Product Survey: Flow Cytometers
Small is Beautiful
by Harald Zähringer, Labtimes 06/2014
Since our last survey on flow cytometers in issue 4 of Lab Times 2012, the race for ever smaller and smarter, easy-to-operate flow cytometers has continued with unimpaired speed.
The main drivers behind the ongoing trend to cheaper, easier-to-operate, flow cytometers with smaller footprints are new concepts for embedded fluidic and optical systems.
In conventional flow cytometers, sheath fluid and cell suspension (sample stream) are pushed towards the flow chamber by compressed air, which differentially pressurises sheath and sample containers. Applying a higher pressure on the sheath container leads to a faster flow of the sheath fluid in relation to the sample. The latter is slowly delivered (at sample rates of approximately 10 to 20 microlitres per minute) into the fast moving sheath and thereby hydrodynamically focused, briefly before it enters the flow cell. As a consequence, a string of single cells with a diameter of approximately five micrometres passes the analysing volume of the flow cell at a speed of up to ten metres per second.
Pressurised fluidic systems have stood the test of time since the early beginnings of flow cytometry but they require considerable amounts of sheath fluid (usually PBS) and depend on a sophisticated, expensive and delicate technical setup.
Hence, manufacturers of small benchtop cytometers have recently turned to non-pressurised systems based on small, inexpensive peristaltic pumps to force the fluids through the flow cell. One pump pushes the sheath fluid to the flow cell, a second one pulls both sheath and sample from the flow cell to the waste container. The hereby created pressure differential finally draws the sample through the flow cell. Installing a peristaltic pump, which per se creates an uneven fluid flow into a cytometer, sounds very ambitious in the first place, however, microprocessor-controlled dampening systems assure a pulsation-free sample flow.
Precise positioning of cells inside the flow cell is still accomplished in the majority of pressurised and non-pressurised flow cytometers by hydrodynamic focusing. Exceptions to this rule are instruments equipped with acoustic and micro-capillary focusing units.
The idea behind acoustic focusing is pretty simple. Ultrasonic waves, created by a piezocrystal attached to the outer walls of the flow cell, transport the cells inside the capillary to a nodal position at the centre of the flow channel. Acoustic focusing has one major advantage. Since the alignment of the cells is decoupled from hydrodynamic forces and sheath flow, sample flow rates may be adjusted to experimental needs. They may be raised up to one millilitre per minute to increase throughput or cut back, to allow the optical system to collect more photons to detect rare events.
Applying a micro-capillary to align cells seems a pretty obvious idea − at least in theory. Simply push the sample with a syringe pump through a clear “straw” with an inner diameter that admits particles to pass only one at a time and detect the sample with a laser beam focused on a small section of the capillary tube that serves as test volume.
Sounds rather easy, however, putting this concept into practise is a completely different story, given all the clogging and optical issues associated with micro-capillaries. It remains the engineers’ secret as to how they’ve solved these problems, however, the major benefits of sheathless micro-capillary systems are rather obvious: you may forget about fluid tanks and giant waste containers, the saved containers and pressure system allow smaller, benchtop-suitable footprints and, last but not least, the capillary-system enables, similar to acoustic focusing, controllable sample flow rates.
Shrinking the capillary system even further and implementing it on a plastic or silicon slide opened the door to microfluidic flow systems. Instead of cylindrical capillaries, however, which are hard to fabricate on plastic or silicon slides, single or multiple rectangular micro-channels are curved out of the chips to serve as flow cells. The cells are aligned in the centre of the micro-channels by two-dimensional hydrodynamic focusing, acoustic waves or other microfluidic related focusing techniques.
One of the most promising features of microfluidic flow systems is speed. Increasing the sample flow rates in conventional flow cytometers in order to detect rare cells leads to high shear forces, which tear the cells apart. In microfluidic flow systems higher velocities may simply be achieved by embedding a whole bunch of microchannels on a chip acting in parallel.
Similar to the parting of microcapillary flow cells and acoustic focusing with decades of hydrodynamic focusing, new optical detection systems based on spectral flow cytometry are challenging conventional systems. In traditional flow cytometers, fluorescent light, emitted from the laser-excited samples, is selected by dichroic mirrors and band pass filters according to its wave length, and is subsequently guided to the respective photomultiplier tubes (PMT) via glass fibres.
This optical regime is well-suited to efficiently separate and detect a small number of different fluorescent signals. Chances are getting better, however, that with every extra colour added, the signals will overlap in the detection channels and must be corrected with elaborate computer programmes.
Substitute the mirrors and band pass filters with a spectrograph made of prisms or gratings, which disperse the whole spectrum of emitted light across the detector array of a single CCD or multichannel PMT and you’ve basically got a spectral flow cytometer. The idea to analyse the complete spectrum of fluorescence-labelled samples passing through the flow cell dates back to the 1970’s but was hampered by technical obstacles.
However, recent improvements in CCDs, multichannel PMTs and data management, have enabled the construction of spectral flow cytometers for routine applications − and opened another exciting door to smarter, smaller, cheaper and faster flow cytometers.
First published in Labtimes 06/2014. We give no guarantee and assume no liability for article and PDF-download.
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