Optical modulation of fluorophores based on dark state photophysics

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Mahoney, Daniel
Dickson, Robert M.
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Fluorescence microscopy is an established technique in chemical and biological imaging, allowing signal of interest from fluorescent molecules to be detected over background. However, autofluorescent background and finite imaging depth limit signal to noise in traditional fluorescence imaging. Amplitude modulation is one way to increase signal to noise, and by modulation and subsequent demodulation of fluorescent signal, but not background, allows for greater signal to noise as well as imaging depth. The properties of molecular photophysics involving a nonfluorescent dark state allow for application of modulation by controlling fluorescence signal intensity. This has been demonstrated previously by work from the Dickson Lab using triplet, photoisomer, and electron transfer dark states. In this work, new pentamethine cyanine derivatives were tested experimentally using single and dual laser modulation techniques to determine fluorescence enhancement and photophysical dark state kinetics. Application of these techniques showed that derivatives with longer alkyl substituents had greater fluorescence enhancement (modulation depth) as well as longer on and off times (longer lived dark states). Molecules with short alkyl chains and halogen substituents on the polymethine bridge exhibited lower modulation depth and shorter on and off times. In the case of these cyanine dyes, increased fluorescence enhancement is correlated with longer-lived dark states, while cyanines with shorter-lived dark states show less enhancement. Longer dark state lifetimes allow for greater dark state buildup leading to greater fluorescence recovery, whereas shorter dark state lifetimes yield less fluorescence recovery. By investigating the mechanism of modulation using experimental and theoretical methods we can determine energetics of the photoisomer dark states as well the photoisomer responsible for the fluorescence modulation. By using dual laser modulation, thermal dark state population can be estimated and used to calculate the dark state-ground state energy difference via the Boltzmann distribution. This is compared to Density Functional Theory calculations of the all trans ground state and various cis photoisomers, showing that isomerization about the middle of the polymethine bridge is most likely responsible for the modulatable dark state, with other states possibly playing a minor role. A new modulation scheme was applied to Merocyanine 540 which has both red-shifted photoisomer and triplet absorptions. Experiments show that photoisomer dark states recover the fluorescent ground state upon dark state recovery while triplet dark states transition to the fluorescent excited state and subsequently fluoresce. This effect allows for optically activated delayed fluorescence and can be utilized for fluorescence recovery by a red-shifted excitation source. The triplet state depends on O2 concentration, so removing molecular oxygen using a nitrogen gas purge, an enzymatic oxygen scavenging system, or by immobilizing in a polymer film will all extend triplet lifetimes. Finally, a protein-binding chromophore was studied using fluorescence modulation. This molecule is an analog of the green fluorescent protein and binds to human serum albumin. Upon binding, the chromophore goes from nonfluorescent in solution to brightly fluorescent with ~40% modulation depth upon longer wavelength secondary co-illumination.
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