We thank the members of the Light Microscopy Facility, Max-Planck Institute of Experimental Medicine, for their expert advice and support.

1. Seeding of HeLa Cells
<ol>
	<li>Prepare 2 mL of 5 &#956;g/mL fibronectin solution in phosphate buffered saline (PBS) and add 450 &#956;L of it into each well of a 4 well chambered coverslip (#1.5). Incubate the slide for 15 min at 37 &#176;C and 5% CO2.</li>
	<li>Rinse asynchronously growing HeLa cells with Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) and incubate with trypsin-EDTA (0.05%: 0.02%; w:v) for 5 min at 37 &#176;C. Stop the enzymatic reaction by the addition of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) at 3:1 (v:v) ratio of added trypsin-EDTA. 
	NOTE: HeLa cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS at 37 &#176;C and 5% CO2 and were routinely passaged once they reached 80&#8211;90% confluency as described above.</li>
	<li>Determine the cell concentration using a Neubauer chamber. Mix a 50 &#956;L aliquot of the cell suspension with trypan blue at 1:1 (v:v) ratio, resuspend, and transfer 10 &#956;L of the suspension into the chamber. Count only the trypan blue-negative cells inside of the four large squares (for details see Phelan et al.47). Derive the cell concentration from the counted cell number using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/60478/60478eq1.jpg" /></li>
	<li>Pellet the cells by centrifugation at 300 x g for 2 min. Resuspend with fresh RPMI 1640 in order to obtain 1 x 106 cells/mL.</li>
	<li>Remove the fibronectin from the chambered coverslip, wash the wells twice with DPBS, and seed 50,000 cells per well.</li>
	<li>Return the chambered coverslip with the cells to the incubator and grow them for 24 h at 37 &#176;C and 5% CO2.</li>
</ol>
2. Expression of pEB3-tdTomato in HeLa Cells
<ol>
	<li>Prepare a 1.5 mL microcentrifuge tube. For each tube, dilute 2 &#956;g of pEB3-tdTomato48 with transfection buffer (synthetic product in aqueous solution) to a final volume of 396 &#956;L.</li>
	<li>Add 4 &#956;L of transfection reagent (non-lipidic, containing polyethylenimine) to the first tube, and vortex the mixture immediately for exactly 10 s.</li>
	<li>Briefly spin down the tube with a microcentrifuge and incubate at room temperature (RT) for 10 min.</li>
	<li>Remove the HeLa cells from the incubator. Dropwise, add 100 &#956;L of the transfection mixture to each well of a 4 well chambered coverslip, and return the cells to the incubator.</li>
	<li>After 4 h of incubation at 37 &#176;C and 5% CO2, supplement the cells with fresh growth medium and incubate for at least 24 h at 37 &#176;C and 5% CO2. 
	NOTE: It is necessary to optimize transfection conditions for each cell type. The expression levels need to be low enough to allow the identification of single MT growing ends. Alternatively, a cell line stably expressing EB3-tdTomato can be used in the experiments; this would reduce variability in expression levels of EB3-tdTomato between preparations and between cells from the same preparation49.</li>
</ol>
3. Synchronization and Live-cell Imaging of pEB3-tdTomato&#8211;expressing HeLa Cells
<ol>
	<li>Prepare a 2.5 &#956;M solution of dimethylenastron (DME) in phenol-red free Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FCS and 2 mM L-glutamine or an alternative glutamine supply.</li>
	<li>Replace the growth medium in the chambered coverslip with 500 &#956;L of the growth medium containing 2.5 &#956;M DME and incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>After 3.5 h of incubation with DME, transfer the cells to the microscope, mount the chambered coverslip into an environmental chamber with dark panels for imaging at 37 &#176;C and 5% CO2, and further incubate until the total incubation time is 4 h. 
	NOTE: The maintenance of temperature at 37 &#176;C without fluctuation is crucial for the experiment.</li>
	<li>Perform the time-lapse imaging on an inverted microscope equipped with a 100x 1.49 N.A. oil immersion objective, a dual spinning disk confocal system, and a reliable autofocus system for continuous maintenance of the focal plane. Define the imaging parameters as follows. 
	NOTE: We use an Electron Multiplying Charge-Coupled Device camera (EM-CCD).
	<ol>
		<li>For EB3-tdTomato excitation, use a 561 nm laser line with 200 ms exposure time. Collect the emitted light through a quadruple bandpass (405, 488, 561, 640 nm) dichroic mirror and a 600/52 nm emission filter. 
		NOTE: Laser power can be adjusted for each imaged cell to prevent image saturation. In all time-lapse movies given here the laser power was set to 5.3 mW.</li>
		<li>Find a cell in prophase and focus in the Z-plane corresponding to the center of the monopolar mitotic spindle. Acquire images every 0.5 s over a total of 1 min with no binning and no illumination between the exposures.</li>
	</ol>
	</li>
</ol>
4. Analysis of the MT Dynamics Using U-Track v2.2.0
<ol>
	<li>To analyze the MT dynamics a numerical computing environment software is required (e.g., MATLAB). 
	NOTE: Basic understanding of the software is sufficient for the analysis. Comprehensive help material and tutorials are available on the developer&#8217;s website (https://uk.mathworks.com/products/&#173;matlab/&#173;getting-started.html).</li>
