Why image with near-infrared wavelengths?
Application Note on Borealis Perfect Illumination Delivery™
- Avoid autofluorescence
- Better contrast - better resolution
- Move into 5+ channel imaging
- Little cross-talk with shorter wavelengths
The sensitivity of fluorescence microscopy measurements, especially in living tissues, is often limited by autofluorescence - the natural emission of light by certain biological molecules . Autofluorescence interferes with and obscures the detection of extrinsic fluorescent contrast agents, causing structures other than those of interest to become visible and manifest as a high background signal in an image that reduces contrast and clarity. Autofluorescence spectra are generally broad and encompass most of the visible spectral range, overlapping with the emission wavelengths of green fluorescent protein (GFP) and other common visible wavelength fluorescent probes. Many other cellular metabolites also exhibit autofluorescence, and since cellular extracts are often crucial components of culture media, such media can also be intensely autofluorescent, thus compounding the problem. We have developed Borealis, a combination of optical techniques resulting in Perfect Illumination Delivery™. One of the many benefits of Borealis is the broadening of the available spectrum for excitation illumination in multi-point microscopy.
Fortunately, at near-infrared (NIR) wavelengths above 700 nm, autoflourescence emission is dramatically reduced. Combined with the development of newer contrast agents or engineered proteins that absorb light (730-800 nm) and emit fluorescence (750-1000 nm) at NIR wavelengths, fluorescence imaging at longer wavelengths where tissue autofluorescence is essentially eliminated is now a viable option for researchers . Many cellular tissues are also relatively transparent to infrared light, exhibiting lower absorbance and scattering by molecules like hemoglobin and skin melanin, and thus fluorescent proteins/probes that can be excited at these wavelengths are useful for deeper-tissue optical imaging.
Existing fluorescence microscopes are ill-equipped to operate at NIR wavelengths. Most current laser scanning confocal microscopes do not deliver infrared laser illumination light to the sample, and metal-halide or gas discharge arc lamps used in regular widefield epifluorescence microscopes have very low output in this wavelength range.
Borealis, with its Perfect Illumination Delivery™ technology enables illumination of microscopic specimens with wavelengths that extend beyond the visible into the ultraviolet (UV) and the NIR regions of the electromagnetic spectrum. The latter is demonstrated in the corresponding figure where the entire 90 mm thickness of an aged rat brain slice containing oligodendrocytes labeled with LiCor®IRDye 800CW NHS Ester has been imaged in 3D with 730 nm excitation and >785 nm emission. Tissues like these accumulate autofluorescent lipofuscin pigments that create a high image background when excited and imaged at more common visible wavelengths. All images were acquired with the same microscope hardware components (60x 1.4 NA oil immersion objective, EMCCD camera)
90 mm thickness of an aged rat brain slice containing oligodendrocytes labeled with LiCor®IRDye 800CW NHS Ester has been imaged in 3D with 730 nm excitation and >785 nm emission.
- Frangioni, J.V., In vivo near-infrared fluorescence imaging. Current Opinion in Chemical Biology, 2003. 7(5): p. 626-634
- Lin, M.Z., Beyond the rainbow: new fluorescent proteins brighten the infrared scene. Nature Methods, 2011. 8(9): p. 726-728.