Micro- and Nanotechnologies
Katarzyna Tokarska
PhD, adjunct
Centre for Advanced Materials and Technology CEZAMAT
Warsaw, Mazowieckie, Poland
Microfluidic approaches in optical imaging techniques for more accurate investigations of the dynamic cellular environment are highly demanded. Herein, we present a novel ultrathin chip combined with holographic tomography (HT) to visualize subcellular structure without exogenous labelling. HT enables non-invasive and high resolution bio-observations based on the refractive index (RI) 3D distribution from multiple 2D holograms measured with varying illumination angle. The RI value enables to dynamically track changes in cell environment at the submicron level.
To address the limitations of HT working distance (due to high NA objective lenses), the overall thickness of the chip is set to around 500μm. This is much thinner than for the typical microfluidic devices. The presented chip is composed of 4 layers which are: i) 1.5H-thick - cover glass (24x60mm) for cell adhesion, ii) silicon pad (300µm thick which flattens under applied pressure) with microfluidic structure, iii) 175μm PMMA foil iv) silicon pad to seal. Microchamber and all microfluidic structures along with the inlet/outlet were made using CO2 laser cutting machine. All layers were tightly squeezed in the two-piece made by micromilling PEEK holder which also includes ports for the tubings prior to cell culture and examination.
A549 and MeWo cancer cells were seeded into cover glass and left overnight. Then, chip layers were precisely adjusted and placed into PEEK holder, of which 2 parts have been firmly twisted together using screws to seal the microchip. Media exchange was performed through the tubings using a peristaltic pump. Cells were cultured in microfluidic conditions and put under observation using custom-built holographic tomography (HT) microscope to visualize their dynamics. 90 projections of the sample were captured with 633nm laser illumination. Gerchberg-Papoulis algorithm with finite object support regularization was used to retrieve 3D RI distribution of analyzed samples from measured projections. The lateral and axial resolution of the 3D reconstruction was 0.32µm and 1.3µm, respectively.
Based on 3D RI maps quantitative on-chip bioimaging was performed. The identification of the subcellular organelles (nuclei, nucleoli, mitochondrial network or lipid droplets) was possible due to the high resolution imaging capability. After cell microfluidic culture and HT observations, high cell viability was confirmed using calcein staining. In conclusion, we demonstrated novel microfluidic approach that can be adapted along with the HT for high resolution 3D label-free quantitative subcellular imaging. Moreover, the geometry and fabrication method of the presented ultra-thin chip makes it a versatile platform that can be readily expanded to various applications, including organoid culture and drug screening and discovery.
Studies were funded by BIOTECHMED-1 project granted by Warsaw University of Technology under the program Excellence Initiative: Research University (ID-UB) under the grant no. 1820/1/Z01/POB4/2020.
SLAS Events