Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection:  Evidence of Two-Step Photolysis

Abstract

The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, p(b), is in the order of 10(-)(6)-10(-)(7) for irradiances below 10(3) W/cm(2). However, a considerable increase of p(b) for irradiances above this level was observed which can only be described by photobleaching reactions from higher excited states (two-step photolysis). In view of these observations, the probability of photobleaching, p(b), as well as a closed expression of its dependence on the CW excitation irradiance considering a five-level molecular electronic state model with the possibility of photobleaching from higher excited electronic states, is derived. From this model, optimal conditions for SMD with respect to the number of emitted fluorescence photons and to the signal-to-background ratio are discussed, taking into account both saturation and photobleaching. The additional photobleaching due to two-step photolysis limits the applicable irradiance.

Conformational changes of the H+‐ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence

“…Fluorescence photons are emitted as bursts when a single enzyme di¡uses through the confocal volume. At the mean excitation irradiance of I H /2 = 4U10 R W/cm P , the mean number of photons emitted by a single TMR £uorophore in water is about 10 T before photobleaching occurs [14]. Since the transit time of the labeled EF I is about 3 ms, the molecule can emit about 30 000 photons during this period [14].…”

“…At the mean excitation irradiance of I H /2 = 4U10 R W/cm P , the mean number of photons emitted by a single TMR £uorophore in water is about 10 T before photobleaching occurs [14]. Since the transit time of the labeled EF I is about 3 ms, the molecule can emit about 30 000 photons during this period [14]. Assuming a detection e¤ciency of about 3% [15], about 900 £uores-cence photons should be detected from a single molecule transit.…”

Iodide as a Fluorescence Quencher and Promoter—Mechanisms and Possible Implications

“…Due to the relatively high irradiances that can be used in FCS experiments, effects of excitation to higher singlet and triplet states (S n and T n states) can sometimes be noticed. In particular, from these states, photooxidation and photo-bleaching is typically strongly enhanced 7,[11][12][13] . To describe the generation of photo-oxidized states in the FCS experiments it is therefore motivated to include higher excited states (S n and T n ) into the electronic state model, as outlined in Figure …”

“…Also the excitation conditions can have a substantial influence [11][12][13][14] . High excitation rates, as often used in SMD or FCS experiments, or in other applications where high read-out rates or high sensitivities are required, can strongly increase the generation of several long-lived, photo-induced states of the dyes, such as triplet states, photo-isomerised states, photo-oxidized states or other states generated by photo-induced charge transfer 8,[12][13][14][15] . The multitude of states generated, and that the effect of the added photo-stabilizers and quenchers may lead to the formation of additional intermediate states increases the complexity.…”

Hidden Markov Model Analysis of Multichromophore Photobleaching

“…The existence of these states in TMR and other organic dyes has been well established in the literature. [38][39][40][41][42][43][44][45] Transitions between the two domains of states are characterized by two rates: photobleaching from bright to dark (k pb ) and recovery from dark to bright k rec ). Reducing the state space using this two-state model brings the problem to a tractable level.…”