Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences

Masahiko Taniguchi; Gongfang Hu; Rui Liu; Hai Du; Jonathan S. Lindsey

doi:10.1117/12.2302709

20 February 2018 Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences

Masahiko Taniguchi, Gongfang Hu, Rui Liu, Hai Du, Jonathan S. Lindsey

Author Affiliations +

Proceedings Volume 10508, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications X; 1050806 (2018) https://doi.org/10.1117/12.2302709
Event: SPIE BiOS, 2018, San Francisco, California, United States

Abstract

Demands in flow cytometry for increased multiplexing (for detection of multiple antigens) and brightness (for detection of rare entities) require new fluorophores (i.e., “colors”) with spectrally distinct fluorescence outside the relatively congested visible spectral region. Flow cytometry fluorophores typically must function in aqueous solution upon bioconjugation and ideally should exhibit a host of photophysical features: (i) strong absorption, (ii) sizable Stokes shift, (iii) modest if not strong fluorescence, and (iv) narrow fluorescence band. Tandem dyes have long been pursued to achieve a large effective Stokes shift, increased brightness, and better control over the excitation and emission wavelengths. Here, the attractive photophysical features of chlorophylls and bacteriochlorophylls – Nature’s chosen photoactive pigments for photosynthesis – are described with regards to use in flow cytometry. A chlorophyll (or bacteriochlorophyll) constitutes an intrinsic tandem dye given the red (or near-infrared) fluorescence upon excitation in the higher energy ultraviolet (UV) or visible absorption bands (due to rapid internal conversion to the lowest energy state). Synthetic (bacterio)chlorins are available with strong absorption (near-UV molar absorption coefficient ε(λ_exc) ~10⁵ M^-1cm^-1), modest fluorescence quantum yield (Φf = 0.05–0.30), and narrow fluorescence band (10–25 nm) tunable from 600–900 nm depending on synthetic design. The “relative practical brightness” is given by intrinsic brightness [ε(λ_exc) x Φ_f] times η_f, the fraction of the fluorescence band that is captured by an emission filter in a multicolor experiment. The spectroscopic features of (bacterio)chlorins are evaluated quantitatively to illustrate practical brightness for this novel class of fluorophores in a prospective 8-color panel.

Citation Download Citation

Masahiko Taniguchi, Gongfang Hu, Rui Liu, Hai Du, and Jonathan S. Lindsey "Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences", Proc. SPIE 10508, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications X, 1050806 (20 February 2018); https://doi.org/10.1117/12.2302709

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