This study was supported by the EU structural Fund (ESF/14-BM-A55-0024/18). In addition, H.L. is supported by the FORUN Program of Rostock University Medical Centre (889001 and 889003) and the Josef and K&#228;the Klinz Foundation (T319/29737/2017). C.I.L. is supported by the Clinician Scientist Program of the Rostock University Medical Center. R.D is supported by the DFG (DA1296/6-1), the DAMP foundation, the German Heart Foundation (F/01/12) and the BMBF (VIP+ 00240).
We thank Madeleine Bartsch for her technical support in iPSC cell culture and cardiac differentiation.

All steps in this protocol involving neonatal and adult mice were performed according to the ethical guidelines for animal care of the Rostock University Medical Centre.
1. Cultivation and dissociation of iPSC-derived cardiomyocytes
<ol>
	<li>Differentiate hiPSC-CMs for 25 days using a 2D monolayer method as described previously17.</li>
	<li>Prewarm dissociation medium to 37 &#176;C and support medium to room temperature.</li>
	<li>Wash cells twice with PBS.</li>
	<li>Add prewarmed dissociation medium to cells and incubate for 12 min at 37 &#176;C and 5% CO2.</li>
	<li>Add support medium to the cells. 
	NOTE: The volume of added support medium needs to be twice the volume of dissociation medium used in step 1.4.</li>
	<li>Dislodge the cells using a 5 mL serological pipette.</li>
	<li>Centrifuge cells at 200 x g for 5 min and resuspend the pellet in 3 mL of hiPSC-CM culture medium17.</li>
	<li>Seed cells in 8 well glass bottom chambers at a cell density of 75,000 cells/well and culture for 3 days.</li>
</ol>
2. Adult cardiomyocyte isolation
<ol>
	<li>Isolation and cultivation of adult cardiomyocytes from NMRI mice was performed as reported previously18.</li>
	<li>Seed cells in 8 well glass chamber slide and culture for one day.</li>
</ol>
3. Isolation and cultivation of neonatal cardiomyocytes
<ol>
	<li>Isolation procedure of neonatal cardiomyocytes, obtained from NMRI mice, was performed as described previously19.</li>
	<li>Seed isolated cell in 8 well glass chamber slide at a cell density of 75,000 cells/well and culture for 3 days in neonatal CM culture medium19.</li>
</ol>
4. Immunofluorescence labeling of the &#945;-actinin network
NOTE: For optimal results, cells are cultured in 8 well glass bottom chambers. Labeling should be performed one day before imaging.
<ol>
	<li>Prewarm 4% PFA at 37 &#176;C.</li>
	<li>Fix iPSC-CM by adding 4% paraformaldehyde directly into the culture media (1:1 dilution) and incubate at 37 &#176;C for 15 min. The final concentration of PFA for fixation is 2%.</li>
	<li>Incubate fixed cells in 0.2% Triton-X, diluted in PBS, for 5 min at room temperature.</li>
	<li>Wash cells twice with PBS, 5 min each.</li>
	<li>Add 1% BSA solution (diluted in PBS) and incubate for 60 min at room temperature.</li>
	<li>Prepare 150 &#181;L of primary antibody solution by diluting &#945;-actinin antibody 1:100 in 1% BSA, containing 0.05% Triton-X. Add to cells and incubate at room temperature for 60 min.</li>
	<li>Wash cells twice with 0.2% BSA solution, 5 min each.</li>
	<li>Prepare 150 &#181;L of secondary antibody solution by diluting goat-anti-mouse Alexa647 antibody 1:100 in 1% BSA, containing 0.05% Triton-X. Add to the cells and incubate at room temperature for 40 min.</li>
	<li>Wash cells twice with 0.2% BSA solution, 5 min each.</li>
	<li>Wash cells twice with PBS, 5 min each. Keep labeled cells in the dark at 4 &#176;C until PALM imaging.</li>
</ol>
5. Preparation of the PALM imaging buffer
NOTE: It is critical to freshly prepare the PALM imaging buffer for each experiment.
<ol>
	<li>Prepare 50% glucose solution by dissolving 25 g glucose in 50 mL of distilled water.</li>
	<li>Prepare basic buffer containing 50 mM Tris-HCl, 10% glucose and 10 mM sodium chloride.
	<ol>
		<li>Adjust the pH level to ~8.0 using hydrochloric acid.</li>
	</ol>
	</li>
	<li>Prepare pyranose oxidase solution by dissolving 0.6 mg pyranose oxidase in 316 &#181;L of basic buffer.</li>
	<li>Prepare catalase solution by dissolving 7 mg of catalase in 500 &#181;L of basic buffer. Mix thoroughly and centrifuge at 10,000 x g for 3 min. Keep the supernatant for further use. Catalase solution can be kept at 4 &#176;C for several days.</li>
	<li>Prepare 500 &#181;L of PALM imaging buffer by mixing 316 &#181;L of pyranose oxidase solution, 25 &#181;L of catalase solution, 100 &#181;L of 50% glucose solution, 50 &#181;L of cysteamine, 5 &#181;L of cyclooctatetraene and 3.5 &#181;L of &#946;-Mercaptoethanol. The final catalytic activity of pyranose oxidase and catalase need to be 7.5 U, 35,00U respectively. 
	NOTE: The PALM imaging buffer provides optimal imaging conditions for 3-5 h. If blinking capability of the fluorescent dye decreases, prepare a new buffer aliquot.</li>
</ol>
6. PALM image acquisition
<ol>
	<li>Switch on the microscope at least 3 h before use and bring the sample to room temperature before imaging to allow thermal equilibration. If the microscope is equipped with an incubation chamber, adjust temperature to 30 &#176;C.</li>
