The benefits of near-infrared imaging, both in vitro and in vivo, have generated intense interest in near-infrared microscopy. Most microscopes are outfitted for detection of visible fluorescent wavelengths and not near-infrared wavelengths, so questions may arise about how to perform microscopy with IRDye near-infrared (NIR) fluorescent dyes.
Although LI-COR does not provide microscopy equipment, we have evaluated the near-infrared detection capabilities of microscopes from several manufacturers, particularly in the ~800 nm wavelength region. We are pleased to provide you with guidelines and recommendations for configuring an Olympus or Zeiss microscope for near-infrared detection. The microscope manufacturer can also offer technical assistance.
Microscopic Evaluations Using IRDye Conjugates and Probes
Figure 5-6: Leica DM RXA epifluorescent deconvolution microscope. Outfitted with xenon light source, IRDye 800 filter set from Chroma Technology (EX: HQ740/35x, DC: 770DCXR, EM: HQ780LP), and Cooke Sensicam CCD camera without extended spectral range (quantum efficiency for IRDye 800 emission ~5-10%). Images courtesy of Mark Winey and Harold Fisk, Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder.
Near-Infrared Fluorescence Offers Advantages for Microscopy
Unlike conventional visible fluorophores, IRDye fluorophores absorb and emit light in the NIR region of the light spectrum. Since most biomolecules have very low autofluorescence in the NIR region, IRDye 800CW Infrared Dye provides a level of performance not available with visible dyes. Emission of IRDye 800CW Infrared Dye is separated by more than 100 nm from most commonly used dyes (Cy5, for example, emits at 670 nm), so there is no risk of spectral overlap or cross-talk between channels. Bright, clear images with extremely clean backgrounds and excellent sensitivity like those shown are typical with LI-COR's IRDye fluorophores.
The Infrared Advantage
Very low autofluorescence produces images with exceptionally low backgrounds
Lower-energy NIR excitation wavelengths cause less sample damage than visible wavelengths
NIR dyes have no spectral overlap with visible dyes
Multiplex and co-localize without concerns about channel cross-talk or overlap
Visualize your NIR dye-labeled agent in cells or tissue sections
Figure 9. Very low autofluorescence is detected at 800 nm: NIH 3T3 cells were fixed in 0.2% glutaraldehyde. These unstained cells were viewed using standard blue, green, and red filters, plus an IRDye 800 Infrared Dye filter set from Chroma Technology Corp. Glutaraldehyde fixation caused strong autofluorescence in the visible fluorescence channels (blue, green, and red). However, no autofluorescent background could be seen in the IRDye 800 Infrared Dye channel, even with a very long exposure (signal would be shown as red in false color).
Image was captured with Leica DM RXA epifluorescent deconvolution microscope, outfitted as follows: xenon light source, IRDye 800 filter set from Chroma Technology (EX: HQ740/35x, DC: 770DCXR, EM: HQ780LP), and Cooke Sensicam CCD camera without extended spectral range. Image courtesy of Mark Winey and Harold Fisk, University of Colorado at Boulder.