Use the fundamental principle for laser amplification to ultra-sensitive biological imaging

It is amazing that they could image LacZ without the need to add substrate!

It used the phenomenon of stimulated emission described by Einstein to “force” the non-radiative decay into some kind detectable signal. Is it accurate? I read the mechanism section twice and still could not understand it :-(

Both reportergene and brainwindows have posts about this paper. Their comments are:

reportergene said: “This open the race to the intelligent design of new chromo-reporters able to produce images of unlabelled, non-fluorescent molecules at sub-diffraction (nanoscale) resolution. Seeing is believing. The potential to kill the field of reporters is easily explained: who ever need a “reporter” when you can spy in a cell the whole bunch of molecular processes with resolutions at googlemeter per googlesecond?”

brainwindows said: “The intersection of both a preferred excitation wavelengh and a preferred stimulated emission wavelength will provide some selectivity, but I suspect this will be most useful for imaging the distribution of fairly highly expressed chromophores in vivo. Distinguishing chromophores with highly overlapping spectra may not be possible. Of course, many, many proteins don’t have distinctive chromophores (tyrosine does not count!) built in to them, so GFP won’t be out of work any time soon. However, this stimulated emission imaging doesn’t require transgenic or small molecule labeling, so it could potentially allow imaging in humans.”

The abstract of the paper

Nature. 2009 Oct 22;461(7267):1105-9.

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy.

Min W, Lu S, Chong S, Roy R, Holtom GR, Xie XS.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.

PMID: 19847261