Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine

Imaging with molecular vibrations The vibrational spectra of biomolecules could in principle image cells and tissue without added markers. Practically, several technical problems need to be overcome to achieve sufficient imaging depths, resolution, and data acquisition speed. Cheng and Xie review emerging bioimaging methods for use in the lab and the clinic. Science, this issue p. 10.1126/science.aaa8870 BACKGROUND Biomolecules can serve as natural labels for microscopy by measuring their molecular vibration spectra in living cells and tissues. However, the transition from spectroscopy of molecules in cuvettes to spectroscopic imaging of living systems requires more than putting spectrometers on microscopes. A series of technical challenges must be addressed, such as delivery of light beams for sample excitation and scattering of signals, which limit the probe depth of spectroscopic imaging. There are also trade-offs in how large a spectral window can be measured at a pixel (the amount of chemical information) in a given amount of time (the recording speed). ADVANCES Several technical advances made by different groups have pushed the boundary of the vibrational spectroscopic imaging field in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and penetration depth. Specifically, coherent Raman scattering microscopy has emerged as a high-speed vibrational imaging platform. Single-frequency coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopes have reached a video-rate imaging speed. Multiplex CARS by broadband excitation has reached a speed of 3.5 ms per pixel; multiplex SRS covering a window of 200 wave numbers has reached a speed of 32 μs per pixel. The marriage of high-speed coherent Raman microscopy and Raman-sensitive tags of large cross sections has enabled real-time imaging of small molecules at micromolar concentration. Nanoscale vibrational imaging has been demonstrated by integration of atomic force microscopy and vibrational spectroscopy. Vibrational imaging of deep tissue has been enabled by acoustic detection of overtone transitions or optical detection of diffuse photons. Biological applications of CARS and SRS microscopy have generated new insights into myelin biology, lipid droplet biology, intracellular drug delivery, and single-cell metabolism. Meanwhile, clinical applications of vibrational spectroscopic imaging are enabling molecule-based diagnosis of cancer and heart disease without the need for any exogenous contrast agent. Examples include intravascular vibrational photoacoustic imaging of lipid-laden plaques and spatially offset Raman spectroscopic detection of cancer margins. OUTLOOK There remain two central challenges facing the field. One is to increase the detection sensitivity of vibrational microscopy to micromolar or even nanomolar levels, so that low-concentration biomolecules in a living system can be mapped. The other is to increase the vibrational imaging depth to tens of centimeters for noninvasive molecule-based medical diagnosis. With continuous developments, high-resolution, high-speed vibrational microscopy will cultivate unexpected discoveries in cell biology. The findings may lead to the development of new therapies for currently incurable diseases. Meanwhile, with further improvement of penetration depth and progressive reduction of instrument size, vibrational spectroscopic imaging devices are expected to become fundamental clinical tools for disease diagnosis and therapy effectiveness evaluation. Molecular fingerprints for biology and medicine through imaging. Recent efforts focused on pushing the fundamental limits of vibrational spectroscopic imaging in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and penetration depth. The resulting platforms are enabling transformative applications in functional analysis of single living cells and noninvasive diagnosis of human diseases with biomarker sensitivity. Vibrational spectroscopy has been extensively applied to the study of molecules in gas phase, in condensed phase, and at interfaces. The transition from spectroscopy to spectroscopic imaging of living systems, which allows the spectrum of biomolecules to act as natural contrast, is opening new opportunities to reveal cellular machinery and to enable molecule-based diagnosis. Such a transition, however, involves more than a simple combination of spectrometry and microscopy. We review recent efforts that have pushed the boundary of the vibrational spectroscopic imaging field in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and imaging depth. We further highlight recent applications in functional analysis of single cells and in label-free detection of diseases.