	<li>Download (https://github.com/DanuserLab/u-track) and install the open-source U-Track v2.2.0 software following the detailed instructions given in the &#34;Readme_u-track.pdf&#34; file50,51,52.</li>
	<li>Launch the numerical-analysis software and add U-Track v2.2.0 folder with subfolders into the software search path.</li>
	<li>From the command window call &#34;movieSelectorGUI&#34;. This opens a dialogue window from which the raw files generated by the image acquisition software at the microscope can be imported (Supplementary Figure 1, Figure 2, Figure 3, Figure 4). 
	NOTE: The U-Track software is compatible with other image data formats. It uses Bio-Formats, which recognizes different life science data formats53.</li>
	<li>The size of each image is read from the metadata automatically. Manually enter the numerical aperture of the objective (in this case 1.49) and the time interval (0.5 s) used for imaging (Supplementary Figure 1B). Additionally, information on the excitation wavelength, the fluorophore, and the exposure time can also be provided, but they are not critical for further analysis.</li>
	<li>Once all the images are loaded, save the entered time-lapse series as a movie list by selecting the &#34;Save As Movie List&#34;. On the right side of the dialogue window select the &#34;U-Track&#34; option and press &#34;Continue&#34; (Supplementary Figure 1C). 
	NOTE: The values are optimized for HeLa cells. If switching to a different cell line, the values should be defined again. Alternatively, use the settings recommended by the software developers. The detailed explanation of each of the parameters and how they should be defined can be found in the technical report provided with the previous version of the software, plusTipTracker50.</li>
	<li>From the pop-up window select &#34;Microtubule Plus-Ends&#34; and press &#34;Ok&#34; (Supplementary Figure 1C). The new dialogue window allows determining the parameters for the three steps of the analysis (Supplementary Figure 1D), which are detection, tracking, and track analysis.</li>
	<li>In step 1 choose &#34;Settings&#34; and from a drop-down menu select &#34;Comet Detection&#34; as a detection method (Supplementary Figure 2B).
	<ol>
		<li>From the new dialogue window define the parameters for the difference of Gaussians filter and the watershed segmentation as follows (Supplementary Figure 2C): Mask process to be used for the detection = None; Low-pass Gaussian standard deviation = 1 pixel; High-pass Gaussian standard deviation = 3 pixels; Minimum threshold = 3 standard deviations; Threshold step size = 0.25 standard deviations. Select &#34;Apply Settings to All Movies&#34; and &#34;Apply&#34;.</li>
	</ol>
	</li>
	<li>In step 2, the parameters for linking, gap closing, merging and splitting, and Kalman filter functions are defined in three steps as highlighted in pink, green, and blue, accordingly (Supplementary Figure 3B). For these steps, select the &#34;Microtubule Plus-end Dynamics&#34; and from the &#34;Setting&#34; option define the values as indicated in Supplementary Figure 3C&#8211;E, respectively.
	<ol>
		<li>For problems with dimensionality, choose &#34;2&#34; from the drop-down menu. Use Maximum Gap to Close = 5 frames; Minimum Length of Track Segments from First Step = 3 frames. As before, select &#34;Apply Settings to All Movies&#34; and click on &#34;Apply&#34;.</li>
	</ol>
	</li>
	<li>In step 3 of the analysis, the detected MT tracks are classified (Supplementary Figure 4). As a track analysis method, choose &#34;Microtubule Dynamics Classification&#34; and define the parameters through the &#34;Setting&#34; button as indicated in Supplementary Figure 4B,C. After that, choose the &#34;Apply Settings to All Movies&#34; box and click on &#34;Apply&#34;.</li>
	<li>Once all the parameters are defined, from the &#34;Control Panel&#8211;U-Track&#34; window (Supplementary Figure 1D) select the &#34;Apply Check/Uncheck to All Movies&#34; and &#34;Run All Movies&#34; boxes and press &#34;Run&#34;. This will initiate the MT analysis of the time-lapse series.</li>
	<li>Once the movie processing is completed, a message &#34;Your movie(s) have been processed successfully&#34; is displayed. Press &#34;Ok&#34;, then &#34;Save&#34;.</li>
	<li>Now it is safe to quit the numerical-analysis software. The results from the movie processing are stored in subfolder structures as m-files in the folder where the raw files are stored.</li>
</ol>
5. Statistical Analysis of the MT Dynamics
<ol>
	<li>Import the m-files into a preferred statistic analysis program. 
	NOTE: In our case, we first import the files in a standard spreadsheet to make them readable. The m-files contain statistical information (median, mean, and standard deviation) on different parameters (e.g., growth speed, MT dynamicity). The detailed list of the parameters is given in the technical report provided with the previous version of the software, plusTipTracker50,52. The generated m-files can also be imported into other data processing software.</li>
	<li>Choose the &#34;growth speed mean&#34; parameter and import it into a table for statistics and display. Enter the information on other parameters, (e.g., &#34;dynamicity&#34;) either in a new table or in a new column of the same grouped table and plot.</li>
</ol>

