Peering Through the NIR Optical Window

Fluorescence is well-ensconced in nearly every laboratory technique that makes use of imaging—from microscopy to gel documentation and plate-based assays to flow cytometry. Coupling fluorophores to antibodies and expressing GFP-coupled proteins in vivo, for example, offer unprecedented opportunities for specificity, sensitivity and multiplexing.

Light from different parts of the spectrum is imbued with different properties. Toward the blue and ultraviolet (UV) regions, for example, these higher-frequency wavelengths are more energetic, making them easier to detect. At the far-red and near-infrared (NIR) end, on the other hand, these longer wavelengths can penetrate samples more deeply. In addition, different objects will preferentially absorb, fluoresce, reflect, refract and scatter one portion of the spectrum more than another. In the case of biologicals, this results in an “optical window” through which objects can most efficiently be imaged.

Here we look at the increasing use of NIR fluorescent dyes in biological research, including the predominant applications for which they’re used, the instrumentation necessary to use them and areas of research beginning to embrace longer wavelengths.

Window of opportunity

Many of the proteins, melanin, hemoglobin and other substances that make up biological tissues preferentially absorb between 200 nm and 650 nm, which includes almost all of the visible range. Many of these will also fluoresce—absorb light at one wavelength and emit it at a typically longer, less energetic wavelength—when exposed to light in the visible spectrum, a phenomenon often termed “autofluorescence” [1].

In contrast, tissue absorbance and autofluorescence are at a minimum between about 650 nm and 1,000 nm—in the far-red and part of the NIR spectra. Many authors call this the “optical window” or “imaging window.” It’s the portion of the spectrum that allows for maximal penetration.

I’m looking through you

“NIR dyes can actually be imaged through a whole mouse, for example,” points out Lori Roberts, principal scientist at Biotium. “No other dye can do that.”

In fact, small-animal imaging is an important application for NIR dyes. A host of vendors, including LI-COR and Revvity, offer instruments capable of in vivo optical imaging that includes the NIR range, often in conjunction with modalities ranging from PET and CT to luminescence. NIR fluorescence allows more than one color—and therefore more than one molecule or structure—to be simultaneously imaged in the same animal.

Labeled antibodies can be used to find their antigens in vivo. And fluors can be used to label other proteins and entities, which can then be injected and tracked. “We make all of our Alexa Fluor NIRs in reactive dyes,” says Brian Almond, senior product manager of cell analysis at Thermo Fisher Scientific. Similarly, whole cells can be labeled in vitro with membrane-specific dyes and then transplanted into the animal to track its migration, Roberts says.

Biodistribution assays that determine where probes go and see if they hit their intended targets, such as core organs, “cannot be done with bioluminescence—it can only be done with IR fluorescence,” says Jim Wiley, director of marketing at LI-COR. “That’s an area I think is going to take off.”

On the slide (plate, flask or dish)

If NIR fluorescence can be used for whole animals, certainly it can be used in microscopy, as well. However, most instruments are not equipped to take full advantage of the dyes—they most often lack the optimal excitation sources and filter sets, says Roberts. “If you have low expression or dim labeling, then laser excitation is required,” points out Almond

But there certainly are some—largely custom-designed instruments and high-content imagers—that can take advantage of the longer-wavelength dyes. The camera technology and illumination has caught up, says Almond. “There are plenty of people out there who are imaging and making movies.”

In addition to greater penetration of thick tissue like brain sections, there are perhaps two principal, interrelated advantages to NIR microscopy.

Most people aren’t doing single-color imaging, and “being able to spectrally separate your fluors is desirable when you’re taking pictures of different colors,” says Almond. “Moving into the NIR opens up more spectral real estate to use for multiplexing with other shorter-wavelength fluors.”

Adding fluors at the other (UV) end of the spectrum can be problematic, because compared with the NIR they are more likely to cause photobleaching and cellular toxicity—perhaps not an issue for taking a snapshot of cells to be discarded, but a real hurdle for applications like live-cell imaging.

On the Western front

Perhaps the main application of NIR fluorescence, surprisingly, is quantitative, multiplex Western blot imaging.

Previously, blots were typically imaged using chemiluminescence resulting from a dynamic enzymatic reaction. There was virtually no background, affording the assay’s high sensitivity. Yet it was difficult if not impossible to normalize the results, because only a single color could be detected at a time. Similarly, to visualize multiple proteins the blot had to be stripped and re-probed (or duplicate blots needed to be run).

The obvious solution should be to use fluorescence—which displays a signal proportional to the amount of protein and allows for multiplexing. This would enable distinction between, for example, a phosphorylated and nonphosphorylated version of the same protein, notes Lisa Isailovic, director of marketing at Azure Biosystems. Yet the typical Western membrane exhibits considerable autofluorescence in the visible range. Low-fluorescence membranes are now available, but because many proteins also autofluorescence in the visible spectrum, it can still be difficult to visualize faint bands.

Using NIR dyes—especially on systems like those from Azure and LI-COR that use laser excitation in the NIR range—solves these issues, as well. These systems even offer the ability to image plates and gels and to read in-cell Western assays.

Another option is to use something like Thermo Fisher’s WesternDot® reagents, which are based on quantum dot technology. They are excited by blue light, so they’re not fully NIR reagents and are subject to some of the caveats about absorption by biological materials. These reagents fluoresce extremely brightly with a choice of emission wavelengths, including 705 nm and 800 nm.

Super (re)agent

Only 15 years ago, NIR imaging was almost unheard of, but the state of the art in commercially available NIR fluorescent probes has been “fairly static” in recent years, with only “incremental innovation,” notes Almond.

There may now be more choices in terms of attributes such as the size, hydrophobicity and charge of the dye. These, says Roberts, can affect the number of dyes that can be conjugated to a protein or perhaps affect the specificity of the affinity reagent.

Yet a lot of the dyes are almost exactly the same, says Wiley. “What’s really important when you’re matching up dyes with the detection system is that the emission wavelengths line up with the detection optics.”

Reference

[1] Pansare, V, et al., “Review of long-wavelength optical and NIR imaging materials: Contrast agents, fluorophores and multifunctional nano carriers,” Chem Mater, 24(5):812-827, 2012. [PMID: 22919122]