	<li>Clean the objective and bottom of the chamber slide using an appropriate cleaning solvent.</li>
	<li>Add 300 &#181;L of PALM imaging buffer into one well of labeled cells and insert the chamber slide into the stage holder of the microscope.</li>
	<li>Set the PALM image acquisition parameters.
	<ol>
		<li>Select 1.57 N.A. 100x oil objective for acquisition.</li>
		<li>Select PALM mode and activate the TIRF settings.</li>
		<li>Adjust the number of frames to be acquired. Usually 5, 000-10, 000 frames are sufficient to obtain optimal results. However, the number of acquired frames strongly depends on the labeling efficiency and the blinking capability of the fluorescent dye and may be adjusted by the user.</li>
		<li>Set UV laser power to 0.1% and 647 laser to 0.2%.</li>
		<li>Set the gain level to 50-100.</li>
		<li>Switch on the laser illumination and select a target cell. The gain level can be increased if signal intensity is to low (depending on fluorescence labeling).</li>
		<li>Switch off the laser illumination</li>
	</ol>
	</li>
	<li>PALM image acquisition
	<ol>
		<li>Reduce gain to 0 and increase the laser power (ex 647) to 100%.</li>
		<li>Bleach the target cell for ~5 s.</li>
		<li>Increase gain to 50 and start PALM image acquisition. 
		NOTE: Gain needs to be adjusted to get sufficient signal intensity while oversaturated pixels should be avoided. If signal intensity reduces, increase the gain level.</li>
	</ol>
	</li>
	<li>Optionally, steadily enhance the UV laser power (0.1%-10%) to increase signal intensity and to promote blinking of the fluorophore.</li>
</ol>
7. Reconstruction of PALM data
<ol>
	<li>Open the Image J software and import PALM data.</li>
	<li>Open Thunderstorm Plugin and &#8220;Run analysis&#8221;
	<ol>
		<li>In the &#8220;Camera setup&#8221; menu enter pixel size and EM gain. 
		NOTE: When using 100x objectives and 1.6x magnification lens, 100 nm pixel size is appropriate. However, as the pixel size depends on the hardware features of the microscope and camera used for PALM imaging, users need to carefully check and adapt this parameter. EM gain values can be obtained from the metadata.</li>
		<li>In the &#8220;Run analysis menu&#8221; set parameters as follows: B-spline order: 3, B-spline scale: 2.0, peak intensity threshold: stf(Wave.F1), fitting radius: 3, initial sigma: 1.6, magnification: 5.0, update frequency: 50, lateral shifts: 2. Confirm by clicking the &#8220;Ok&#8221; button.</li>
	</ol>
	</li>
	<li>Post processing of reconstructed PALM image
	<ol>
		<li>In the &#8220;Plot histogram&#8221; menu select the &#8220;Sigma&#8221; parameter.</li>
		<li>Use the &#8220;Rectangle&#8221; tool to select a ROI, excluding possible artefacts and apply ROI to the filter. ROI values will appear in the filter command box.</li>
		<li>Add &#8220;&#38; uncertainty &#60;25&#8221; to the ROI values. A possible filter command will look like this: &#8220;(sigma &#62; 48.6821 &#38; sigma &#60; 1117.40) &#38; uncertainty &#60;25&#8221;. Apply selected sigma values.</li>
		<li>In the &#8220;Remove duplicates&#8221; menu, enter a distance threshold of &#8220;10 nm&#8221; and apply.</li>
		<li>In the &#8220;Merging menu&#8221;, set maximum distance to &#8220;20&#8221;, maximum frames per molecule to &#8220;0&#8221; and maximum off frames to &#8220;1&#8221;. Apply settings.</li>
		<li>In the &#8220;Drift correction menu&#8221;, select cross correlation and set &#8220;Number of bins&#8221; to &#8220;5&#8221; and &#8220;Magnification&#8221; to &#8220;5.0&#8221;. Apply drift correction settings.</li>
		<li>Save the final PALM image and export post processed data if desired.</li>
	</ol>
	</li>
</ol>
8. Analysis of sarcomere filaments
<ol>
	<li>Analysis of sarcomere length
	<ol>
		<li>Open Image J software and import the reconstructed PALM image.</li>
		<li>Draw a line between selected sarcomere structures perpendicular to the z-disc to measure the shortest distance between actinin filaments.</li>
		<li>Select &#8220;Plot profile&#8221; in the &#8220;Analyze&#8221; menu and acquire the length between two peaks. As sarcomere length may vary within one cell, a minimum of 20 sarcomeres should be measured in different areas of the target cell.</li>
	</ol>
	</li>
	<li>Analysis of z-Disc thickness
	<ol>
		<li>Open Image J software and import the reconstructed PALM image.</li>
		<li>Convert the reconstructed PALM image into an 8-bit mode image.</li>
		<li>Open the ridge detection plugin and enter the following parameters: line width: 20, high contrast: 230, low contrast: 10, sigma: 0.79, lower threshold: 25.84, minimum line length: 20.</li>
		<li>Set &#8220;Estimate width&#8221;, &#8220;Extend line&#8221; and &#8220;Display results&#8221;.</li>
		<li>Click &#8220;Ok&#8221; and use &#8220;Mean line width&#8221; from results table for further analyses.</li>
	</ol>
	</li>
</ol>

