While seeing is believing, biological samples are far more complex than meets the eye. And though conventional methods in optical microscopy, such as phase and fluorescence, are popular because they are so powerful, complementary imaging modalities also have much to offer biologists seeking to understand the structure and function of their samples, from the subcellular to the organismal scale.

Three-dimensional SRS metabolic nanoscopy of Zika virus-infected mammalian cells displayed in depth color-coded projection. Credit: Haonan Lin

In this issue we highlight bond-selective imaging approaches and the types of studies to which they are well suited. By bond-selective imaging we mean vibrational imaging, including Raman and infrared microscopy. Raman spectroscopy and its variants, including coherent Raman spectroscopy (CARS) and stimulated Raman spectroscopy (SRS), are well established in both spectroscopy and microscopy. When used in a label-free manner, these methods offer spatial mapping of the chemical compositions of tissues, with clear delineations between lipids, proteins, nucleic acids and metabolites. Infrared microscopy offers similar advantages and is gaining popularity in life sciences research because of its relative simplicity, its sensitivity and the availability of commercial microscopes. These methods have been shown to be advantageous structurally mapping samples, studying metabolism across scales, and even quantifying the abundance of specific subcellular components.

Two historical Comments in this issue highlight the development of vibrational imaging technologies. The first1, from Xiaoliang (Sunney) Xie, describes the development of coherent Raman microscopy over the past 25 years. In addition to describing the roles of major players in the field, Xie explains how his research team serendipitously achieved CARS imaging. Xie further shares how subsequent innovations led to advances in CARS and the invention of SRS imaging, and how this work has set the stage for making Raman microscopy a vital part of biological research.

The second historical Comment2 comes from Ji-Xin Cheng and colleagues and describes their 20-year effort to develop vibrational photothermal microscopy. Photothermal microscopy is a newer form of vibrational imaging that offers advantages over conventional vibrational microscopy, such as high sensitivity. In photothermal imaging, molecules absorb light and release heat into the environment. This heat causes a local change in refractive index that gives the contrast for imaging. Cheng recounts how some failures in photoacoustic vibrational microscopy led to their pioneering work in infrared photothermal imaging and how subsequent work from his team and others have propelled this technology into an advanced tool for discovery.

A comprehensive introduction to and summary of advanced vibrational imaging with practical guidance for interested users is presented in a Review from Ji-Xin Cheng and colleagues3. This piece covers key technologies, how they work, how they compare, and their ideal application ranges. We hope this stands as a landmark piece that will connect biologists with the cutting edge of these approaches.

And while vibrational imaging has been historically considered label-free, there is an entire world of Raman and infrared-sensitive probes that can be imaged with vibrational microscopy, much as fluorescent probes stand out from background signal in fluorescence microscopy. A Review from Wei Min and colleagues4 offers a thorough introduction to probes for vibrational microscopy, how they work, and their numerous biological applications. This piece highlights these probes’ unique potential for multiplexed imaging and how they offer complementary benefits to hyperspectral imaging of endogenous biomolecules in diverse applications.

Given the many unique advantages of vibrational imaging, one might wonder why these methods have not gained more of a foothold among researchers — even those who are microscopists — in the life sciences. From an editorial perspective, it has been exciting to watch these methods develop at a rapid pace over the past 20-plus years, but we have not seen the kind of broad adoption by biologists that often happens as cutting-edge methods truly hit their stride in terms of ease and performance. Although these technologies are certainly gaining momentum, much of the cutting-edge work, even in terms of biological applications, comes from labs that are also developing the technology.

This gap between biologists and technology could be due to a number of factors. Perhaps researchers think of vibrational spectroscopy as something they had to muddle through in organic chemistry class rather than a bona fide technology for biology research. It is possible that issues with speed, sensitivity, resolution and lack of hyperspectral imaging that plagued early iterations of this technology still persist in the minds of researchers. A relative dearth of affordable commercial instruments still limits the field, and it could be that developers have not made a strong enough case that biologists need these methods, especially since fluorescence microscopy can already show us so much.

Whatever the reasons, we hope this Focus issue can help narrow this gap by introducing readers to what the latest technologies can deliver and how they can give new views into old biological problems. A Comment5 from Meng Wang showcases how vibrational microscopy is offering unprecedented views of metabolism in vivo, highlighting how the field of spatial metabolomics has immediately benefited from these methods. A research Article6 from Ji-Xin Cheng’s lab describes ultrasensitive reweighted visible stimulated Raman scattering (URV-SRS), an approach that combines advances in both hardware and denoising algorithms to enable multiplexed super-resolution imaging of intracellular metabolites. A final Comment from Katsumasa Fujita and colleagues7 provides a unique and forward-looking view of the field. It covers challenges and limitations in the technology but, most importantly, discusses how valuable these approaches can be for exploratory biology, as well as how the field can benefit from advances in computer vision and integration with spatial omics technologies.

We learned a lot while developing this Focus issue and are more enthusiastic than ever about the possibilities of vibrational imaging, especially for revealing chemical complexity that is easily missed with fluorescence. We invite you to explore these pieces and consider whether bond-selective imaging could play a role in your future research.