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Microscopy

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.

Deconvolved image of A431 cells

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.

Deconvolved image of IRDye 800CW RGD peptide binding to A431 (epidermoid carcinoma) cells

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.

Deconvolved image of IRDye 800CW RGD peptide binding to A549 (lung carcinoma) cells

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.

Deconvolved image of IRDye 800CW EGF binding to an A431 cell

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.

Three-color image of a mitotic cell

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.

Staining of duplicated centrosomes

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.



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