operation, but the cost and complexi-
ty often cannot be justified for multila-ser OEM instrumentation applications.
Instead, some manufacturers used extra-cavity tripling of passively mode-locked
lasers that have pseudo-C W output with
extremely high peak power. Of concern,
there are questions about the potential
negative impact of using high peak power UV on live cells.
Optically pumped semiconductor laser
(OPSL) technology is excellent for gen-
erating true-CW ultraviolet because it
is does not suffer from green noise. The
reason is that the upper state lifetime of
the semiconductor gain medium is con-
siderably shorter than the round trip time
of the laser cavity, so there is no stored
gain to support dynamic mode competition. OPSL technology is also power scalable. As a result, true-C W, OPSL lasers
at 355 nm now offer output powers exceeding 20 m W.
Multiple wavelengths
in flow cytometry
Another trend in flow cytometry is the simultaneous use of an increased number of
fluorochromes to support more complex
analyses. For example, ten and more color assays have been shown to provide the
sensitivity and specificity for important
applications such as determining minimal
residual disease (MRD) analysis in various types of leukemia. One approach to
increasing the number of fluorochromes
that can be independently sensed is to use
fluorochromes with narrower emission
bands, i.e., less spectral overlap.
Another approach is to increase the
instrument’s spectral bandwidth, which
can then be divided between more indi-
vidual fluorescence channels. The exci-
tation bandwidth can be expanded by
use of UV lasers, or by filling in key gaps,
such as the one between 561 and 630 nm.
These gaps occurred because of the lim-
ited available wavelengths from DPSS la-
sers and laser diodes.
OPSL technology addresses this be-
cause it is wavelength scalable; the fun-
damental wavelength output by the semi-
conductor gain chip can be customized
by changing the composition and size
of the active quantum wells. As a result,
OPSLs have been developed at several
new strategic wavelengths in the yellow
and orange, specifically 552, 577, and
588 nm, as well as 568 nm (which was
previously available but only from bulky
krypton lasers).
Near-infrared OPSLs (e.g., 833 nm)
are also of interest for extending instru-
ment bandwidth on the longer wavelength side. All these wavelengths are
available in plug and play formats such as
the Coherent OBIS series, simplifying integration and tasks such as hot swapping.
Lasers optimized for super-
resolution microscopy
In microscopy, the diffraction limit was
long considered unsurmountable, limiting resolution to around 200 nm in the
xy plane and 500 nm in the z axis of
confocal microscopes. The 2014 Nobel
Prize in Chemistry—jointly awarded to
Eric Betzig, Stefan W. Hell, and William
E. Moerner for pioneering super-resolution microscopy techniques—proves how
different and complementary techniques
have surpassed the diffraction limit.
All optical super-resolution or nanoscopy techniques involve optically and re-
versibly preparing states of a fluorescence
label that differ in their emission characteristics (e.g., a bright ON and a dark
OFF state). Optical nanoscopy methods
can be loosely divided into two groups:
those that directly improve microscope ef-
fective spatial resolution by deterministi-cally photoswitching labels in space and
time and those that achieve the higher res-
olution by stochastically switching single-
molecule fluorescence on and off in space.
Examples of the first group include
stimulated emission depletion (STED) mi-
croscopy, ground-state depletion (GSD)
microscopy, and reversible saturable optical fluorescence transition (RESOLF T)
microscopy. These techniques use two
overlapping laser beams. The first beam
bleaches fluorescence from a part of the
area excited by the second probe beam.
The easiest to visualize is STED, which
uses a donut-shaped beam. The extent
