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Polycold® is a trademark of Brooks Automation, Inc.
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is located at the bottom of each waveguide (see Fig. 3).
Laser illumination occurs through the
silica and can only penetrate about 30 nm
into each waveguide, so millisecond pulses of fluorescence can only be efficiently
collected from the nucleotide as they are
being added to the DNA. Importantly, in
the company’s PacBio RS II system, up to
150,000 sequencing reactions are monitored simultaneously, in real time, by dividing the lasers into that many individual diffraction-limited spots targeted to
the individual ZMWs. Moreover, the
company claims single continuous read
lengths as long 40,000 bases.
A wide number of methods are also
targeted at profiling. Rather than record-
ing every individual base, these meth-
ods look for specific sequences, for ap-
plications ranging from epidemiology to
forensics and pre-natal screening. One
clever method here first inserts fluores-
cently labeled target sequences, which
are incorporated within the DNA dou-
ble helix. The treated samples are forced
through ultra-narrow channels on mi-
croarray plates so that the DNA tertia-
ry structure is stretched to leave a string-
shaped molecule with various different
fluorescent points along its length. These
can be read like a colored barcode using
a laser and detector.
In terms of laser power, requirements
vary from a few milliwatts for techniques
using a tight focus to multiple watts for
techniques using wide-field illumina-
tion. Similarly, some applications need
a perfect TEM00 output whereas others
work with a lower cost, multimode laser. And, several of these highly automated techniques have run times of several
hours or even days, so long-term power and pointing stability are very impor-
tant. Reliability is also critical, as a laser problem will ruin the entire data run.
Preferred laser wavelengths are still
dominated by argon ion and DPSS legacy
wavelengths (488, 514, 532 nm). But the
wavelength scalability of OPSL technolo-
gy is creating interest in new wavelengths,
such as 640 nm. In particular, the devel-
opment of 505 nm OPSLs is potentially
disruptive; as the arithmetic mean of 488
and 514 nm, this new wavelength can be
used to simultaneously excite some of
the fluorophores previously optimized
for these two separate wavelengths, sim-
plifying instrument design and lowering
instrument costs.
Marco Arrigoni is director of marketing-scientific market, Nigel Gallaher is senior
product line manager, Dan Callen is product
line manager for direct-diode-laser systems,
Darryl McCoy is director of product marketing, and Matthias Schulze is director of
marketing, OEM components and instrumentation, at Coherent, Santa Clara, CA. Volker
Pfeufer is senior product line manager for
diode-pumped solid state lasers at Coherent
GmbH, Luebeck, Germany. Contact
email: marco.arrigoni@coherent.com; www.
coherent.com.
