Flow cytometry is a technology for cell counting and cell sorting. It presently is an essential tool in biomedical applications such as DNA sequencing or diagnosis of health disorders. The current cytometers rely on exciting a stream of suspended cells by up to three lasers, each emitting at different wavelengths, and recording the light from scattering and fluorescence of the cells, which are tagged with specific fluorescent bio-markers. The recorded light is then processed to extract information related to cell counting, sorting and structure.
A key performance parameter in a cytometer is the laser power and its spectral coverage. Having a large set of visible and UV lasers available allows increasing the instrument flexibility, inspecting more physical and chemical properties of the particles at a time. So far, the common laser technology is limited in providing both sufficient power density and spectral coverage. Furthermore, having more than three different lasers implies an increase in costs and complexity.
The intent of the present work is to explore a new laser source developed by the company GLOphotonics and coined CombLas. This radiation source emits a comb of laser lines in the UV-VIS and NIR. It is based on Stimulated Raman Scattering (SRS) in a hydrogen-filled Hollow-Core Photonic Crystal Fibre (HC-PCF). This work reports on the properties and the generation of the rotational and vibrational Stokes and anti-Stokes lines. Such a Raman comb is generated and analyzed using two types of HC-PCFs: a Kagome lattice HC-PCF and a Tubular lattice HC-PCF, which are pumped with a compact 532nm DPSS laser.
The conversion efficiency to the higher order Raman lines is proportional to the pump laser pulse energy, which in the case of our laser decreases of repetition rate. On the other hand, the cytometer signal strongly depends on the laser average power, and hence on the repetition rate. Hence, this requires a trade-off in operating in sufficiently high energy to generate a strong Raman comb, and sufficiently low so to have the required high repetition rate. In the case of our system, when the DPSS laser is emitting its maximum energy of 115 μJ at 50kHz, the Raman comb is well populated by a total number of 31 rotational and vibrational Stokes and anti-Stokes lines, and spanning from 368 nm to 1075 nm. However, at a repetition rate of 210kHz, the maximum pulse energy is reduced to 22 μJ, and 2 only vibrational lines survive.
Two higher order Raman lines have been studied and sent in the cytometer: the first consists of the first order rotational Stokes @549nm and the second consists of first order vibrational anti-Stokes @435nm. Those have been extracted both using a grating and bandpass filters. In the second case, the quality and power of laser beam proved to be better for our intents. The measurements were conducted on running Rainbow Fluorescent particles in the Flow Cytometer and the relation between power, repetition rate and correct beads illumination has been investigated. Fluorescence histograms show that a comb generated with a pump operating at a repetition rate as low as 180kHz and average power as low as 4 mW provided good results.
In comparison with state-of-the-art and cytometry requirements, the current results from this novel source are promising and demonstrate the viability of such Raman comb Raman for Flow Cytometry applications. However, improvements must be done to further increase the repetition rates whilst still generating a strong comb, allowing to illuminate the greater number of cells possible.