CF 680 and CF 680R are two outstanding near-IR dyes excitable at about 680 nm with emission at about 700 nm. The two dyes each have unique properties suitable for different application needs. CF 680 is a highly water-soluble cyanine-based dye with a molecular weight of ~3000.This dye is excellent for labeling antibodies, producing the brightest fluorescence and highest signal-to-noise ratio in immunostaining among spectrally similar dyes, such as Cy 5.5, Alexa Fluor 680, DyLight 680 and IRDye 680. However, because of its relatively large molecular size, CF 680 is not recommended for labeling nucleic acids or relatively small biomolecules, for which our CF 680R is better suited.CF 680R is a novel rhodamine-based dye with a relatively small molecular weight of 912. The dye is highly fluorescent and, more importantly, extremely photostable. Rhodamine dyes are traditionally known to be bright and photostable. However, it has been a synthetic challenge to make functional rhodamine dyes with long wavelengths and high water solubility necessary for bio-labeling.Scientists at Biotium have overcome the challenge and invented a new way to make highly water soluble, bright and photostable rhodamine dyes with wavelengths ranging from red to near-IR. These dyes are excellent for labeling proteins, nucleic acids and small bio-molecules. They are ideal for confocal microscopy, single molecule imaging and other applications that demand both brightness and photostability. A full selection of reactive dyes, secondary antibodies, antibody labeling kits, and other bioconjugates including phalloidins, Annexin V and -bungarotoxin are available for CF dyes. Figure 2. Jurkat cells were stained with isotype or mouse anti-human intracellular CD3 antibody followed by 1 mg of goat anti-mouse IgG conjugated with Cy 5.5, Alexa Fluor 680, CF 680 or DyLight 680. Fluorescence was detected by a BD FACS Calibur in the FL4 channel. The bars represent the signal-to-noise ratio of CD3 positive fluorescence to isotype using similar degrees of labeling (DOL). Figure 3. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF 680R (Biotium), Alexa Fluor 680 (Invitrogen) or Cy 5.5 (GE Healthcare) goat anti-rabbit IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy 5 filter set and CCD camera. Images were taken at t=0, 1 min and 5 min. Figure 4. Photostability comparison by microscopy. Jurkat cells were fixed, permeabilized and stained with rabbit anti-CD3 (Abcam) followed by CF 680R, CF F680, Cy 5.5 or Alexa Fluor 680 (AF680) goat anti-mouse IgG conjugates. Cells were imaged using an Olympus mercury arc lamp microscope equipped with a Cy 5 filter set and CCD camera. The graph illustrates the relative fluorescence intensities of sequential images taken every 10 seconds for 5 minutes. Figure 5. HeLa cells were stained with mouse a-tubulin antibody followed by CF 680 goat anti-mouse IgG. Images were captured using an Olympus mercury arc lamp microscope equipped with a Cy5 filter set, CCD camera and ImagePro Express software. Figure 6. Near-IR CF 680 and CF 770 for two-color Western blotting. Two-fold dilutions of HeLa cell lysate were run on an acrylamde gel, transferred to a nitrocellulose membrane and probed with mouse alpha-tubulin and rabbit COX IV primary antibodies followed by goat antimouse CF 770 or IRDye 800 (green) and goat anti-rabbit CF 680 or IRDye 680 (red) at the same final concentrations. After probing, membranes were dried and scanned using an Odyssey infrared imaging system (LI-Cor Biosciences). Quantitation of the bands showed approximately a 3.5-fold higher fluorescence intensity of CF dyes compared to the respective IRDye secondary antibodies (LI-Cor ).