Benutzer:Kathrin Brenker/Optogenetic Flow Cytometry

Optogenetic flow cytometry

Optogenetic flow cytometry refers to the analysis of optically controlled substances in a flow cytometer. This method was developed by the University of Freiburg and led to the formation of the Spin-off opto biolabs.

Optogenetics

The term “optogenetics” was coined by neuroscientists and originally referred to a variety of different techniques to image and regulate neuronal functions. 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.

Dronpa expressing cells were photoswitched multiple times using flashlight devices and analyzed using flow cytometry. Illumination with 500 nm light leads to a slow decrease of Dronpa mean fluorescence intensity (MFI). Ilumination with 400 nm light leads to a fast increase in Dronpa MFI. The illumination using flashlight devices is inhomogenous as can be seen by the inhomogenous decrease of Dronpa MFI.

Flow Cytometry

A flow cytometer is a powerful analytical machine for  biological research. It 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, the combination of optogenetics with flow cytometry would significantly increase the efficiency of optogenetic research and broaden the experimental repertoire.

Optogenetic Flow Cytometry

Optogenteic flow fytometry describes the analysis of optogenetic cell samples in a flow cytometer. Optogenetic originated from the field of neurobiology, hence fluorescence microscopy is the most commonly used analytical method for optogenetic research. Using a confocal fluorescence microscope, small areas of e.g. neuronal membrane can be optically activated to observe synaptic transmission. An equally powerful method is flow cytometry. Yet, it has so far hardly been used for optogenetic research. This can be explained by the lack of suitable illumination devices. Currently, LED-flashlight devices are used. A novel, quickly spreading approach is to use specialized illumination devices designed for optogenetic flow cytometry.

Cells were adjusted to 37°C and illuminated with LED flashligh devices

Flashlight devices

So far, optogenetic flow samples have been illuminated only with flashlight devices. Depending on the angle and distance of the flashlight to the sample, substantial variability in the amount of illumination is expected between experiments. Furthermore, there is a limit to the number of flashlights a single person can operate in an experiment. This restricts the experimental repertoire and reproducibility. An essential pitfall of flashlight devices is that they do not offer temperature control which is essential especially for illumination with light of longer wavelengths. Minor changes in temperature can have substantial effects on the behavior of cells, like changes in membrane potential. Major temperature changes can induces apoptosis.

Cells were adjusted to 37°C

Specialized illumination devices

A suitable illumination device for flow cytometry requires homogenous, radial illumination, easy control of light wavelength and intensity, temperature control as well as convenient usage during experimental procedures.

One such device was developed by a Spin-off from the University of Freiburg called opto biolabs. Their first prototype, called the pxONE, is a temperature-controlled LED illumination device used for optogenetic research. The advantages of such a specialized illumination device are digital control of light intensity and exposure time, also allowing the analysis of fast switching optogenetic proteins. Radially mounted LED lights surround the FACS tube and allow for homogenous illumination that is easily reproduced between experiments.

Cells were illuminated using specialized illumination devices for optogenetics

Photoswitching experiments with different illumination devices

A simple way to analyze the illumination capability of optogenetic illumination devices are photoswitching experiments. Some optogenetic proteins change their fluorescent properties during photoswitching. Hence, recording the fluorescent intensity of these proteins using different approaches of optogenetic flow cytometry is the simple way to compare different illumination devices. The Dronpa protein is a suitable optogenetic protein since it is only fluorescent in its dimerized state, induced by 400 nm illumination. In its dissociated state, induced by 500 nm illumination, it is not fluorescent.