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Optogenetic tools have been gaining popularity, in part because they can be used to decipher the wiring of signaling pathways. They are based on the ability of photoactivatable proteins to change their conformation and binding affinity when illuminated with light. Fusing these proteins to signaling elements allows for the specific regulation of a single player within complex intracellular signaling pathways. Consequently, a signaling pathway can be studied with high temporal and spatial resolution.
Most cell-based optogenetic studies utilize microscopy-based methods combined with culturing in the presence of light, followed by biochemical analysis. In contrast, a flow cytometer singularizes cells along a capillary and measures cell size, granularity and fluorescence intensities. This method has major advantages over microscopy or biochemical methods, including the ability to analyze thousands of living cells at single cell resolution in a very short time. Hence, it is desirable to combine optogenetics with flow cytometry. To our knowledge, there is no established protocol for optogenetic flow cytometry. A broadly accepted procedure is to manually illuminate cells from outside the reaction tube with flashlight devices. However, manual illumination in the flow cytometer requires the light to pass through the reaction tube and, for live cell imaging, a cylindrical, heated water chamber. This causes substantial light scattering and loss of light. Moreover, the light intensity provided by manual illumination is not reproducible between experiments (angle, distance, etc.) and there is a practical limit to the number of wavelengths in one experiment. By constructing the pxONE prototype, we were able to overcome these limitations. With this device, cells can be illuminated with specific wavelengths in a temperature-controlled manner during flow cytometric measurements. This allows for precise and reproducible amounts of light within and between experiments.
To demonstrate the utility of our device, we recorded the fluorescence signal of Dronpa in Ramos B cells during photoswitching. Ramos B cells are derived from a human Burkitt's lymphoma. Dronpa is a fluorescent protein that exists as a monomer, dimer or tetramer. In its monomeric form, it is non-fluorescent. Illumination with 400 nm light induces dimerization and tetramerization and renders the Dronpa protein fluorescent. This process can be reversed by illumination with 500 nm light. The Dronpa protein has been used to control the function and location of signaling proteins.
We expressed a Dronpa-Linker-Dronpa protein in Ramos B cells to study photoswitching of Dronpa in a flow cytometer. Using our device, we were able to efficiently and reproducibly photoswitch Dronpa while recording its fluorescence intensity in real time. This method provides substantial advantages over current illumination protocols with manual illumination and significantly broadens the experimental repertoire for optogenetic tools and cage compounds. The first publication using our device was recently published and shows optogenetic regulation of T cells (https://www.biorxiv.org/content/early/2018/10/01/432740).
Using our technology will significantly simplify and accelerate the discovery and development of novel optogenetic tools.
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