Description
Springer Biophysics And The Challenges Of Emerging Threats by Joseph Puglisi
Single-Molecule Techniques Eliminate Ensemble Averaging Thus Revealing Transient Or Rare Species In Heterogeneous Systems [1-3]. These Approaches Have Been Employed To Probe Myriad Biological Phenomena Including Protein And Rna Folding [4-6] Enzyme Kinetics [7 8] And Even Protein Biosynthesis [1 9 10]. In Particular Immobilization-Based Fluorescence Te- Niques Such As Total Internal Reflection Fluorescence Microscopy (Tirf-M) Have Recently Allowed For The Observation Of Multiple Events On The Millis- Onds To Seconds Timescale [11-13]. Single-Molecule Fluorescence Methods Are Challenged By The Instability Of Single Fluorophores. The Organic Fluorophores Commonly Employed In Single-Molecule Studies Of Biological Systems Display Fast Photobleaching Intensity Fluctuations On The Millisecond Timescale (Blinking) Or Both. These Phenomena Limit Observation Time And Complicate The Interpretation Of Fl- Rescence Fluctuations [14 15]. Molecular Oxygen (O) Modulates Dye Stability. Triplet O Efficiently 2 2 Quenches Dye Triplet States Responsible For Blinking. This Results In The For- Tion Of Singlet Oxygen [16-18]. Singlet O Reacts Efficiently With Organic Dyes 2 Amino Acids And Nucleobases [19 20]. Oxidized Dyes Are No Longer Fluor- Cent; Oxidative Damage Impairs The Folding And Function Of Biomolecules. In The Presence Of Saturating Dissolved O Blinking Of Fluorescent Dyes Is Sup- 2 Pressed But Oxidative Damage To Dyes And Biomolecules Is Rapid. Enzymatic O -Scavenging Systems Are Commonly Employed To Ameliorate Dye Instability. 2 Small Molecules Are Often Employed To Suppress Blinking At Low O Levels.