Using photon statistics to boost microscopy resolution.
Explore the source record for details and available documents.
DNA↗
Biomedical subjects
Xavier Michalet
Publications and source records attributed to Xavier Michalet.
Single-molecule fluorescence studies of protein folding and conformational dynamics.
Explore the source record for details and available documents.
Energy Transfer↗
Advances in fluorescence imaging with quantum dot bio-probes.
After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.
Chromogenic Compounds↗
Thoughtful peer review is worth the time it takes.
Explore the source record for details and available documents.
Peer Review, Research↗
Protein-protein interactions as a tool for site-specific labeling of proteins.
Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.
Cysteine↗
Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.
Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and x-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.
Biophysics↗
New light on quantum dot cytotoxicity.
As quantum dots are beginning to be used for in vivo imaging, the question of their long-term effect on cell viability is becoming critical. In this issue of Chemistry & Biology, Lovrić and colleagues examine the likely role of reactive oxygen species in quantum dot cytotoxicity .
Animals↗
Femtomole mixer for microsecond kinetic studies of protein folding.
We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 mus and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Forster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.
Acyl Coenzyme A↗
The power and prospects of fluorescence microscopies and spectroscopies.
Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.
Equipment Design↗