Biomedical subjects

Julian Borejdo

Publications and source records attributed to Julian Borejdo.

3 recordsLinked to original sources

PubMed2006

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy.

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.

Animals↗

PubMed2006

Measuring rotations of a few cross-bridges in skeletal muscle.

The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.

Fluorescent Dyes↗

PubMed2002

Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body.

Adenosine Triphosphate↗