Use of single-molecule spectroscopy to tackle fundamental problems in biochemistry: using studies on purple bacterial antenna complexes as an example

2009 ◽  
Vol 422 (2) ◽  
pp. 193-205 ◽  
Author(s):  
Richard J. Cogdell ◽  
Jürgen Köhler
Keyword(s):  
Electronic Structure ◽  
Energy Transfer ◽  
Single Molecule ◽  
Physical Structure ◽  
Single Molecule Detection ◽  
Transfer Reactions ◽  
Single Molecule Spectroscopy

Optical single-molecule techniques can be used in two modes to investigate fundamental questions in biochemistry, namely single-molecule detection and single-molecule spectroscopy. This review provides an overview of how single-molecule spectroscopy can be used to gain detailed information on the electronic structure of purple bacterial antenna complexes and to draw conclusions about the underlying physical structure. This information can be used to understand the energy-transfer reactions that are responsible for the earliest reactions in photosynthesis.

Towards Single-Molecule Diagnostics Using Microfluidic Manipulation and Quantum Dot Nanosensors

2007 ◽  
Author(s):  
Hsin-Chih Yeh ◽  
Christopher M. Puleo ◽  
Yi-Ping Ho ◽  
Tza-Huei Wang
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Detection ◽  
Specific Analysis ◽  
Low Volume

In this report, we review several single-molecule detection (SMD) methods and newly developed nanocrystal-mediated single-fluorophore strategies for ultrasensitive and specific analysis of genomic sequences. These include techniques, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis, which allow separation-free detection of low-abundance DNA sequences and mutational analysis of oncogenes. Microfluidic approaches developed for use with single-molecule detection to achieve rapid, low-volume, and quantitative analysis of nucleic acids, such as electrokinetic manipulation of single molecules and confinement of sub-nanoliter samples using microfluidic networks integrated with valves, are also discussed.

scholarly journals λ/2 Fabry Pérot micro-resonators in single molecule spectroscopy

2018 ◽  
Vol 190 ◽  
pp. 02007
Author(s):  
Alfred J. Meixner ◽  
Frank Wackenhut ◽  
Alexander Konrad ◽  
Michael Metzger ◽  
Marc Brecht
Keyword(s):  
Energy Transfer ◽  
Quantum Yield ◽  
Quantum Dot ◽  
Single Molecule ◽  
Single Molecule Spectroscopy ◽  
Radiative Relaxation ◽  
Fabry Perot ◽  
Förster Energy Transfer ◽  
Micro Resonator ◽  
Dye Molecule

Embedded in a tuneable λ/2-FabryPérot micro-resonator the radiative relaxation of a dye molecule or quantum dot can reproducibly be modified allowing to determine their quantum yield, control Förster energy-transfer or localize them with nanometer precision.

Incorporation of STED technique into single-molecule spectroscopy to break the concentration limit of diffusing molecules in single-molecule detection

2018 ◽  
Vol 54 (69) ◽  
pp. 9667-9670
Author(s):  
Namdoo Kim ◽  
Jiwoong Kwon ◽  
Youngbin Lim ◽  
Jooyoun Kang ◽  
Sohyeon Bae ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Detection ◽  
Concentration Limit ◽  
Single Molecule Spectroscopy

Incorporation of STED into ALEX-FRET increases the concentration limit of single-molecule detection by 100-fold to 5 nM.

scholarly journals Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay.

2021 ◽  
Author(s):  
Derrick Lau ◽  
Chloe Magnan ◽  
Kathryn Hill ◽  
Antony Cooper ◽  
Yann Gambin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Size Exclusion ◽  
Single Molecule Detection ◽  
Single Molecule Spectroscopy ◽  
Parkinsons Disease ◽  
Exclusion Chromatography ◽  
Particle Level ◽  
Amplification Assay ◽  
Multiple Species

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinsons disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes

2006 ◽  
Vol 39 (3) ◽  
pp. 227-324 ◽  
Author(s):  
Richard J. Cogdell ◽  
Andrew Gall ◽  
Jürgen Köhler
Keyword(s):  
Quantum Mechanics ◽  
Energy Transfer ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Light Harvesting ◽  
Structural Information ◽  
Purple Bacteria ◽  
Theoretical Background ◽  
Single Molecule Spectroscopy

1. Introduction 2292. Structures 2342.1 The structure of LH2 2342.2 Natural variants of peripheral antenna complexes 2422.3 RC–LH1 complexes 2423. Spectroscopy 2493.1 Steady-state spectroscopy 2493.2 Factors which affect the position of the Qy absorption band of Bchla 2494. Regulation of biosynthesis and assembly 2574.1 Regulation 2574.1.1 Oxygen 2574.1.2 Light 2584.1.2.1 AppA: blue-light-mediated regulation 2594.1.2.2 Bacteriophytochromes 2594.1.3 From the RC to the mature PSU 2614.2 Assembly 2614.2.1 LH1 2624.2.2 LH2 2635. Frenkel excitons 2655.1 General 2655.2 B800 2675.3 B850 2675.4 B850 delocalization 2736. Energy-transfer pathways: experimental results 2746.1 Theoretical background 2746.2 ‘Follow the excitation energy’ 2766.2.1 Bchla→Bchla energy transfer 2776.2.1.1 B800→B800 2776.2.1.2 B800→B850 2786.2.1.3 B850→B850 2796.2.1.4 B850→B875 2806.2.1.5 B875→RC 2806.2.2 Car[harr ]Bchla energy transfer 2817. Single-molecule spectroscopy 2847.1 Introduction to single-molecule spectroscopy 2847.2 Single-molecule spectroscopy on LH2 2857.2.1 Overview 2857.2.2 B800 2867.2.2.1 General 2867.2.2.2 Intra- and intercomplex disorder of site energies 2877.2.2.3 Electron-phonon coupling 2897.2.2.4 B800→B800 energy transfer revisited 2907.2.3 B850 2938. Quantum mechanics and the purple bacteria LH system 2989. Appendix 2999.1 A crash course on quantum mechanics 2999.2 Interacting dimers 30510. Acknowledgements 30611. References 307This review describes the structures of the two major integral membrane pigment complexes, the RC–LH1 ‘core’ and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to ‘power’ the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

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