Biomedical subjects

Harshad D Vishwasrao

Publications and source records attributed to Harshad D Vishwasrao.

5 recordsLinked to original sources

PubMed2005

Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution.

Water-soluble quantum dots (qdots) are now being used in life sciences research to take advantage of their bright, easily excited fluorescence and high photostability. Although the frequent erratic blinking and substantial dark (never radiant) fractions that occur in all available qdots may interfere with many applications, these properties of individual particles in biological environments had not been fully evaluated. By labeling Qdot-streptavidin with organic dyes, we were able to distinguish individual dark and bright qdots and to observe blinking events as qdots freely diffused in aqueous solution. Bright fractions were measured by confocal fluorescence coincidence analysis (CFCA) and two-photon cross-correlation fluorescence correlation spectroscopy (FCS). The observed bright fractions of various preparations were proportional to the ensemble quantum yields (QYs), but the intrinsic brightness of individual qdots was found to be constant across samples with different QYs but the same emission wavelengths. Increasing qdots' illuminated dwell time by 10-fold during FCS did not change the fraction of apparently dark qdots but did increase the detected fraction of blinking qdots, suggesting that the dark population does not arise from millisecond blinking. Combining CFCA with wide-field imaging of arrays of qdots localized in dilute agarose gel, the blinking of qdots was measured across five orders of magnitude in time: approximately 0.001-100 s. This research characterizes photophysical pathologies of qdots in biologically relevant environments rather than adhered on dielectric surfaces and describes methods that are useful for studying various bioapplicable nanoparticles.

Biophysical Phenomena↗

PubMed2005

Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy.

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism.

Animals↗

PubMed2004

Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis.

We have found that two-photon fluorescence imaging of nicotinamide adenine dinucleotide (NADH) provides the sensitivity and spatial three-dimensional resolution to resolve metabolic signatures in processes of astrocytes and neurons deep in highly scattering brain tissue slices. This functional imaging reveals spatiotemporal partitioning of glycolytic and oxidative metabolism between astrocytes and neurons during focal neural activity that establishes a unifying hypothesis for neurometabolic coupling in which early oxidative metabolism in neurons is eventually sustained by late activation of the astrocyte-neuron lactate shuttle. Our model integrates existing views of brain energy metabolism and is in accord with known macroscopic physiological changes in vivo.

Animals↗

PubMed2004

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy.

We describe a technique for imaging enzyme activity through steady-state fluorescence anisotropy measurements on a per-pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 microm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy-FL-labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five-fold. Fluorescence lifetime and time-resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy-FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy-containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.

Animals↗

PubMed2003

Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy.

Microtubule (MT) ensemble polarity is a diagnostic determinant of the structure and function of neuronal processes. Here, polarized MT structures are selectively imaged with second-harmonic generation (SHG) microscopy in native brain tissue. This SHG is found to colocalize with axons in both brain slices and cultured neurons. Because SHG arises only from noninversion symmetric structures, the uniform polarity of axonal MTs leads to the observed signal, whereas the mixed polarity in dendrites leads to destructive interference. SHG imaging provides a tool to investigate the kinetics and function of MT ensemble polarity in dynamic native brain tissue structures and other subcellular motility structures based on polarized MTs.

Animals↗