Nearly 20 years since the introduction of GFP as the first genetically encoded fluorescent marker, biologists have had at their disposal an ever-growing palette of fluorescent proteins with colors that extend across the blue, green, orange and, increasingly, red and near-infrared parts of the electromagnetic spectrum. Tools that absorb and emit in the near infrared (NIR) are particularly promising for probing deep into living tissue.

A guiding theme for delivering light deep into living tissue is that, up to a point, redder is better. The near-infrared window that extends from 650 to 900 nm is ideal: in this region, the combined absorption from hemoglobin and water are minimal and tissue is maximally transparent to light.

Near-infrared probes could help biologists see deeper into living tissue. Credit: Marina Corral

In recent years we have seen notable advancements in the development of GFP-like fluorescent proteins that absorb and emit in the low 600-nm range, but further red-shifting of the fluorescent proteins' chromophores has proven hard thus far. Similarly, the activation wavelengths of currently available rhodopsin-based optogenetic tools are limited to 600–630 nm, which is below the NIR tissue transparency window.

For the design of NIR optical tools, researchers have started to explore the use of alternative proteins known as phytochromes that plants and bacteria use for sensing light. Bacterial phytochromes are particularly promising because they naturally absorb red and near-infrared light and because they use biliverdin as a chromophore, which is endogenously produced in mammalian cells. Newly engineered phytochrome-based NIR fluorescent proteins—IFP1.4, iRFP and Wi-Phy—have excitation and emission maxima in the low 700s (nm) and hold promise for deep in vivo imaging.

Bacterial phytochromes could also serve as templates for building NIR biosensors, extending the possibilities of existing tools, such as for deep calcium detection or multicolor imaging. One day, we may also see these proteins develop into optogenetic modulators of biological functions.

Much work will be needed before the full potential of bacterial phytochromes is really demonstrated. Surely matching the qualifications of monomeric, nonperturbative GFP will be hard. But the potential for highly penetrating nontoxic biological exploration is worth the effort.