Stokes shift BODIPY

Fluorochromes have been introduced that offer excitation and emission spectra similar to those of fluorescein, but that overcome some of fluorescein s limitations. BODIPY FL has a short Stokes shift, but offers higher fluorescence intensity, and is claimed to be more photostabile and less pH-sensitive than fluorescein. Oregon Green 488 and the newly introduced Alexa 488 fluorochromes have spectra nearly identical to those of fluorescein, but are considerably more photostabile, and produce less quenching of fluorescence with higher numbers of fluorochromes per antibody than does fluorescein. 

The emission spectra of BODIPY derivatives normally display narrow bandwidths, providing intensely fluorescent labels for biomolecules. Unfortunately, they also have very small Stokes shifts, typically on the order of only 10—20 nm. Excitation at the optimal wavelength may cause some interference in measurements at the emission wavelength due to light scattering or cross-over from the wide bandwidth of the excitation source. The dyes usually require excitation at suboptimal wavelengths to prevent this problem. 

The excitation maximum for the molecule occurs at 5 35 nm and its emission at 552 nm. Its Stokes shift is slightly greater than some of the other BODIPY fluorophores, but a 17-nm shift still may not be enough to avoid completely problems of excitation-light interference in emission measurements. BODIPY 530/550 C3 has an extinction coefficient in methanol of about 62,000 M cm 1 at 535 nm. 

The excitation maximum for BODIPY 530/550 C3-SE occurs at 533 nm and its emission at 550 nm. Its Stokes shift is relatively small and may not be enough to avoid... 

The excitation maximum for BODIPY 493/503 C3 hydrazide occurs at 498 nm, and its emission at 506 nm. As this is an extremely small Stokes shift, it may be difficult to avoid completely problems of excitation-light interference in critical emission measurements unless suboptimal excitation wavelengths are used. The molecule has an extinction coefficient in methanol of about 92,000 M-1cm-1 at 493 nm. 

The excitation maximum for Br-BODIPY 493/503 is 515 nm and its emission occurs at 525 nm when dissolved in methanol. On coupling to a sulfhydryl compound, however, the excitation wavelength of the adduct decreases to 493 nm and its emission drops to 503 nm. The very small 10-nm Stokes shift may be a problem, particularly in avoiding interference due to excitation-light scattering in critical emission measure-... 

Fluorescein isothiocyanate (FITC) and dansyl-chloride were among the first extrinsic fluorescent labels for proteins used for immunofluorescence microscopy and polarization measurements. Fluorescein and rho-damine labels were extensively employed due to their bright emission in the visible range. These probes have drawbacks including hydrophobicity, small Stokes shifts, and sensitivity to pH and photobleaching, which led to the development of new dyes such as Alexa, Bodipy, and cyanine dye families (see Table 1). These dyes cover a broad... 

FITC+FAM (fluorescein derivatives). ECFP, EGFP, EYFP (enhanced—cyan, green and yellow fluorescent protein, respectively, Clontech Laboratories, Inc., USA). Bodipy is a trademark of Invitrogen Ltd., UK. Attoxxxis a trademark of ATID-TEC GmbH, Gennany. A A S, and A J), A are the one- and two-photon absorption and emission maximum, respectively. QY is the fluorescence quantum yield, SS the Stokes shift, e the molar extinction coefficient and Tf the fluorescence lifetime. Further information regarding two-photon measurements can be found In Chapter 3. 

The rDA reaction of the peripheral BCOD moiety occurred below 235 °C, whereas extmsion of ethylene from the connecting BCOD moiety between BODIPY and fluoranthene took place above 270 °C and settled down until 310°C [128]. Bis(benzofluorantho)BODIPYs 149 showed the longest absorptions at the NIR range above 760 nm with large molar extinctions of 1.66-2.00 x lO M Vm and strong emissions with small Stokes shifts (16-18 nm) and with high quantum yields (0.32-0.35) (Scheme 15.36). 

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