Dr. Hai Du Profile

Dr. Hai Du

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Proceedings Article | 28 May 2024 Paper

Appropriate handling of fluorescence spectra for accurate spectral overlap (J) values in Förster energy transfer (FRET) calculations

Masahiko Taniguchi, Hai Du, Jonathan Lindsey

Proceedings Volume 13083, 130831J (2024) https://doi.org/10.1117/12.3025086

KEYWORDS: Fluorescence, Fluorescence resonance energy transfer, Absorption spectrum, Fluorescence intensity, Absorption, Chromophores, Energy transfer, Photochemistry, Light absorption

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Fluorescence (or Förster) Resonance Energy Transfer (FRET) describes the flow of energy from a donor fluorophore to an acceptor chromophore. Among the parameters in determining FRET efficiency is the donor–acceptor energy matching, given by the overlap of the donor fluorescence spectrum and the acceptor absorption spectrum (J value). Calculations of the J value typically rely on experimentally acquired spectra. When a fluorescence spectrum is converted from the wavelength (nm) scale to the wavenumber (cm^-1 ) scale, the Y-axis intensity needs to be corrected by the square of the wavelength (termed the λ² correction), because fluorescence spectra (but not absorption spectra) are collected with a fixed wavelength bandpass. The λ² correction causes the peak intensity of the short-wavelength (long-wavenumber) side of the fluorescence spectrum to decrease, or the peak intensity of the long-wavelength (short-wavenumber) side to increase. This issue has been known for ⪆60 years but the impact remains little appreciated. The relatively new availability of libraries of spectral data enabled assessment here of the λ² correction for various donor–acceptor pairs (i.e., donor fluorescence spectra and acceptor absorption spectra). The magnitude of error introduced upon omitting the λ² correction increases with the width of the spectra. A meta-analysis of recent literature reveals trends in usage of wavenumber or wavelength scales. While either scale can be used, errors in calculating the ostensibly simple J term are not uncommon. Best methods are articulated here. The software program PhotochemCAD 3, which incorporates the λ² correction for FRET calculations and also contains diverse spectral libraries, is freely downloadable at www.photochemcad.com.

Proceedings Article | 20 February 2018 Paper

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 Lindsey

Proceedings Volume 10508, 1050806 (2018) https://doi.org/10.1117/12.2302709

KEYWORDS: Luminescence, Absorption, Flow cytometry, Near infrared, Bandpass filters, Chromophores, Ultraviolet radiation, Quantum efficiency, Zinc, Multiplexing

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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.

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