- Two-color solid-state lasers could aid confocal microscopy
Diode-pumped solid-state lasers are available at emissions near the
488-nm argon-ion laser line, which can be used for excitation in confocal microscopy
and other fluorescence-based detection and imaging techniques. The solid-state lasers
are small, use little energy and are very stable, making them an attractive alternative
to gas lasers.
Still, solid-state lasers generally cost more
than gas lasers and typically don’t provide multiline capability, which is
important because of a growing demand for fluorophore multiplexing.
Researchers can incorporate multiple
excitation sources into their experimental setups, but this leads to still higher
costs as well as added complexity of the setups.
Hjalmar Brismar, an investigator with
the Royal Institute of Technology in Stockholm, Sweden, was looking for a 488-nm
solid-state laser to provide excitation, mostly for cell volume measurements. He
was aware of these limitations but found that the Dual Calypso laser made by Cobolt
AB, also of Stockholm, offered multiline capability in a single diode-pumped solid-state
device. He and his colleagues, therefore, tested the laser to determine whether
it could replace the conventional 488-nm argon laser they had been using.
A new solid-state laser addresses some of the limitations of gas lasers and, furthermore,
provides simultaneous blue and green emission from the same instrument, which could
be useful in experiments requiring multiplexing of fluorophores.
Indeed, this was the primary motivation
for developing the laser. The company’s researchers chose to combine two well-known
and widely available diode-pumped solid-state lasers (491 and 532 nm), “mainly
because we knew this would give us power scalability, very nice beam and no issues
in supply of components,” said Håkan Karlsson, Cobolt’s vice president
of business development and technology.
The design also enables simultaneous
green emission from the same laser, which can replace the 488/514 lines from an
argon laser and is less expensive and complex than using two solid-state lasers
at 488 and 532 nm, especially because no beam-combination optics are needed.
“An important part of our design
is the use of a single pump source for both laser crystals, and the fact that they
share one common cavity mirror,” Karlsson noted. “This ensures spatial
overlap and codirectional propagation of the two lines. It also makes the laser
easy to manufacture.”
Studies have shown that when employing
simultaneous emission of blue and green light in fluorescein detection, for example,
the fluorescence can be contaminated with the green from the excitation source.
However, placing a 532-nm filter in front of the detector yields good resolution
for fluorescein as well as improved detection for longer-wavelength fluorophores,
according to Karlsson.
Brismar and his colleagues, including
Mårten Stjernström and Fredrik Laurell, tested the laser by integrating
it into a confocal laser scanning microscope made by Zeiss, which is typically outfitted
with an argon and a HeNe laser. They imaged fluorescently labeled bovine pulmonary
artery endothelial cells. The emissions from the cells clearly showed F-actin filaments
stained with fluorescein and mitochondria stained with rhodamine, demonstrating
the potential of fluorophore multiplexing with tight spectral distinctions using
the laser. In fact, they noted that the images may even have been of higher quality
than those obtained with conventional gas lasers.
Researchers tested the laser by imaging fluorescently labeled bovine
pulmonary artery endothelial cells. The emissions from the cells revealed parts
of the cell stained with fluorescein and rhodamine, confirming the laser’s
potential for multiplexing even with tight spectral distinctions.
The investigators plan to employ the
laser generally for studies of salt and water balance in epithelial cells. Currently,
they are using it for studies of aquaporines, which are water channel proteins.
For this, they use a confocal technique to explore the water permeability of the
plasma membrane in living cells and to test how this permeability might be regulated
via the aquaporines and their regulation.
The laser could be useful for other
applications, particularly those that benefit from wavelength multiplexing. Indeed,
Brismar believes that, in the not-too-distant future, solid-state lasers such as
this could replace argon lasers in all new confocal systems.
“We already see that Carl Zeiss
uses 488- and 532-nm solid-state lasers as the main lasers in their new LSM5 Live
systems,” he pointed out.
To extend the potential of its laser,
Cobolt plans to expand the range of available wavelengths. New models might offer
excitation in the orange and ultraviolet regions, for example.
Brismar has some additional ideas.
Incorporating the lasers into the microscope’s scan head and introducing fast
optical modulation would produce more compact confocal microscopes, with more lines
and simpler excitation beam paths, he said.
Contact: Håkan Karlsson, Cobolt
AB, Stockholm, Sweden; e-mail: firstname.lastname@example.org. Hjalmar Brismar, Royal
Institute of Technology, Stockholm; e-mail: email@example.com.
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