Product Survey: Benchtop Flow Cytometers

Channelling Cells
by Harald Zähringer, Labtimes 06/2016

Microfluidic chips allow the construction of ever smaller bench top flow cytomers and cell sorters. But microfluidics is not only about downsizing, it also enables engineers to implement interesting new cell-detection features.

Microfluidic chips are revolutionising flow cytometers and cell sorters. Photo: Stocker lab, ETH Zürich

When flow cytometry pioneers, Lou Kamentsky and Mike (Myron) Melamed, constructed their seminal spectrophotometric cell sorter in 1965, the world saw one of the first applications of microfluidics – without even having heard of “microfluidics”.

To select single cells from various types of cell populations, the two pathologists pumped a cell suspension via a stepper motor-driven infusion pump through a cross-shaped microfluidic channel, drawn into a quartz microscope slide with an ultrasonic cutter and sealed with a quartz slide. The cells entered the 100 µm wide channel at a rate of 0.5ml/min from one arm of the cross and were illuminated at the cross junction with a mercury lamp.

Light emitted from the irradiated cells was collected with an objective lens and conducted via four dichroic mirrors onto four photo multiplier tubes (PMTs). The electronic signals of the PMTs were amplified and visualised on an oscilloscope screen. To sort specific cells at the cross junction into the sample container, the signal of the detected cell induced a slight movement of the stepper motor, which, in turn, caused a fluid pulse that pushed the selected cell into the sample channel.

Kamentsky’s and Melamed’s microfluidic flow cytometer prototype was way ahead of its time. Although, it was put on hold, when much faster and more accurate fluorescence-activated cell sorters (FACS) and flow cytometers, based on pressurised sheath fluid systems, emerged in the early seventies.

Microfluidic channel system

FACS cytometers and sorters are still the top dogs in clinical laboratories and cytometer research facilities. But the bulky, expensive instruments, requiring expert knowledge for operation, are increasingly rivalled by a pack of small, affordable and easy-to-operate benchtop (personal) flow cytometers, based on sophisticated microfluidic flow systems.

The basic set-up of modern microfluidic benchtop flow cytometers is still inspired by Kamentsky’s and Melamed’s prototype, with the microfluidic chip or flow cell at the very heart of the instruments. The microfluidic channel design by Kamentsky and Melamed allowed the detection of mammalian cells at rates up to 1,000 cells per second. Not bad for a first try but pretty lousy compared to 50,000 cells or events per second measured by conventional FACS cytometers. Further issues were the fairly wide channel diameter of 100 µm and the simple flow of the cell suspension through the channel. Assuming a size of approximately 20 µm for mammalian cells, it is pretty obvious that the cells rather tumbled through the detection zone of the microfluidic cell, like a drunken sailor, instead of passing it perfectly aligned one-by-one in a straight line.

Hence, Kamentsky’s and Melamed’s successors zoomed in on techniques that keep the cells in line and came up with different strategies for sample focussing. The most simple, mono dimensional (1D) hydrodynamic focussing, is adopted from the sheath fluidic technique of conventional flow cytometers. The sample fluid arrives at a fork-like junction in the central micro channel of the fork, while the sheath fluids flow from the left and right channels of the fork in a pointed angle into the junction.

Multiple sheath inlets

It is easy to imagine that the sample fluid is compressed by the fast-flowing sheath fluids to a slim line. But there is still a problem here: the sample fluid is only narrowed down from the left and right sites but not in the vertical direction, i.e., in the second dimension (2D). To overcome this problem, engineers designed microfluidic chips with multiple sheath inlets, to completely shield the sample fluids by 2D hydrodynamic focussing.

But cells may also be focussed or separated in microfluidic flow cytometers by acoustic waves. The most common types of waves currently applied in acoustofluidic chips of experimental or commercial µflow cytometers are bulk acoustic standing waves and Standing Surface Acoustic Waves (SSAW).

Bulk acoustic standing waves are generated by ultrasonic waves, with wave lengths matching the diameter of the microchannel. Cells flowing through the channel are drawn by the acoustic radiation force of the ultrasound to the nodes of the standing waves and aligned into a sharp line. SSAWs are induced by so called interdigitated transducers and prolong along the floor of the microchannel. Cells flowing through the channel are guided by the acoustic forces of the SSAW to a defined position inside the channel.

Acoustic forces also come in very handy in cell sorting. The San Diego-based company, NanoCellect, for example, incorporated an acoustic sorting mechanism in their recently launched Wolf Cell Sorter. The acoustic sorting principle of the Wolf sorter, which has been developed by NanoCellect’s CTO Sunghwan Cho during his thesis work at the UC San Diego, is pretty smart. The cells are injected via the sample inlet into a microchannel, carved out of a tiny polydimethylsiloxane (PDMS) chip. A sheath flow drives the cells through the main channel towards a three-way junction that splits the channel into a central waste channel, and two collection channels on the left and right, respectively.

Acoustic separation

Cho has also integrated a piezoelectric actuator a few micrometres before the junction. Every time the detection system registers a fluorescence signal from a specific cell, the actuator receives a voltage pulse that immediately bends the piezoelectric crystal. This, in turn, leads to a fluid wave that pushes the respective cell into the collection channel.

That’s not the only interesting detail that Cho integrated into his cell sorter. His device is actually one of the finest examples of a modern microfluidic cell sorter; still deeply rooted in Kamentsky’s and Melamed’s ideas but with a lot of new twists. Sure enough, he replaced the old mercury-lamp of the historic µflow cell sorter with a laser to detect fluorescently-labelled cells – but not in the way it is usually done.

Similar to traditional FACS cytometers or sorters, fluorescent cells are illuminated in most microfluidic flow cytometers by a point-shaped laser beam perpendicularly directed against the flowing cells. Cho, in contrast, guides the laser light via an optical fibre into the central microchannel of his device, which is coated in its whole length, including waste and collecting channel, with Teflon amorphous fluoropolymers (Teflon AF).

Teflon AF acts as an optofluidic waveguide that transmits the laser light via total internal reflection from the beginning of the microchannel to its very end. Due to this clever trick, fluorescent cells can be excited everywhere in the channel and may be detected at any position. The optofluidic system enabled Cho to implement a very inventive new detection system into his µFACS sorter, based on a technique, which Cho calls space-time coding. The fluorescence signal of a cell that travels through the optical interrogation zone is collected by a 20X objective lens and directed onto a spatial filter mask that modulates the light signal into different waveforms of photocurrents that represent different locations of the fluorescent cell in the channel. The different digital waveforms, encoding the locations of the fluorescent cells, are subsequently recorded by a single photo multiplier tube (PMT).

Colour-code signal

That’s clever enough but Cho has extended the space-time coding approach into a technique he dubbed colour-space-time coding (COST) that gives him the possibility to detect multiple fluorescence colours with a single PMT. During the COST process, the space-time coded fluorescence colours pass additional red, green and blue waveguide arrays that produce a supplemental colour-code signal. The complete COST-coded output signal is finally registered by a single PMT.

Hence, it’s no surprise, that all components of the Wolf sorter are assembled in a cubic box with a footprint of a little less than forty centimetres.

First published in Labtimes 06/2016. We give no guarantee and assume no liability for article and PDF-download.

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