The aim of the PROCHIP project was to study cellular heterogeneity in large populations, allowing the classification of isolated cancer samples based on their chromatin architecture. To this end, we aimed at developing a super-resolution microscope with high throughput capabilities, able to acquire thousands of cellular samples.
We exploited laser writing technology, Femtosecond Laser Irradiation followed by Chemical Etching (FLICE) to develop a light sheet fluorescence microscope (LSFM) on a chip, equipped with structured illumination microscopy (SIM). FLICE allows fabricating optofluidic components as waveguides, microchannels and lenses in a glass substrate. By combining these components, a mm-scaled lab-on-chip with integrated illumination path and sample delivery has been realized.
In details the objectives of the project were:
1) development of a miniaturized three-dimensional fluidic network integrated on a glass chip and its relative pumping system for automatic sample scanning under a microscope.
2) development of a super-resolution microscope, whose illumination and sample scanning components are integrated in a lab-on-chip (super-resolution microscope on-chip).
3) identification of the protocols for isolation of primary and metastatic tumor cells suitable for high-throughput imaging and data analysis at super-resolution based on the image simulator.
4) assessment of cancer cell heterogeneity by querying chromatin domains as functional biomarkers.
We realized different prototypes of microscopes on chip, in which the sample flowing in a microchannel is optically sectioned by a uniform or by a patterned light sheet, capable of automatic sample scanning at high throughput.
The first challenge was the achievement of a stable flow control, to scan the samples in an automated manner. The second was to realize a patterned light sheet within a microchannel for the optical sectioning of a flowing sample with structured light to enhance the spatial resolution. A third challenge concerned the realization of software for setup simulation and design, for remote controlling of the system during the measurement acquisition and for the image reconstruction and analysis.
In conclusion, although phenotypic profiling of chromatin domain has not been completed, all the means necessary for its successful realization have been developed, ranging from the development of protocols for sample preparation and analysis to the realization of an optofluidic microscope on chip capable of high throughput 3D imaging of fluorescent single cells with enhanced resolution.