The authors have nothing to disclose.

Here, we describe a modification of a method first established by Ertych et al.44. Along with several other modifications, we combine this technique of MT dynamics analysis with dual spinning disk confocal imaging. The use of the dual spinning disk improves the resolution of growing MTs while reducing phototoxicity36. We further reduce the photobleaching and laser light-induced damage of the cells by switching to a longer wavelength fluorescent reporter. The tdTomato fluore...

Microtubules (MTs) are highly dynamic structures found in virtually all eukaryotic cells and in some bacteria1. Together with actin and intermediate filaments, they sculpt the cytoskeleton2,3. Cell division4, molecule transport5, flagellar beating6, the sensation of the surrounding environment through primary cilium7, hearing (kinocilium)8,9, embryogenesis10,11,12, invasion and metastasis13,14, and even memory formation15,16,17,18, and many other processes primarily rely on MTs. Participation of MTs in all these events would be impossible without their remarkable ability to rapidly switch between growth (polymerization) and shrinkage (depolymerization). This property is described as dynamic instability19. MT dynamicity is altered in many pathological conditions20,21,22. Hence, determining the nature of this property can help to understand disease mechanisms and subsequently their treatment.
A long list of methods has been developed for MT dynamics analysis, most of which are based on imaging techniques23. Initially, wide field light microscopes were used for observing the formation of tubulin polymers in vitro24. The discovery of end-binding (EB)-proteins that collect at MT plus-ends and the development of methods to fluorescently label proteins made it possible to observe the behavior of MTs directly in living cells with wide field and confocal fluorescence microscopes25,26,27. One EB-protein is end-binding protein 3 (EB3)28; by overexpressing and tracking EB3 fused to a fluorescent protein, MT plus-end assembly rates can be determined29,30.
Confocal laser scanning fluorescence microscopy (CLSM) is frequently used to follow MT dynamics. However, this imaging technique poses a high risk of phototoxicity and photobleaching, two undesirable processes for live cell and dim sample imaging31. In order to obtain a better signal-to-noise ratio, the laser power and the exposure duration should be high enough while not damaging the samples, and this requires sacrificing resolution in exchange for speed. A suitable alternative to CLSM is spinning disk microscopy32. This imaging modality is based on the use of a Nipkow disk33, which consists of a moving disk bearing an array of pinholes, and works equivalently to many CLS microscopes imaging the same sample simultaneously34. Therefore, the light from the laser will illuminate several regions in the sample simultaneously but retain the confocal nature. The Nipkow disk, therefore, allows obtaining images similar to CLSM but faster and using less laser power. The Nipkow disk was further improved by Yokogawa Electric, which introduced a second disk with an array of microlenses on it that individually direct light into a respective pinhole, further reducing phototoxicity and photobleaching35. Thus, spinning disk laser scanning microscopy became a method of choice for live cell imaging, and it makes it possible to obtain images with high signal-to-noise ratio at a high speed31,36, which is crucial for resolving signals such as those from the fast-growing MT ends.
MT dynamics differ temporarily. For example, the mitotic MTs are more dynamic than the interphase ones37,38. Similarly, differences in the growth rate and shrinkage have been observed even within the same cell cycle phase, such as mitosis39,40. Therefore, to avoid false data collection, the measurement of MT dynamics should be limited to a narrow time-window during the cell cycle. For example, measurement of MT dynamics in prometaphase can be achieved by treating the cells with dimethyl&#173;enastron (DME), a monastrol analogue that inhibits the motor kinesin Eg541 and prevents the formation of the bipolar mitotic spindle42. Inhibition of cells at prometaphase with Eg5 inhibitor DME and other monastrol derivatives does not affect the MT dynamics43,44,45, which makes DME a useful tool for studying MT dynamics both in fixed and live cells44.
