HoLoC – holography on a chip. A label-free method of mitochondrial dysfunction imaging in the Lab on Chip microsystem.
The main goal of the project is to study the process of mitochondrial dysfunction induction in hepatocyte cells resulting from their interaction with pharmacological substances. Drug-induced mitochondrial dysfunction is postulated as an important determinant of drug-induced liver damage. In this aspect, it is crucial to design an approach that allows tracking this type of damage with high resolution without introducing additional factors at the level of subcellular compartments. Therefore, it is planned to examine the potential hepatotoxic effects of selected drugs on mitochondrial structures, which can be observed using label-free phase analysis (2D) and three-dimensional refractive index distribution (3D RI).
The planned research will include (i) development of the microfluidic LOC system for hepatocyte culture and its integration with holographic microscope (MH) and holographic tomography (TH) systems, and (ii) development and implementation of data analysis methodology. Microsystem for HEP G2 and Clone-9 cell culture will be fabricated from biocompatible materials. The cells will be treated with pharmaceutical substances with hepatotoxic potential. Integrated LoC-MH and LoC-TH systems will allow long-term cell culture and simultaneous quantitative 2D (in real-time) and 3D phase imaging (at selected moments in time with increased resolution) to obtain information about dry mass distribution and an entire 3D RI map, respectively. The developed supporting software, including algorithms for mitochondria segmentation from received phase distribution or RI maps, will be used to obtain selected parameters of the studied cells. As part of the validation the appropriate MitoTracker staining (confocal microscopy) will be performed. In order to verify the correct mitochondria identification by the holographic tomography system, the results after segmentation will be overlayed with the obtained images from the confocal microscope. Ultimately, 3D RI analysis will allow the monitoring of potential drug-induced changes in mitochondrial network with high resolution. The extensive spatial-temporal and statistical analysis of the biophysical parameters of the tested cells will allow to link the onset of the mitochondrial dysfunction with cell death and the final hepatotoxic response.
The planned interdisciplinary research combines elements of chemistry, biotechnology, materials engineering and photonics. It will allow to formulate conclusions that will be a source of knowledge for further considerations and research on the use of holographic tomography associated with microfluidic systems to track biological processes in the microscale.
As part of the BIOTECHMED-1 Research Grant “Excellence Initiative – Research University” Research Area – Biotechnology and Biomedical Engineering, WUT
Warsaw University of Technology, Centre for Advanced Materials and Technologies CEZAMAT
Institute of Micromechanics and Photonics of the Warsaw University of Technology
200 000 pln
Katarzyna Tokarska, PhD Eng.