow and turnover measurements. Thus, the quality of the quantitative analysis will depend on locking in the focus for the duration of the time lapse.Various strategies for maintaining focus are detailed in the text for this demonstration will be using the Nikon Perfect focus system. Here are various examples of micro injected cells. The left panel shows an under micro injected cell with a low concentration speckle image.The middle panel shows an over micro injected cell with a too high concentration speckle image, and the right panel shows an optimally injected cell. For FSM with a good speckle image. We are just shown you how to micro inject fluorescently labeled acton into epithelial cells for fluorescent speckle microscopy.When doing this procedure, it's extremely important to remember that the quality of your labeled acton, as well as the microinjection technique, will dictate the overall quality of your speckle images. So that's it and good luck with your experiments.

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Research

JoVE Journal

Biology

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

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.

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

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on Förster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

Here, we present methods for preparing quasi-two-dimensional (2D) entangled, cross-linked, and liquid crystal actin assemblies from purified proteins.

Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

Learn fluorescent speckle microscopy to study F-actin dynamics in living cells using microinjection and specialized imaging chambers. This technique enables quantitative analysis of cytoskeletal protein kinetics.

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

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