IRDye Infrared Dyes - See Beyond the Visible

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 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 a microscope for near-infrared detection. The microscope manufacturer can also offer technical assistance.

A. Deconvolved image of A431 cells. pEGFR was detected with appropriate primary antibody and IRDye 800CW goat anti-rabbit polyclonal (PN 926-32211), represented in green. Total ERK was detected with appropriate primary antibody and IRDye 680 goat anti-mouse polyclonal (PN 926-32220) represented in red. Cell nuclei were detected with Sytox Green (Invitrogen), represented in blue .

Image captured using an Olympus IX71/IX81 microscope¹.

B. Deconvolved image of IRDye 800CW RGD peptide binding to A431 (epidermoid carcinoma) cells. Red signal represents IRDye 800CW RGD (LI-COR Biosciences); green signal represents Sytox Green nuclear stain (Invitrogen).

Image captured using an Olympus IX71/IX81 microscope¹.

C. Deconvolved image of IRDye 800CW RGD peptide binding to A549 (lung carcinoma) cells. Blue signal represents IRDye 800CW RGD (LI-COR Biosciences); green signal represents Sytox Green nuclear stain (Invitrogen).

Image captured using an Olympus IX71/IX81 microscope¹.

D. Deconvolved image of IRDye 800CW EGF binding to an A431 cell. Red represents IRDye 800CW EGF (PN 926-08446); green represents Sytox Green nuclear stain (Invitrogen).

Image captured using a Zeiss AxioImager microscope².

E. Three-color image of a mitotic cell. NIH 3T3 cells were stained with anti-tubulin primary and IRDye 800 secondary antibody (shown in red), human CREST serum to show kinetochores (Texas Red™, green (false color)), and Hoechst for chromosomal DNA (blue).

Image captured with a Leica DM RXA microscope³.

F. Staining of duplicated centrosomes. Condensed chromosomes are stained with DAPI (blue). The two centrosomes (red dots) are stained with a primary antibody against pericentrin (a centrosomal component) and IRDye 800 secondary antibody.

Image captured with a Leica DM RXA microscope³.

¹Images A-C: Olympus IX71/IX81 inverted epifluorescent deconvolution microscope. Outfitted with xenon light source, standard Cy7 filter set used for imaging of IRDye 800CW, and CCD camera with extended spectral range.

²Image D: Zeiss AxioImager epifluorescent deconvolution microscope. Outfitted with xenon light source, IRDye 800CW custom filter set from Chroma Technology (EX: HQ760/40x, DC: 790DCXR, EM: HQ830/50m), and CCD camera with extended spectral range.

³Images E-F: 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.

Ready to try IRDye infrared dyes on your epifluorescent microscope?

First, check the microscope’s specifications to make sure it is properly configured. Things to consider:

• Light source

  Recommended Configurations
  for Leading Microscope Manufacturers:

  Olympus • Zeiss

  Your lamp must provide excitation light in the NIR range. Xenon lamps provide low-intensity but very even excitation light over a broad range, including the NIR. Tungsten-halogen lamps emit very strongly in the NIR, and a combination-arc lamp (such as metal-halide) may also be an option. Mercury lamps have very strong emission in the visible region, but their NIR emission is much weaker.

  
  

  To read more about excitation sources, please visit:
  http://www.microscopyu.com/articles/livecellimaging/automaticmicroscope.html

  
  

  Check with the manufacturer of your microscope to find out if your lamp housing has a heat-blocking NIR/IR filter designed to suppress longer-wavelength light. A xenon lamp may have a filter, and a tungsten-halogen lamp is very likely to have one. These filters may interfere with excitation of 800 nm fluorophores.

• Camera
  Some CCD cameras, particularly older models, may have poor response (quantum efficiency) at NIR wavelengths. Newer cameras often have a wider response range and are able to detect NIR light with better efficiency. A camera with higher quantum efficiency will give you the brightest signal possible and allow you to use the shortest exposures, thereby reducing the chance of photobleaching or heating of the sample. Some cameras may contain an IR cutoff filter that rejects longer wavelength light. Check with the manufacturer to see if your camera has an IR filter, and if it can be removed.

