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How to probe the function and repair of cells using fs laser-based nanosurgery

10 May | By Biophotonics.World
How to probe the function and repair of cells using fs laser-based nanosurgery
Figure 1 Femtosecond laser nanosurgery is used to probe sarcomere repair in cardiomyocytes. Single Z-discs are ablated via the femtosecond laser system. Cell repair is recorded over time.

How do cells regenerate and repair? A classical biology approach would be to look at specific proteins and genes of interest. The usually applied methods include a knock-out or knock-down of expression and equally affect all targeted proteins in the cell. In contrast, we are interested to tackle only specific subcellular organelles in the cell. Instead of targeting a specific protein, we aim to inactivate this subcellular structure via laser-based ablation. Femtosecond (fs) laser-based nanosurgery is the ideal tool for these studies, as it presents a highly localized approach for subcellular manipulation. Via nonlinear ionization, the subcellular structures’ molecules in the focal volume are (mechanically) ablated and it is possible to follow the cellular repair over a period of several hours to days using multiphoton or confocal imaging.

In two of our recent studies, we have used this approach to better understand muscle physiology. The smallest contractile units of muscle cells are so called sarcomeres, which are a buildup of I-band, A-band, M-line, and Z-disc. In skeletal muscle cells, already extensive exercise can lead to microdamage, for example, in the Z-disc region. In the heart, altered Z-disc morphologies and dysfunctions have also been detected in severe diseases, for instance, in the heterogeneous group of cardiomyopathies. We aimed to better understand this microdamage in muscle cells using femtosecond laser-based cell ablation.

To obtain suitable laser parameters, we first optimized laser-based nanosurgery by evaluating its effect on cells, with special emphasis on cell staining and cell state. These experiments were performed in C2C12 skeletal muscle cells, which can be differentiated from myoblasts to myotubes. We performed nanosurgery in the cytoplasm with and without cell staining and in the undifferentiated myoblasts and differentiated myotubes. This analysis allowed us to determine a robust parameter set for laser-based nanosurgery in our following experiments. These experiments are published in the Journal of Biophotonics (https://doi.org/10.1002/jbio.201700344).

In the next step, we concentrated on heart muscle cells. Cardiomyopathies are one of the major burdens today. Therefore, it is of vital importance to understand the consequences of malfunctioning sarcomeres in detail. However, the role and function of single elements in the cytoskeletal orchestra of native versus diseased heart muscle cells (cardiomyocytes) are difficult to address. To extend the knowledge, we used our laser setup to induce microdamage in the cytoskeleton of cardiomyocytes (see Figure 1). 

Therefore, the sarcomeric pattern was disrupted using femtosecond laser-based nanosurgery and the physiological consequences as well as the endogenous repair potential of neonatal rat and human pluripotent stem cell (hPSC) derived cardiomyocytes was quantified over 24h. In particular, a single Z-disc, which forms the lateral border of sarcomeres, per cell was ablated and the cell viability, morphology, and calcium homeostasis were analyzed. The metabolic activity in treated and untreated cells was comparable with ≥ 73 % between viable hPSC cardiomyocytes and ≥ 78 % of viable neonatal rat cardiomyocytes. As the viability of cardiomyocytes was unaffected, we analyzed the potential effect of laser treatment in terms of morphological changes and calcium homeostasis. Therefore, cell area, perimeter, major and minor axis as well as the intracellular calcium level were measured before and after ablation of a single Z-disc. Most cardiomyocytes showed constant morphological integrity and no significant abnormalities in the calcium homeostasis were observed. Hence, successful recovery of the ablated Z-disc could be conceivable. The manipulated Z-disc pattern was compared with the original pattern over a time period of 24h. More than 68% of viable hPSC and more than 42% of neonatal rat cardiomyocytes showed a recovered Z-disc pattern.  The results were recently published in Nature Scientific Reports (https://doi.org/10.1038/s41598-019-40308-z). This demonstrates the good recovery capacity of muscle cells. In the following studies, we aim to extend these measurements to cells under physiological stress or under pharmaceutical treatment. For further questions, please feel free to contact us.

Contact and further information: 

Dominik Müller (d.mueller@iqo.uni-hannover.de)

Dr. Stefan Kalies (kalies@iqo.uni-hannover.de)

Prof. Dr. Alexander Heisterkamp (heisterkamp@iqo.uni-hannover.de)


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