research highlights
microscopy
© 2009 Nature America, Inc. All rights reserved.
Seeing in the dark
An imaging platform based
says Xie, “and this allows us
on stimulated emission helps
to visualize molecules that
researchers to lead nonfluor­
were otherwise undetectescent chromophores out of
able before.” As an initial
the shadows.
proof of concept, they used
For years now, fluorescent
stimulated emission microsreagents and proteins have
copy to detect the distribureigned as the undisputed
tion of green fluorescent
champions of the biological
protein− der ived chromicroscopy world. Lightmophores gtCP and cjBlue
absorbing molecules receive
in live Escherichia coli and to
a jolt of energy upon illu- Stimulated emission
visualize the indigo byprodmination at an appropriate microscopy of microvasculature
uct of X-gal cleavage by the
in ex vivo mouse skin,
wavelength, which kicks an with microcapillaries (red)
enzyme b-galactosidasea
electron in their structure visualized from the intrinsic
widely used reporter for
from the relaxed ‘ground contrast of hemoglobin. Scale
gene activityat otherwise
state’ into a transient ‘excit- bar, 50 mm. Image courtesy of
undetectable basal levels of
Wei Min, Sijia Lu and Shasha
ed state’; fluorescent comenzyme activity.
pounds get their glow when Chong of the Xie lab.
Stimulated emission may
this energy is discharged in
likewise prove helpful for
the form of an externally detectable photon as
imaging of fluorescent targets that have a
the excited electron returns to ground state, a
tendency to disappear when they ‘blink’.
process known as ‘spontaneous emission’.
“Green fluorescent proteins will go to a dark
The problem, points out Har vard state, where they don’t emit but still absorb;
University’s Sunney Xie, is that only a relative quantum dots do the same thing,” says Xie.
handful of molecules truly shine as fluores- “We could now have this new mechanism
cence reagents. “Many molecules absorb but that allows people to see the dark state.”
do not fluoresce or have undetectable fluor­
This method also holds promise for cliniescence, such as hemoglobin, cytochrome, cal applications. For example, stimulated
retinal and melanin,” he says. “Upon photoex- emission enabled visualization of the uptake
citation, those molecules quickly return to the of the photosensitizing therapeutic agent
ground state not through spontaneous emis- toluidine blue O by cancer cells. Xie’s group
sion but via the release of energy as heat.”
exploited their capacity to image hemoglobin
As a means to visualize these nonfluores- as a means for visualizing the microvasculacent chromophores, his team has made use ture within the ear of a mouse, with individof stimulated emission, a principle described
ual red blood cells clearly visible.
by Albert Einstein nearly a century ago. Xie’s
These preliminary findings establish a wide
system, which he developed along with range of potential applications for stimulatpostdoctoral fellow Wei Min and graduate ed emission microscopy, and Xie is eager to
student Sijia Lu, makes use of two ultrafast move forward with this platform. “We have
lasers. The first fires a pulse that bumps an collaborators in the skin care industry, and
electron within the target chromophore into we work with surgeons and neurologists,” he
an excited state; this is closely followed by a says. “The study of melanin in the skin and
pulse from the second laser, which knocks the the distribution of microcapillaries around
electron back into the ground state in a man- tumors or neurons [is] based on the contrast
ner that creates an additional photon with
of hemoglobin; we’ll be able to visualize them
the same energy and direction as the initial, with this label-free imaging method and folstimulatory photons.
low them in real time.”
This process of light amplification via Michael Eisenstein
stimulated emission lies at the core of laser
Research Papers
technology and creates a signal boost for othMin, W. et al. Imaging chromophores with
erwise-invisible chromophores. “It’s the first undetectable fluorescence by stimulated emission
time that stimulated emission has been used microscopy. Nature 461, 1105–1109 (2009).
as a contrast mechanism for microscopy,”
866 | VOL.6 NO.12 | DECEMBER 2009 | nature methods