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B., Sainlos, M., Thoumine, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+organization+of+synaptic+adhesion+proteins+revealed+by+single-molecule+localization+microscopy.">Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy.</a> Neurophotonics. 3 (4), 041810 (2016).</li><li>Ma, H., Fu, R., Xu, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+cost-effective+setup+for+super-resolution+localization+microscopy.">A simple and cost-effective setup for super-resolution localization microscopy.</a> Scientific Reports. 7 (1), 1-9 (2017).</li><li>Olivier, N., Keller, D., G&#246;nczy, P., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+Doubling+in+3D-STORM+Imaging+through+Improved+Buffers.">Resolution Doubling in 3D-STORM Imaging through Improved Buffers.</a> PLoS One. 8 (7), 0069004 (2013).</li><li>Swoboda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+oxygen+scavenging+for+photostability+without+ph+drop+in+single-molecule+experiments.">Enzymatic oxygen scavenging for photostability without ph drop in single-molecule experiments.</a> ACS Nano. 6 (7), 6364-6369 (2012).</li><li>Mikhaylova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+bundled+microtubules+using+anti-tubulin+nanobodies.">Resolving bundled microtubules using anti-tubulin nanobodies.</a> Nature Communications. 6 (1), 1-7 (2015).</li><li>Venkataramani, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+labeling+density+and+whole-cell+3D+dSTORM+imaging+by+repetitive+labeling+of+target+proteins.">Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.</a> Scientific Reports. 8 (1), 1-7 (2018).</li><li>Erd&#233;lyi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+compensation+of+imaging+artefacts+in+localization-based+super-resolution+microscopy.">Origin and compensation of imaging artefacts in localization-based super-resolution microscopy.</a> Methods. 88, 122-132 (2015).</li><li>McGorty, R., Schnitzbauer, J., Zhang, W., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+depth-dependent+aberrations+in+3D+single-molecule+localization+and+super-resolution+microscopy.">Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy.</a> Optics Letters. 39 (2), 275 (2014).</li><li>Ribeiro, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cardiomyocyte+show+of+force:+A+fluorescent+alpha-actinin+reporter+line+sheds+light+on+human+cardiomyocyte+contractility+versus+substrate+stiffness.">A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.</a> Journal of Molecular and Cellular Cardiology. 141, 54-64 (2020).</li><li>Pioner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+mechanical+measurements+of+myofibrils+from+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Isolation and mechanical measurements of myofibrils from human induced pluripotent stem cell-derived cardiomyocytes.</a> Stem Cell Reports. 6 (6), 885-896 (2016).</li></ol>

The authors declare no conflict of interest.

