0
Facebook
Volume 87 Issue 43 | p. 5 | News of The Week
Issue Date: October 26, 2009

Seeing Molecules With A New Light

Microscopy: Technique relies solely on light that nonfluorescent compounds absorb
Department: Science & Technology
Keywords: stimulated emission microscopy
[+]Enlarge
Cell Visualization
Cell Visualization Stimulated emission image of hemoglobin (red) in blood cells (inset) in mouse-ear capillaries.
Credit: Nature
8743notw1_XieSE1
 
Cell Visualization
Cell Visualization Stimulated emission image of hemoglobin (red) in blood cells (inset) in mouse-ear capillaries.
Credit: Nature
[+]Enlarge
Xie (from left), Min, and Lu adjust their stimulated emission microscopy apparatus.
Credit: Marcus Halevi
8743notw1_xie1
 
Xie (from left), Min, and Lu adjust their stimulated emission microscopy apparatus.
Credit: Marcus Halevi

A new microscopy technique makes it possible to visualize nonfluorescent molecules based only on their ability to absorb light.

The light that objects absorb and reflect is responsible for the colorful world people see. But because molecular-level absorption is often too weak to detect under a microscope, imaging of molecules typically relies on fluorescence. The new approach overcomes this sensitivity problem, allowing imaging of nonfluorescent compounds that would be difficult or impossible to label with fluorescent tags.

The technique depends on stimulated emission (photon-induced electron de-excitation), which was introduced in 1917 by physicist Albert Einstein and is the basis for lasers. Postdoc Wei Min, grad student Sijia Lu, spectroscopist X. Sunney Xie, and coworkers at Harvard University demonstrate for the first time that stimulated emission can be used as a contrast mechanism for molecular microscopy (Nature 2009, 461, 1105).

The method could make it possible to image biomolecules like hemoglobin, cytochrome, and melanin in living cells and organisms. Such molecules have undetectable fluorescence, and their low abundance in microscopic samples often leads to absorption too weak to be detected by conventional microscopy. The technique can visualize drugs as well. Xie and coworkers used the method to image hemoglobin in blood cells and transdermal delivery of a nonfluorescent drug.

"It's an impressive outcome," says Stefan W. Hell of the Max Planck Institute for Biophysical Chemistry, in Göttingen, Germany, adding that he has "a lot of admiration" for Xie and coworkers. "They have opened up a new way to detect molecules that would otherwise be left in the dark."

"What I love about this technique is its simplicity," comments spectroscopist and microscopist Martin T. Zanni of the University of Wisconsin, Madison. "It is just a standard stimulated-emission experiment, albeit very carefully done. It could have been done five or 10 years ago, which is one of the things that makes it so beautiful."

In conventional absorption spectroscopy, a molecule raised to an excited state relaxes back to its ground state principally by converting that energy to heat. In the new technique, a stimulation beam de-excites a molecule and converts its excitation energy into a photon in an amplified stimulated emission beam. This signal is generated at the common foci of two laser beams, which are scanned across or through the sample to build 2- or 3-D images. Xie and coworkers use intensity modulation, timing adjustments, and lock-in amplification to manipulate pulsed excitation and stimulation beams in such a way that the stimulated emission can be detected.

"The technique potentially will allow in vivo imaging with micron-scale resolution deep into intact thick tissues," says Chris B. Schaffer of Cornell University, whose group uses optical techniques to observe and manipulate biological systems. For example, it could make it possible to study blood oxygenation by monitoring hemoglobin in single brain capillaries or to study redox changes in cytochrome c during the respiratory cascade, he says. "The idea would be to do stimulated emission microscopy at two wavelengths, where the amount of emission is different for two different states, such as the oxygenation or redox states of a molecule."

The technique is less sensitive than fluorescence microscopy "but it's four to five orders of magnitude more sensitive than would normally be possible with absorption," Schaffer adds. "It's an enormous gain in sensitivity."

 
Chemical & Engineering News
ISSN 0009-2347
Copyright © American Chemical Society