Far-red organic fluorophores contain a fluorescent impurity

Abstract

Far-red organic fluorophores commonly used in traditional and super-resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near-red emission. This near-red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super-resolution localization microscopy.

Keywords: fluorescence; fluorescent probes; live cells; quantitative imaging; super-resolution imaging.

Figure 1. Near- and far-red emission is observed with simultaneous 640nm and 561nm excitation

a) CH27 cells are labeled with f(Ab)1 anti BCR conjugated to the indicated dye and observed through simultaneous illumination with 561 nm and 640 nm laser light. Emission was separated using a dichroic and bandpass filter set, and reconstructed images of near-red emission (576 nm–630 nm) are shown in green and far-red emission (660 nm—740 nm) shown in red. Scale bars are 3 µm. b) The number of events per second per µm² detected during the simultaneous excitation as bar plots. c) The average event intensity per second for mEos3.2, Alexa 647, and Cy5 excited by 561nm laser light. Laser intensity and camera settings were kept constant between measurements. Error bars indicate one standard deviation.

Figure 2. Near- and far-red emission is observed with simultaneous 640nm and 532nm excitation

a) CH27 cells are labeled with f(Ab)1 anti BCR that was conjugated to the indicated dye and observed through simultaneous illumination with 532 nm and 640 nm laser light. Emission was separated using a dichroic and bandpass filter set, and reconstructed images of near-red emission (545 nm—620 nm) are shown in green and far-red emission (660 nm—740 nm) shown in red. Scale bars are 3 µm. b) The number of events per second per µm² detected during the simultaneous excitation as bar plots. c) The average event intensity per second for Alexa 532, Alexa 647, and Cy5 excited by 532 laser light. Laser intensity and camera settings were kept constant between measurements. Error bars indicate one standard deviation.

Figure 3. Near-red emission is observed in bulk fluorescence measurements

a) The fluorescence emission spectrum of Alexa 647 either as the unconjugated NHS ester (800nM) or conjugated to f(Ab)1 fragments (50 µg/ml). Samples are excited by 530 nm light with identical gain and bandwith. The magnitude of near-red emission and the overall shape of the spectra are comparable between the two samples. b) Fluorescence emission spectra of the four dyes with excitation at 530 nm. All dyes are diluted to 1 µM in PBS and the near-red portion of the spectrum is shown. c) Fluorescence excitation and emission spectra of the near-red moiety in Alexa 647 NHS ester. The excitation spectrum is acquired by monitoring emission at 575±2 nm over a range of excitation wavelengths. The emission spectrum is acquired by exciting at 530 nm. The gain is held constant within but not between each subfigure.

Figure 4. Fluorescence and absorbance spectra are not concentration dependent

a) Fluorescence emission spectra at 530 nm excitation of unconjugated Alexa 647 NHS ester diluted into PBS at various concentrations. Fluorescence intensity is normalized by the concentration of the dye. b) Absorbance spectra of unconjugated Alexa 647 NHS ester diluted into PBS at various concentrations. Absorbance is normalized by the concentration of the dye.

Figure 5. FCS correlation curves for unconjugated Alexa 647

Time correlation curves for Alexa 647 far-red and near-red emission were determined by diluting Alexa 647 to 8 nM and exciting with 630 nm light, or by diluting Alexa 647 to 4 µM and exciting with 530 nm light. Dotted black lines are fits to Eq. 1 from Methods, and are used to extract fit parameters for Table 1.