The generation of functional iPSC-derived CM in vitro is important for regenerative therapies, disease modeling and the development of drug-screening platforms. However, insufficient maturity of these CM is a major obstacle in cardiovascular research20. In this regard, high resolution imaging techniques are needed that enable monitoring of the structural maturation state of iPSC-derived CM. At the same time, super resolution microscopy can be a valuable tool to precisely analyze the function of sp...

Cardiovascular diseases (CVD) such as myocardial infarction or cardiomyopathy remain the major cause of death in the western world1. As the human heart possesses only poor regenerative capacity, there is a need for strategies to promote the recovery from CVDs. This includes cell replacement therapies to replenish lost cardiomyocytes (CM), as well as the development of new anti-arrhythmic drugs for efficient and safe pharmaceutical intervention. Induced pluripotent stem cell (iPSC) have been shown to be a promising cell source for the unlimited generation of human CM in vitro, suitable for regenerative therapies, disease modeling, and for the development of drug screening assays2,3,4.
Although many different cardiac differentiation protocols exist, iPSC-derived CM still lack certain phenotypical and functional aspects that impede the in vitro and in vivo application5,6. Beside electrophysiologic, metabolic, and molecular changes, the cardiac maturation process involves the structural organization of sarcomeres, which are the fundamental units required for force generation and cell contraction7. While adult CMs exhibit a well-organized contractile apparatus, iPSC-derived CMs commonly demonstrate disarranged sarcomere filaments, associated with a reduced contraction ability and altered contraction dynamics8,9. In contrast to mature CM that show uniaxial contraction pattern, the disoriented structures in immature CM results in a radial contraction of the whole cell or promote the appearance of contraction focal points9,10.
For improving cardiac maturation, multiple approaches have been applied, including 3D cell culture methods, electrical and mechanical stimulation, as well as the use of extracellular matrices mimicking in vivo conditions11,12,13. To evaluate the success and efficiency of these different culture conditions, techniques are needed to monitor and estimate the degree of the structural maturation of iPSC CM, e.g., by microscopic techniques. In contrast to conventional confocal imaging, the resolution in case of photoactivated localization microscopy (PALM) is approximately 10x higher. This technique in turn allows for a more accurate analysis, detecting even subtle alterations of cellular structures14. Considering the high resolution of PALM-based imaging, the overall goal of this method was the microscopic evaluation of sarcomere maturity in iPSC-derived CMs by precise determination of z-Disc thickness and sarcomere length. In previous studies, these structural features have been shown to be appropriate parameters to assess cardiac maturity15. For example, diseased iPSC-CM lacking full length dystrophin exhibit reduced sarcomere length and z-band width when compared to wild type cells16. Likewise, the length of individual sarcomeres was measured to investigate the impact of topographic cues on cardiac development16. Hence, we applied this approach to evaluate the structural maturation of the sarcomere network in iPSC-CM by quantitatively measuring the sarcomere length and z-disc thickness.

<table>
	<tbody>
		<tr>
			<td>human iPSC cell line</td>
			<td>Takara</td>
			<td>Y00325</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well Glass Bottom</td>
			<td>ibidi</td>
			<td>80827</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5ml eppendorf tube</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiomyocyte Dissociation Kit</td>
			<td>Stem Cell Technologies</td>
			<td>05025</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma Aldrich</td>
			<td>C40-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctatetraene</td>
			<td>Sigma Aldrich</td>
			<td>138924-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteamine</td>
			<td>Sigma Aldrich</td>
			<td>30070-10g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Ca2+ and Mg2+</td>
			<td>Thermo Fisher</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2-Goat anti-Mouse IgG Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21237</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image processing software (Image J)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Carl Roth</td>
			<td>X997.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 780 ELYRA PS.1 system</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>8187150100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyranose oxidase</td>
			<td>Sigma Aldrich</td>
			<td>P4234-250UN</td>
			<td></td>
		</tr>
		<tr>
			<td>sarcomeric &#945;-actinin antibody [EA-53]</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile water</td>
			<td>Carl Roth</td>
			<td>3255.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the &alpha;-actinin network in human ipsc-derived cardiomyocytes using single molecule localization microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

For estimating the degree of structural maturation of CM, neonatal, fully mature adult, and iPSC CM were initially labeled the CM with &#945;-actinin antibody to visualize the sarcomere network. Following PALM acquisition, images were reconstructed, and z-disc thickness was measured using plugin-based image processing software for the automatic detection of the width of individual filaments. Sarcomere length was calculated by measuring the distance between two adjacent intensity peaks, corresponding to neighboring filame...

Watch this Scientific Journal Video about Analyzing the ÃŽÂ±-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy at JoVE.com

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

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6.6K Views.  Rostock University Medical Center. The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Video: Analyzing the α-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.