• Optics
  Some objectives may have anti-reflective coatings that could block NIR light. Also, not all lenses are corrected for the chromatic aberration that may occur because of wavelength-dependent variation in the refractive index of glass. This could lead to problems with focus. Older lenses are more likely to be uncorrected for longer wavelengths, and may not give optimal performance. Newer, NIR-capable objectives will offer better light throughput and wavelength correction. To learn more about chromatic aberration, contact the manufacturer of your optics or visit http://www.microscopyu.com/tutorials/java/aberrations/chromatic/index.html

• Filters
  Check with the manufacturer to be sure that the emission side of your filter cube does not have a coating that could block NIR light. An empty filter position is required to hold the appropriate NIR filter set.

IRDye 800CW: Several filter sets have reportedly been used successfully to image this dye.

1. IRDye^® 800 Infrared Dye Filter Set: Chroma Technology Catalog #41037
   Exciter HQ740/35x
   Dichroic 770DCXR
   Emitter HQ780LP 2

2. IRDye 800CW Custom Filter Set: composed of three individual filters from Chroma Technology
   Exciter HQ760/40x
   Dichroic 790DCXR
   Emitter HQ830/50m

3. Cy7 Standard Filter Set: Chroma Technology Catalog #41009
   Exciter HQ710/75x
   Dichroic Q750LP
   Emitter HQ810/90m

   All three sets have been found to work with IRDye 800CW. Set number 2 appears to be the best spectral match, but the other sets are acceptable substitutes.

IRDye 680 and IRDye 700DX: For imaging of these dyes, the standard Cy5.5 filter set stocked by Chroma Technology is a good spectral match.

1. Cy5.5 (Red Shifted) Standard Filter Set: Chroma Technology Catalog #41022
   Exciter HQ665/45x
   Dichroic Q695LP
   Emitter HQ725/50m

• Other considerations
  You cannot see near-infrared light when you look through the microscope oculars – it is not visible to the eye. Use a DNA stain such as DAPI or Sytox Green, or co-stain with a visible fluorophore such as Cy3, so you can easily locate cells and focus on them. Then, switch to the NIR channel and use the CCD camera and computer monitor to visualize the NIR image in pseudocolor.

Infrared Advantage

Cell biologists can now add a new "color" to multi-color imaging, with IRDye® infrared dye reagents. Unlike conventional visible fluorophores, IRDye fluorophores absorb and emit light in the near-infrared (NIR) region of the light spectrum.

The Infrared Advantage:

• Very low autofluorescence for exceptionally low backgrounds.

• Lower-energy NIR excitation wavelengths cause less sample damage than visible wavelengths.

• No spectral overlap with visible dyes, allowing you to.

• Multiplex and co-localize without concerns about channel cross-talk or overlap.

• Ability to visualize your NIR-labeled agent in cells or tissue sections.

Very Low Autofluorescence

Primary and secondary antibodies conjugated with LI-COR's IRDye® 800CW Infrared Dye produce images with very low background. IRDye® 800CW is part of LI-COR's exclusive family of NIR dyes that fluoresce at approximately 800 nm. In some instances, autofluorescence may limit the detection of weak signals with visible fluorophores. 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. Bright, clear images with extremely clean backgrounds and excellent sensitivity like those shown are typical with LI-COR's IRDye fluorophores.

No Spectral Overlap With Visible Dyes

The emission maximum of IRDye® 800CW infrared dye is at 789 nm in aqueous solution.

Since the 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), there is no risk of spectral overlap or cross-talk between channels.

Low Autofluorescence at NIR wavelengths

Very low autofluorescence 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 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.

PubMed

Publishing with LI-COR data?

• Kovar, J et al.
  Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models.
  Anal Biochem. 384(2): 254-62 (2009)

• Roy, EJ et al.
  Imaging membrane intercalating near infrared dyes to track multiple cell populations.
  J Immunol Methods. Epub (2009)

• Johnson, ND et al.
  Intrathecal delivery of fluorescent labeled butyrylcholinesterase to the brains of butyrylcholinesterase knock-out mice: visualization and quantification of enzyme distribution in the brain.
  Neurotoxicology. 30(3):386-92 (2009)

• Sampath, L et al.
  Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer.
  J Nucl Med. 48(9):1501-10 (2007)