Here we combine the method of MT dynamics analysis in prometaphase cells described by Ertych et al.44 with dual spinning disk imaging. This method allows measurement of the MT dynamics in prometaphase cells collected from a single focal plane with a higher imaging rate, yet without photobleaching and minimal phototoxicity. Furthermore, as a fluorescent reporter, we use tandem dimer Tomato fluorescent protein (tdTomato) which has improved brightness and photostability in comparison to the green fluorescent protein (EGFP) and is excited with lower energy light46. Therefore, tdTomato requires less laser power for excitation and is less phototoxic. Altogether, we further improve the method by reducing the phototoxicity and improving the resolution and postprocessing required for the MT dynamics analysis. Additionally, we create a basis for future modifications of the method by combining it with other synchronization techniques.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dimethylenastron</td>
			<td style="width:123px;">Merck</td>
			<td style="width:344px;">324622</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM w/o phenol red</td>
			<td>Gibco</td>
			<td>31053-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum</td>
			<td>Biochrom</td>
			<td>S0415</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fibronectin Bovine Plasma</td>
			<td>Merck</td>
			<td>F4759</td>
			<td>Sterile powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Stable glutamine substitutive</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">jetPRIME</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EB3-TdTomato</td>
			<td>Addgene</td>
			<td>plasmid #50708</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Merck</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin/EDTA solution</td>
			<td>Biochrom</td>
			<td>L2143</td>
			<td>0.05% / o.02 % w/o calcium and magnesium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#181;-slide</td>
			<td>Ibidi</td>
			<td>80426</td>
			<td>4-well slide with #1.5 coverslip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eclipse Ti Inverted microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective</td>
			<td>Nikon</td>
			<td>MRD01991</td>
			<td>CFI Apo TIRF 100XC Oil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ACAL Laser Excahnger</td>
			<td>Nikon</td>
			<td></td>
			<td>Laser box. 405, 458, 488, 514, 561 and 647nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spinning disk module</td>
			<td>Andor</td>
			<td>CSU-W</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Camera</td>
			<td>Andor</td>
			<td>iXon Ultra 888</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Environmental Chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>Dark chamber equipped with CO2 supply, tmeperature control and humidifier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HeLa Cells</td>
			<td>DSMZ</td>
			<td>ACC-57</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NIS Elements v4</td>
			<td>Nikon</td>
			<td></td>
			<td>Spinning disk microscope. Acquisition Software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>Computing environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td>Statistical analysis and display software</td>
		</tr>
	</tbody>
</table>

measurement of microtubule dynamics by spinning disk microscopy in monopolar mitotic spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Following the given protocol outlined in Figure 1A, the pEB3-tdTomato plasmid was transiently expressed in asynchronously growing HeLa cells. The cells were synchronized 48 h after the transfection at prometaphase through DME treatment (Figure 1B). This step ensured that the measurement of MT dynamics was always done at the same phase of the cell cycle. The time-lapse movies were further processed and analyzed with U-Track v2.2.0 as described in its supplementar...

Watch this Scientific Journal Video about Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles at JoVE.com

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

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.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

measurement-microtubule-dynamics-spinning-disk-microscopy-monopolar

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6.1K Views.  Max-Planck Institute of Experimental Medicine. 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.

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Methods Article

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