Endogenous Dark Chromophore Imaging via Modulated Stimulated Emission

Here is an interesting paper, Imaging chromophores with undetectable fluorescence by stimulated emission microscopy, from Sunny Xie’s group.  They pump the sample with a excitation laser while simultaneously hitting it with a longer wavelength laser to induce stimuated emission. The pump laser is modulated at a high frequency which they can pick up and amplify with a lock-in amplifier.

Theory and illumination schematic

In two examples of imaging from a mouse ear, (above) shows the distribution of TBO, a photodynamic therapy drug, following drug administration, (below, red) shows the distribution of hemoglobin in blood vessles.

How specific is the detection of endogenous chromophores?  They report that 60nm is the absolute detection limit, but this is for a pure chromophore in water.  In real cells there will be many other endogenous chromophores at various concentrations. For example, endogenous background fluorescence of flavins is often easily seen when imaging at CFP wavelenghts. Watt Webb has been imaging those types of chromophores for years. 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.

Do the absorbance and emission spectra and the excited state lifetime provide sufficient selectivity to detect low concentrations of chromophores in vivo?

Thanks goes to Reporter Gene for the story tip.`