Label-free image produced by the older techniques of two-photon and second- and third-harmonic-generation microscopy.

It would be difficult to deny that recent developments in fluorescent probes—most notably the development of fluorescent proteins—have resulted in spectacular growth in the use of light microscopy in the life sciences. But the enormous benefits provided by these probes for observing the location and dynamics of biomolecules are offset by the fact that they invariably require the attachment of the probe to the biomolecule of interest. Unfortunately, if the goal is to image endogenous molecules in vivo, labeling may not be feasible. Furthermore, labels may perturb the function of biomolecules, particularly small ones.

Label-free microscopy methods that rely on a variety of different photophysical processes to generate light signals from biological molecules have been around for many years, but they have seen limited use for answering biological questions. Two-photon microscopy can detect some prevalent autofluorescent cellular species. Second- and third-harmonic-generation methods can distinguish fibrillar structures and lipid bodies. Raman microscopy can detect specific types of chemical bonds and is capable of determining the chemical makeup and abundance of lipids, but its use is hindered by high background.

About ten years ago, the development of coherent anti-Stokes Raman scattering (CARS) microscopy by Sunney Xie's group significantly improved the Raman signal and the usability of the method, but identification of specific molecules was still challenging. In the past two years, however, Xie and colleagues have reported promising new developments in label-free imaging that make it clearly a method to watch.

At the end of 2008, Xie's group reported a new Raman-based imaging method that overcomes some of the problems with CARS microscopy. Their stimulated Raman scattering (SRS) microscopy makes it easier to identify specific target molecules and provides very high sensitivity. The relatively poor molecular specificity of the label-free methods in general has been one of their major drawbacks compared to fluorescent labeling. SRS promises to help bridge this divide while being particularly amenable to smaller biological molecules like lipids, which have proven difficult to productively tag with fluorescent labels. And in 2009 the Xie group developed a different label-free imaging method, based on stimulated emission, that coaxes light from nonfluorescent molecules.

Although none of these methods will replace fluorescence microscopy or fulfill the needs of every label-free imaging experiment, it appears that we are reaching a turning point in label-free microscopy that holds great promise for the future of these methods.