Exploring Life by Single-Molecule Fluorescence Spectroscopy

Abstract

onsidering all the effort scientists put into visualizing individual atoms and molecules, it is amazing that it only takes a standard fluorescence microscope with high collection efficiency and suitable filters to detect the fluorescence emission of a single fluorophore. In <20 years, several innovations and technical breakthroughs have brought us to the point where we can study matter on a molecular scale-a skill unimaginable 50 years ago. Parallel to these developments, biochemistry and molecular and cellular biology have grown from infancy into prominent disciplines. New, exciting insights into biomolecular structure, self-assembly, dynamics, and regulation within living systems are results of refined methodological and experimental developments in biochemistry and molecular biology.We can now directly visualize and track individual molecules in their native environment; this allows us to study biological systems on a molecular scale. For example, single-molecule techniques are used to visualize the enzymatic mechanisms of DNA polymerase, which is responsible for the replication of genetic information. DNA polymerase synthesizes a DNA strand by incorporating nucleotides while moving like a locomotive on the complementary DNA template. We no longer calculate incorporation rates by looking at average DNA lengths; rather, we directly track the individual enzyme that is incorporating the nucleotides. In addition, we gain new insights into the working mechanism and performance of the enzyme.To see the difference between single-molecule and ensemble experiments, imagine that you are at a large station observing hundreds of passengers arriving on an unknown number of trains. From such an observation, you cannot answer questions about the individual routes the trains took, how many passengers boarded the train at which stations and at what times, or how many stops each train made. You observe only an average and can conclude only that trains typically transport hundreds of passengers at a time. If single-molecule spectroscopy is used to monitor biological reactions, individual properties can

Fluorescent proteins for single‐molecule fluorescence applications

“…Recently, fluorescent proteins have also entered the field of single-molecule spectroscopy and microscopy [7][8][9][10][11][12]. Single-molecule fluorescence applications of fluorescent proteins are particularly attractive because they enable the visualization of proteins of interest within living cells without any synchronization procedure [13][14][15]. Although single-molecule fluorescence studies of GFP-or yellow fluorescent protein (YFP)-fusion proteins were demonstrated, their complicated photophysics and low photostability render their application in single-molecule experiments still very difficult [16][17][18][19][20].…”

A Close Look at Fluorescence Quenching of Organic Dyes by Tryptophan

“…[9] The PET-based quenching mechanism lays the foundation for a single-molecule sensitive tool that allows probing of conformational changes in macromolecules through a reporter dye that is in a fluorescent or nonfluorescent state, depending on the proximity of a quencher molecule. [23][24][25] The use of PET interactions to study conformational dynamics in biomolecules is in many aspects different from that of fluorescence resonance energy transfer (FRET). Neglecting through-bond electron-transfer mechanisms in labeled biopolymers, efficient PET between an oxazine dye and Trp residue can only occur if the dye and quencher are at van der Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies.…”

“…Contact formation induced quenching, where the resulting complex can be considered nonfluorescent, simplifies data analysis by allowing the use of a two-state model to describe fluorescence switching between an on and off state. [24,25] In addition, the transitions can be observed with high signal-to-noise ratios, allowing single-molecule experiments. [10,24,25] Fluorescence correlation spectroscopy (FCS) measures fluorescence fluctuations arising from fluorescent molecules as they pass the observation volume.…”

“…[24,25] In addition, the transitions can be observed with high signal-to-noise ratios, allowing single-molecule experiments. [10,24,25] Fluorescence correlation spectroscopy (FCS) measures fluorescence fluctuations arising from fluorescent molecules as they pass the observation volume. FCS can be used to obtain information about any dynamic process at the molecular level that results in fluctuations of the fluorescence intensity.…”

In Situ Single-Molecule Imaging with Attoliter Detection Using Objective Total Internal Reflection Confocal Microscopy

“…Zero-mode waveguides are not scanned, but they define a tiny illuminated volume on the sample surface. Either near-field method produces detection volumes of ∼1 attoL Total internal reflection microscopy (TIRM) 1 produces an evanescent field on the water side of the glass/water interface made by a coverslip contacting aqueous solution (15). Excitation is delivered through the glass side by propagating light incident on the interface at angles beyond the critical angle for total internal reflection (16).…”