Flow Cytometry & Cell Sorting (FCCF)

To increase the number of parameters of cells that can be detected by flow cytometry, we are investigating cytometric pulse shape analysis in collaboration with the Berlin-based company APE. In this method, the resulting pulse shape of the scattered light from a cell is detected in an angle-dependent manner and classified by data processing. This allows the discrimination of cell properties based on the pulse shape alone, i.e. it is not necessary to label the cells with a surface marker, for example. In a further step, this method will be extended to enable cell sorting.

Also in cooperation with the Berlin-based company APE, we have developed the LED-based calibration tool for flow cytometers (quantiFlashTM). It enables us to quantify the sensitivity of flow cytometers.

We introduced an experimental strategy, in which discontinuous emission spectra were acquired and analyzed by principal component analysis (PCA). In a proof-of-principle study we could show, that the simultaneous detection of up to 4 fluorochromes, which were all excited by a 488 nm Argon-laser could be resolved by the detection of a discontinuous emission spectrum. In other words we use a standard cytometer just by changing the optical filters as a multispectral cytometer. No further expensive modifications as described in the literature are required.

We have developed a flux reactor that sterilizes the sheath fluid at the point of use. The flux reactor consists of an array of 6 UV-C LEDs. Such UV-C radiation source is also used in water treatment and disinfection. The module is currently installed in an INFLUX cell sorter and allows to reduce the number of microorganisms by up to 1000 times. The module was manufactured in cooperation with APE.

We developed a cooling device in order to keep the sheath fluid temperature on a FACS Aria constant. We found that the foccusing of the side-streams on a FACS Aria are temperature sensitve. Room and instrument temperature shifts of more than +/- 5K results in a defocusing of the side-streams and the recovery of sorted cells decreased drastically. Furthermore, the sensitivity of delayed lasers decreased due to instable laser-delay.
To overcome this effects we hold the sheath-fluid constant at 17°C by using our device.

During long-term sorts cells often sediment at the bottom of the sample tube. In consequence, the threshold rate are instable or interupted and cells in the settlement are exposed to cytotoxin. In cooperation with Advalytix we modified their universal acoustic mixing platform “SAWStation4” so that we can implement it in a FACS Aria.
In our study we could show that the yield of sorted cells was about 5% higher compare to cells mixed by the original FACS Aria agitator. Moreover, we found less dead cells in sorted populations.