The use of organic fluorescent probes in biochemical and biophysical applications of single molecule spectroscopy and fluorescence microscopy techniques continues to increase. As single molecule measurements become more quantitative and new developments in super-resolution imaging allow researchers to image biological materials with unprecedented resolution, it is becoming increasingly important to understand how the properties of the probes influence the signals measured in these experiments. In this review, we focus on the photochemical and photophysical processes of organic fluorophores that affect the properties of fluorescence emission. This includes photobleaching, quenching, and the formation of non-emissive (dark) states that result in fluorescence blinking in a variety of timescales. These processes, if overlooked, can result in an erroneous interpretation of the data. Understanding their physical origins, on the other hand, allows researchers to design experiments and interpret results so that the maximum amount of information about the system of interest can be extracted from fluorescence signals.

Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.

10Chirped-Pulse millimetre-Wave (CPmmW) rotational spectroscopy provides a new class of information about photolysis transition state(s). Measured intensities in rotational spectra determine species-isomervibrational populations, provided that rotational populations can be thermalized. The formation and detection of S 0 vinylidene is discussed in the limits of low and high initial rotational excitation. CPmmW 15 spectra of 193 nm photolysis of Vinyl Cyanide (Acrylonitrile) contain J=0-1 transitions in more than 20 vibrational levels of HCN, HNC, but no transitions in vinylidene or highly excited local-bender vibrational levels of acetylene. Reasons for the non-observation of the vinylidene co-product of HCN are discussed. A. Introduction 20Chirped-Pulse millimetre-Wave (CPmmW) spectroscopy 1-3 is capable of determining the relative species-conformervibrational level populations of all polar products of a photolysis reaction. These experimentally determined populations encode the structures of the transition states 25 that are most important at each photolysis wavelength or combination of wavelengths. 4 The isomer-conformervibrational level population information obtained from a CPmmW spectrum is more complete than what is obtainable by mass spectrometry 5 and free of the need for 30 the transition strength and quantum yield determinations that are required for most laser-based population measurements. However, difficulties exist in the use of populations determined by CPmmW spectroscopy to characterize transition states. B. Chirped Pulse SpectroscopyChirped Pulse Fourier Transform Microwave Spectroscopy 55 is a revolutionary technique developed in the research group of Brooks Pate at the University of Virginia. 2,3 In its initial implementation, the frequency of a microwave pulse is chirped linearly in time over several GHz. This microwave pulse is broadcast into a gas phase molecular 60 sample, polarizing all two-level systems with frequencies within the spectral interval of the chirped pulse. These polarizations relax by Free Induction Decay (FID), which is a voltage vs. time signal that is collected, downconverted by mixing with a local oscillator (heterodyne where  is the electric dipole moment,  0 is the peak microwave electric field, N 1,2 is the population density 75 difference between levels 1 and 2,  is the chirp rate, A is

The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.

Irradiation of solutions of the cyanine dyes Cy3, Cy3B, and Cy5 in the presence of Mn(2+) causes an increase in the yield of formation of the triplet state of the dye. This results in increased photobleaching and triplet blinking. Experiments with other divalent ions and paramagnetic molecules suggest that the enhancement in the intersystem-crossing rate is related to the paramagnetic nature of the Mn(2+) cation. The results are consistent with a model in which the formation of a weak collisional complex between the dye and the ion results in mixing of the singlet and triplet states of the dye. These findings are particularly significant in single-molecule spectroscopy and super-resolution imaging methods, in which photobleaching and blinking play an important role.