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

This method can be used for the quantitative evaluation of sarcomere maturation in iPSC-derived cardiomyocytes. Using this super resolution-based approach, it is possible to detect even subtle alterations in sarcomere organization, which is not possible with conventional confocal imaging. At least three hours before use, switch on the microscope and bring the sample to room temperature.

When the microscope is ready, add 300 microliters of PALM imaging buffer to a well of labeled cells and insert the chamber slide into the stage holder of the microscope. Select the 1.57 NA 100X oil objective and use the PALM mode in the imaging software and activate the TIRF settings. Set the number of frames to 5, 000 to 10, 000, the UV laser power to 0.1%and the 647 laser to 0.2%and the gain level to 50 to 100.

When all of the acquisition parameters have been set, switch on the laser illumination and select a target cell. To acquire an image, increase the 647 laser power to 100%and reduce the gain to zero. Bleach the target cell for about five seconds, then increase the gain to 50 and initiate the PALM image acquisition.

For reconstruction of the PALM data, at the end of the acquisition, open the data in ImageJ and open the Thunderstorm plugin. Select run analysis in the plugin and open the camera setup menu. Enter the pixel size and the electromagnetic gain.

In the run analysis menu, set the B-spline order to three, the B-spline scale to two, the peak intensity threshold to stfwave. f1, the fitting radius to three, the initial sigma to 1.6, the magnification to five, the update frequency to 50, and the lateral shifts to two, and click OK.After reconstruction, select sigma in the plot histogram menu and use the rectangle tool to select a region of interest, excluding possible artifacts. Add the region of interest to the filter and add and uncertainty less than 25 to the region of interest values.

In the remove duplicates tab, enter a distance threshold of 10 nanometers. In the merging tab, set the maximum distance to 20, the maximum frames per molecule to zero, and the maximum off frames to one. In the drift correction tab, select cross-correlation and set the number of bins and the magnification to five, then save the final PALM image and export the post-process data.

To analyze the sarcomere length, import the reconstructed PALM image of interest into ImageJ and use the line tool to draw a line between the selected sarcomere structures perpendicular to the Z-disc to measure the shortest distance between the actin filaments. In the analysis menu, select plot profile and acquire the length between the two peaks. To analyze Z-disc thickness, convert the reconstructed PALM image into an eight-bit mode image and open the Ridge Detection plugin.

Set the line width to 20, the high contrast to 230, the low contrast to 10, the sigma to 0.79, the lower threshold to 25.84, and the minimum line length to 20. Select estimate width, extend line, and display results, then click OK and use the mean line width from results table for further analysis. iPSC-derived cardiomyocyte and neonatal cells exhibit a similar alpha actinin pattern with irregular disarranged sarcomere structures.

Quantitative assessment demonstrates that the length and thickness of the alpha actinin filaments are almost identical between the two groups of cells indicating a premature developmental state of iPSC-derived cardiomyocytes. In contrast, adult mature cardiomyocytes exhibit a regular sarcomere network with a slightly increased sarcomere length and reduced Z-disc thickness. A comparison of conventional confocal imaging and PALM reveals no significant difference in sarcomere length.

However, a profound reduced Z-disc thickness is detected when iPSC cardiomyocytes are subjected to PALM imaging. A gain in resolution is observed when PALM is applied, which is supported by the corresponding intensity plots. Notably, sarcomere structures acquired using a low quality buffer appear to be thicker compared to structures captured under optimal imaging conditions.

This lack of data accuracy is due to the reduced blinking properties of the fluorophore, resulting in fewer detected photons per localization event. Moreover, localization precision is decreased, lowering the overall resolution of the reconstructed PALM image. In addition, sample drift can affect the precise localization of the fluorescent molecules resulting in blurry images.

To acquire proper PALM images, it is very important to allow thermal equilibration of the entire imaging system to avoid excessive sample drift during imaging. Following this procedure, other cellular structures such as mitochondria can be imaged by PALM to acquire additional parameters of cardiomyocyte maturation.