Fluorescence saturation imaging microscopy: molecular fingerprinting with a standard confocal microscope.

Molecular specificity in fluorescence imaging of cells and tissues can be increased by measuring parameters other than intensity. For instance, fluorescence lifetime imaging became a widespread modality for biomedical optics. Previously, we suggested using the fluorescence saturation effect at pulsed laser excitation to map the absorption cross-section as an additional molecular contrast in two-photon microscopy [Opt. Lett.47(17), 4455 (2022).10.1364/OL.465605]. Here, it is shown that, somewhat counterintuitive, fluorescence saturation can be observed under cw excitation in a standard confocal microscopy setup. Mapping the fluorescence saturation parameter allows obtaining additional information about the fluorophores in the system, as demonstrated by the example of peptide hydrogel, stained cells and unstained thyroid gland. The suggested technique does not require additional equipment and can be implemented on confocal systems as is.

Excited States in Single-Stranded and i-Motif DNA with Silver Ions.

We have studied the excited states and structural properties for the complexes of cytosine (dC)10 chains with silver ions (Ag+) in a wide range of the Ag+ to DNA ratio (r) and pH conditions using circular dichroism, steady-state absorption, and fluorescence spectroscopy along with the ultrafast fluorescence upconversion technique. We also calculated vertical electronic transition energies and determined the nature of the corresponding excited states in some models of the cytosine-Ag+ complexes. We show that (dC)10 chains in the presence of silver ions form a duplex stabilized by C-Ag+-C bonds. It is also shown that the i-motif structure formed by (dC)10 chains is destabilized in the presence of Ag+ ions. The excited-state properties in the studied complexes depend on the amount of binding ions and the binding sites, which is supported by the calculations. In particular, new low-lying excited states appear when the second Ag+ ion interacts with the O atom of cytosine in the C-Ag+-C pairs. A similar picture is observed in the case when one Ag+ ion interacts with one cytosine via the N7 atom.

Exciton Absorption and Luminescence in i-Motif DNA.

We have studied the excited-state dynamics for the i-motif form of cytosine chains (dC)10, using the ultrafast fluorescence up-conversion technique. We have also calculated vertical electronic transition energies and determined the nature of the corresponding excited states in a model tetramer i-motif structure. Quantum chemical calculations of the excitation spectrum of a tetramer i-motif structure predict a significant (0.3 eV) red shift of the lowest-energy transition in the i-motif form relative to its absorption maximum, which agrees with the experimental absorption spectrum. The lowest excitonic state in i-(dC)10 is responsible for a 2 ps red-shifted emission at 370 nm observed in the decay-associated spectra obtained on the femtosecond time-scale. This delocalized (excitonic) excited state is likely a precursor to a long-lived excimer state observed in previous studies. Another fast 310 fs component at 330 nm is assigned to a monomer-like locally excited state. Both emissive states form within less than the available time resolution of the instrument (100 fs). This work contributes to the understanding of excited-state dynamics of DNA within the first few picoseconds, which is the most interesting time range with respect to unraveling the photodamage mechanism, including the formation of the most dangerous DNA lesions such as cyclobutane pyrimidine dimers.