a) b)
1 µm 1 µm
of bleaching, and hence the constriction
of the probed area, is varied by changing
the power of the S TED beam (see Fig. 1).
Examples of stochastic switching
methods include (direct) stochastic opti-
cal reconstruction microscopy, known as
(d)S TORM and (fluorescence) photoacti-vation localization microscopy, known as
(f)PALM. Here, extensive sample bleaching by a wide-field light source leaves
just a few isolated unbleached molecules.
The samples are imaged by a low-
noise camera and algorithms then con-
vert each isolated “blob” into a point
location, based on the optical transfer
function of the microscope. Repeating
the on/off reversible bleaching process
results in another random set of un-over-lapped blobs. Eventually an entire image
is built up of the point locations.
These applications require new wavelengths, particularly orange and yellow,
for photobleaching without crosstalk
with the fluorescence excitation wave-
lengths. They also need low noise and
higher power—as high as 2 W at the
laser output—and the ability to adjust
the power to optimize the various super-resolution bleaching mechanisms. OPSL
technology is ideal for this purpose;
unlike other solid state lasers, OPSLs
do not suffer from thermal lensing, so
the power can be dynamically adjust-
ed (from 10% to 100% power) with
no effect on beam quality and point-
ing. Figure 2 shows gated STED im-
ages acquired with one of these yellow
OPSLs at 577 nm.
Divergent multiphoton
microscopy techniques
Multiphoton microscopy is another dynamic area because of the continued development of diverse methods. Key needs
here are femtosecond lasers that com-
bine versatility and industrial reliability
for multiuser facilities, higher power at
wavelengths longer than 1 µm, and dual-
wavelength capability to support exciting
developments in optogenetics, in which
light-sensitive proteins are used to optically stimulate/inhibit specific neurons.
A second laser wavelength is then used to
map activity in con-
nected neurons us-
ing two-photon ex-
citation of calcium
indicators.
While Ti:sapphire
lasers continue to
be the workhorse
sources for multi-
photon microscopy,
femtosecond lasers
based on ytterbium
technology have re-
cently gained popu-
larity. Used directly
at their wavelength of 1055 nm, they excel
in activating red-shifted fluorescent pro-
teins, photoactivators, and Ca indicators.
They can also be used to pump an inte-
grated OPO to achieve broader tunabili-
ty (680–1300 nm) than Ti:sapphire lasers.
The combination of a 1–2 W, 1055 nm
fixed output with the tunable output of
the OPO or a Ti:sapphire laser perfectly
addresses optogenetics, where dual-color
excitation can independently excite/inhib-
it and detect action potentials in neurons.
These dual-wavelength sources are also
well-suited to other methods, including
coherent Raman techniques like coher-
ent anti-Stokes Raman scattering (CARS)
and stimulated Raman scattering (SRS).
Lasers for DNA sequencing
and profiling
Automated sequencing and profiling
are still in their infancy, and sequencing methods vary significantly among
instrument developers. Most of these
utilize laser-excited fluorescence, but
the challenge for our industry is that
laser performance (beam quality, wavelength, and power) packaging, and cost
requirements are as widely different as
the techniques themselves.
A comprehensive survey of sequenc-
ing methods is beyond the scope of this
article. (For a more detailed discussion,
see “Laser fluorescence powers sequenc-
ing advances” in the January/February
2015 issue of BioOptics World.) Here,
we present a brief overview designed to
show some of the technical diversity.
The successful Human Genome Project
relied on automating the so-called Sanger
method, in which DNA is first cut into
strands that are a few hundred bases long.
These are then copied in vitro using DNA
polymerase in the presence of normal bas-
es (adenosine, cytosine, guanidine, and
thymidine) as well as a small amount
of chemically modified bases that “jam”
the synthesis process when incorporated
into the new copy. Each of these modified bases is also labeled with a different
fluorophore for each base type (ACGT).
The result is a mix of every possible
length, from a few bases all the way to
the original hundreds of bases, with a
fluorescent label identifying what base it
ends with. These are then run through a
capillary electrophoresis column to sepa-
rate them according to length; the column
exit is excited with a focused 488 nm laser. The sequence is then read by record-
ing which fluorophore appears next and
so on. Only a few hundred bases can be
read in this way, so sequencing the en-
tire human genome (~ 109 bases, circa
35,000 genes) was a massive collaborative challenge costing roughly $3 billion.
NGS and emerging platforms
In just 20 years, the cost per entire hu-
man genome has experienced a reduction
of seven orders of magnitude. So-called
next-generation sequencing (NGS) and
emerging third-generation instruments
have made this possible by the ability to
simultaneously sequence up to hundreds
of thousands of lengths of DNA and/or
to analyze longer cut strands.
FIGURE 2. Gated STED (gSTED) imaging with a 577 nm OPSL.
Scanning confocal and gSTED images of a) 40 nm fluorescent
yellow-green beads and b) microtubule in mammalian cells
immunostained with Alexa 488. Spatial resolution in A is 50 nm.
