Because they can tag and illuminate biologically-important proteins, fluorescent proteins (FPs) have revolutionized optical microscopy, allowing scientists to view single proteins as they migrate through living cells or observe previously invisible biological processes. Among the most useful FPs are those that fluoresce in the near-infrared (NIR) portion of the electromagnetic spectrum (650 nm to 900 nm)—passing right through hemoglobin, melanin, and water and allowing for imaging of tissues deep within human and other mammals.
In two Nature papers published in May 2022, Vladislav Verkhusha, Ph.D., professor of genetics and co-director of the Gruss Lipper Biophotonics Center at Albert Einstein College of Medicine, reported recent major advances in NIR imaging and the use of light to control cellular functions.
Small NIR-FPs are needed for tagging proteins in imaging applications. That’s because FPs are fused to proteins of interest—and since standard FPs are large compared to proteins they’re attached to, FPs can sometimes interfere with protein activity. Dr. Verkhusha’s development of a small NIR-FP was published online on May 23 in Nature Methods, where an accompanying “news & views” commentary described it as “by far the best NIR-FP available to date.” The new NIR-FP brightly fluoresces in mammalian cells and allows for deep-brain imaging. In addition, Dr. Verkhusha and his colleagues inserted the new NIR FP into nanobodies—small, engineered antibody fragments designed to target particular parts, or epitopes, of proteins. The resulting molecules, termed NIR-Fbs, brightly fluoresce when bound to antigen; but they quickly degrade in the absence of epitopes to which they’re designed to bind and consequently do not produce unwanted background fluorescence. Beyond their benefits in imaging of intracellular proteins, the NIR-Fbs proved to be useful molecular tools to degrade targeted proteins, control protein expression, and modulate enzymatic activities.
Optogenetics and deep tissue imaging depend on the use of phytochromes—naturally occurring photoreceptors that plants, bacteria, and fungi use to detect light. In the paper published online on May 19 in Nature Communications, Dr. Verkhusha and colleagues developed a transgenic mouse whose genome encodes a near-infrared absorbing bacterial phytochrome, BphP1, that can be expressed in specific tissues. The researchers were able to activate this phytochrome to carry out two different processes: photoacoustic tomography and optogenetic manipulation. In photoacoustic tomography, tissue is illuminated with laser pulses and the absorbed laser energy is converted into heat; the heat causes the targeted tissue to briefly expand and emit ultrasound waves that are detected by ultrasonic transducers and yield images when analyzed. Using photoacoustic tomography, the researchers obtained deep-tissue images of the mouse, including images of different internal organs, developing embryos, and regenerating livers. In the second process, optogenetic manipulation, near-infrared light was used to penetrate deeply into the mice and non-invasively trigger transcriptional activity in the livers of the animals. The researchers showed that this optogenetic activation can efficiently initiate the transcription of any protein desired, ranging from fluorescent proteins to proteins that affect cell biochemistry, tissue metabolism, or animal physiology.
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Posted on: Thursday, May 26, 2022