Time-resolved pump–probe (PP) spectroscopy has revolutionized mechanistic investigations in chemistry. In the present work, we introduce a new approach, fluorescence-detected pump–probe (F-PP) spectroscopy, that overcomes several limitations of traditional PP. F-PP suppresses excited-state absorption, provides background-free detection, removes artifacts resulting from pump-pulse scattering, from non-resonant solvent response or from coherent pulse overlap, and allows unique extraction of excited-state dynamics under certain conditions. Despite incoherent detection, time resolution of F-PP is given by the duration of the laser pulses, independent of the fluorescence lifetime. We describe the working principle of F-PP and provide its theoretical description. Then we illustrate specific features of F-PP by direct comparison with PP, theoretically and experimentally. For this purpose, we investigate, with both techniques, a molecular squaraine heterodimer, core-shell CdSe/ZnS quantum dots, and fluorescent protein mCherry. F-PP is broadly applicable to chemical systems in various environments and in different spectral regimes.
https://ift.tt/3j772U1
A novel multi-label synthetic platform for a functional dual fluorescence–spin label probe enables the non-destructive simultaneous quantification and visualization of molecules in biological tissue. Multiplexed FLIM and EPR spectroscopy avoids analytical inconsistencies between both techniques. Beside tissue applications, molecular spectroscopic studies of biomolecular conformation, structure, dynamics, and microenvironment are feasible.
Abstract
Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label-mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof-of-principle penetration experiments in human skin ex vivo.
https://ift.tt/3wCWOOs
Journal of the American Chemical SocietyDOI: 10.1021/jacs.1c01077
Bidyut Sarkar, Kunihiko Ishii, and Tahei Tahara
https://ift.tt/2RpVK1J
Journal of the American Chemical SocietyDOI: 10.1021/jacs.1c00542
Lukas Whaley-Mayda, Abhirup Guha, Samuel B. Penwell, and Andrei Tokmakoff
https://ift.tt/2OEFyrq
A π‐spacer blocks electronic communication between two ionic rhodamine dyes in a dyad isolated in vacuo as it screens for the electric field that each dye senses from the other. Energy transfer based on the dipole–dipole interaction is thus less efficient than that for systems with alkyl spacers.
Abstract
While many key photophysical features are understood for electronic communication between chromophores in neutral compounds, there is limited information on the effect of charges in practically relevant ionic chromo/fluorophores. Here we have chosen positively charged rhodamines and prepared a selection of homo‐ and heterodimers with alkyl or π‐conjugated, acetylenic bridges. Protonated molecules were transferred as isolated ions to gas phase where there is no solvent screening of charges, and fluorescence spectra were measured with a custom‐made ion‐trap setup. Our work reveals strong polarization of the π‐spacer (induced dipole/quadrupole) when it experiences the electric field from one/ two dyes. Hence, π‐spacers provide efficient shielding of charges by reducing the Coulomb interaction, whereas two dye cations polarize each other when connected by an alkyl. The screening influences the Förster resonance energy transfer efficiency that relies on the dipole–dipole interaction.
https://ift.tt/3iGAP2l
Looking for eosin: In situ mapping of fugitive red lake pigments (eosin and carmine lakes) in a van Gogh painting was achieved with molecular fluorescence imaging spectroscopy. The high sensitivity of this method enabled lower concentrations of red lakes to be detected than by elemental X‐ray fluorescence imaging spectroscopy.
Abstract
Vincent van Gogh used fugitive red lake pigments that have faded in some paintings. Mapping their distribution is key to understanding how his paintings have changed with time. While red lake pigments can be identified from microsamples, in situ identification and mapping remain challenging. This paper explores the ability of molecular fluorescence imaging spectroscopy to identify and, more importantly, map residual non‐degraded red lakes. The high sensitivity of this method enabled identification of the emission spectra of eosin (tetrabromine fluorescein) lake mixed with lead or zinc white at lower concentrations than elemental X‐ray fluorescence (XRF) spectroscopy used on account of bromine. The molecular fluorescence mapping of residual eosin and two carmine red lakes in van Gogh's The Olive Orchard is demonstrated and compared with XRF imaging spectroscopy. The red lakes are consistent with the composition of paint tubes known to have been used by van Gogh.
https://ift.tt/38tYT3a
A novel multi-label synthetic platform for a functional dual fluorescence–spin label probe enables the non-destructive simultaneous quantification and visualization of molecules in biological tissue. Multiplexed FLIM and EPR spectroscopy avoids analytical inconsistencies between both techniques. Beside tissue applications, molecular spectroscopic studies of biomolecular conformation, structure, dynamics, and microenvironment are feasible.
Abstract
Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label-mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof-of-principle penetration experiments in human skin ex vivo.
https://ift.tt/3wCWOOs
Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label‐mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof‐of‐principle penetration experiments in human skin ex vivo .
https://ift.tt/2MtGq1B
While many key photophysical features are understood for electronic communication between chromophores in neutral compounds, there is limited information on the effect of charges in practically relevant ionic chromo/fluorophores. Here we have chosen positively charged rhodamines and prepared a selection of homo‐ and heterodimers with alkyl or π‐conjugated, acetylenic bridges. Protonated molecules were transferred as isolated ions to gas phase where there is no solvent screening of charges, and fluorescence spectra were measured with a custom‐made ion‐trap setup. Our work reveals strong polarization of the π‐spacer (induced dipole/quadrupole) when it experiences the electric field from one / two dyes. Hence, π‐spacers provide efficient shielding of charges by reducing the Coulomb interaction, whereas two dye cations polarize each other when connected by an alkyl. The screening influences the Förster Resonance Energy Transfer efficiency that relies on the dipole‐dipole interaction.
https://ift.tt/3iGAP2l
