The spatial organization and functional properties of organs or tissues are preferably studied in the intact state. Several emerging techniques achieve this goal either in fixed or in living samples.

Imaging deep within intact tissue samples or animals. Credit: Katie Vicari/Nature Publishing Group

The opacity of biological material complicates imaging deep within a specimen. 'Clearing' the tissue can overcome this hurdle in fixed samples with protocols such as CUBIC, iDISCO or PACT, all of which preserve fluorescent immunolabeling signals. These techniques were established recently and await application to a variety of different questions.

Clearing techniques are not available for live specimens. Alternative approaches can reduce light scattering and improve transparency in these circumstances: for example, by employing nonlinear excitation such as with two-photon microscopy, which does not excite molecules along the path of incident light, or by imaging with near-infrared illumination, which is poorly scattered by biological material. The advent of genetically encoded near-infrared probes and nanoparticles has been particularly useful for non-invasive cancer studies or cell tracking in vivo. At the same time, progress has been made in shifting the imaging window to longer wavelengths, allowing for even deeper tissue penetration.

Another strategy for dealing with light scattering is to forgo imaging with light. In photoacoustic or optoacoustic imaging, incident light is absorbed within a tissue and converted into ultrasound via thermoelastic expansion. Ultrasound is less sensitive to scattering than light, enabling imaging at greater depths than with conventional microscopy while maintaining good resolution. The different absorption properties of biological materials make label-free imaging possible. In addition, absorption differences between saturated and unsaturated hemoglobin allow for functional imaging in the brain.

Finally, the characteristics of different biological materials introduce imaging aberrations, a problem that worsens with depth. Adaptive optics is used in astronomy to correct for optical aberrations, and it has recently been used to image transparent biological specimens such as the zebrafish embryo. Efforts in this direction may soon allow for use of adaptive optics in less transparent organisms.

Researchers now have an unprecedented choice of techniques for imaging tissue deep within a specimen. We hope that further developments in this area will spur the analysis of cells and tissues in their native context.