Decreasing photobleaching by silver island films: application to muscle

Abstract

Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10 −18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-to 5-fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible. Keywords Photobleaching; Silver island films; MicroscopyRecently it has become possible to study single protein molecules in a cell. This avoids problems associated with averaging responses from an assembly of molecules with different kinetics and problems associated with diluting of proteins in vitro. The difficulties of studying large assemblies of molecules are well illustrated by the problem of muscle contraction. Contraction results from the interactions of myosin cross-bridges with actin. Myosin crossbridges act asynchronously, i.e., at any time during muscle contraction each one is in a different part of the mechanochemical cycle. Therefore measurement taken from an assembly of crossbridges at any time during contraction is an average value, likely to obscure details of crossbridge kinetics. Furthermore, it is desirable to detect signal from a working muscle, because contractile proteins may behave differently in solution and in whole muscle, where protein ⋆ Dedicated to Professor Avraham Oplatka on the occasion of his birthday. To be able to obtain information from the individual molecules in muscle, it is necessary to collect data from an extremely small volume, small enough to...

Metal enhanced fluorescence on silicon wafer substrates

“…As expected, no difference in the emission spectra were recorded, and estimated enhancement in fluorescence brightness is above 300%, which is close to enhancement observed on a glass cover slip for Rhodamine-phalloidin (4 fold) [23]. …”

“…We previously observed increased photostability of fluorophores deposited on silver nanoparticels [23–27]. Both, decrease in the fluorescence lifetime and increase in the quantum yield lead to a significant reduction of photodegradation.…”

Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes

“…In the presence of SIFs and some ZMWs lower excitation powers yield the same fluorescence intensities as higher excitation powers in the absence of the metal constructions (Muthu et al, 2007; Rigneault et al, 2005; Wenger et al, 2005). The lower excitation powers help to guard against excessive photobleaching.…”

“…In addition to limiting detection volumes, metallic devices, in particular silver island films (SIF) and some ZMW, can also serve to enhance fluorescence emission (Muthu et al, 2007; Rigneault et al, 2005; Wenger et al, 2005). The quantum yield (Q) of fluorophores is determined by the nonradiative (k nr ) and radiative (Γ) rate constants as Q = Γ/(Γ + k nr ).…”

Fluorescence quenching/enhancement surface assays: Signal manipulation using silver-coated gold nanoparticles

“…In some cases, very low analyte concentrations can be detected on the surface only in presence of silver colloid nanoparticles [26]. It should be noted, that shorter lifetime reduces potential photodegradation of fluorophore [27][28][29].…”