Researchers at JILA have demonstrated the use of infrared laser light to quickly
and precisely heat the water in "nano bathtubs"-tiny sample
containers-for microscopy studies of the biochemistry of single molecules
Described in a new paper,* the JILA technique is faster, more controllable,
and less prone to damaging expensive optics or accidentally altering chemistry
than conventional methods using electric currents for bulk heating of microscope
stages, optics and samples. The demonstration extends a technique used to study
single living cells to the field of single-molecule microscopy. Fast, noncontact
heating of very small samples is expected to enable new types of experiments
with single molecules. For example, sudden, controlled jumps in temperature
could be used to activate molecular processes and observe them in real time.
JILA is jointly operated by the National
Institute of Standards and Technology (NIST) and the University of Colorado
at Boulder (CU).
The JILA "bathtubs" consist of about 35 picoliters (trillionths of
a liter, or roughly one-thirtieth of a nanoliter) of water on a glass slide.
Gently focused infrared laser light is used to heat a nanoscale column of water.
By moving the laser beam, this column can be made to warm single RNA molecules
attached to the slide. The samples are mounted above an inverted fluorescence
microscope, used to study folding of tagged RNA molecules (See for example,
"JILA Study of RNA Dynamics May Help in Drug Design," NIST Tech Beat,
July 13, 2005 at http://www.nist.gov/public_affairs/techbeat/tb2005_0713.htm#JILA).
The researchers simultaneously heated and observed folding of the molecules,
comparing results obtained with the laser heating technique to measurements
obtained with bulk heating.
The heating laser is directed at the samples from above, with the beam focused
to a spot size of about 20 micrometers. The near-infrared light is just the
right wavelength to excite vibrations in chemical bonds in the water molecules;
the vibrations quickly turn into heat. The laser offers a much larger dynamic
temperature range (20 to 90 degrees Celsius, or 68 to 194 degrees Fahrenheit)
than bulk heating methods, according to the paper. In early trials, the technique
controlled bathtub heating to an accuracy of half a degree Celsius in less than
20 milliseconds across a micrometer-scale sample area.
"Exact sizes of the laser beam and sample area don't matter," says
NIST/JILA Fellow David Nesbitt, senior author of the paper. "What's important
is having time and temperature control over volumes of fluid small enough to
be able to look at single molecules."
The research is funded in part by the National Science Foundation, NIST, and
the W.M. Keck Foundation initiative in RNA sciences.
* E. D. Holmstrom and D.J. Nesbitt. Real-Time Infrared Overtone Laser Control
of Temperature in Picoliter H2O Samples: "Nanobathtubs" for Single
Molecule Microscopy. Journal of Physical Chemistry Letters 1, pages 2264–2268.
Published online: July 7, 2010.