Biomedical subjects

Sripad Ram

Publications and source records attributed to Sripad Ram.

3 recordsLinked to original sources

PubMed2006

Beyond Rayleigh's criterion: a resolution measure with application to single-molecule microscopy.

Rayleigh's criterion is extensively used in optical microscopy for determining the resolution of microscopes. This criterion imposes a resolution limit that has long been held as an impediment for studying nanoscale biological phenomenon through an optical microscope. However, it is well known that Rayleigh's criterion is based on intuitive notions. For example, Rayleigh's criterion is formulated in a deterministic setting that neglects the photon statistics of the acquired data. Hence it does not take into account the number of detected photons, which, in turn, raises concern over the use of Rayleigh's criterion in photon-counting techniques such as single-molecule microscopy. Here, we re-examine the resolution problem by adopting a stochastic framework and present a resolution measure that overcomes the limitations of Rayleigh's criterion. This resolution measure predicts that the resolution of optical microscopes is not limited and that it can be improved by increasing the number of detected photons. Experimental verification of the resolution measure is carried out by imaging single-molecule pairs with different distances of separation. The resolution measure provides a quantitative tool for designing and evaluating single-molecule experiments that probe biomolecular interactions.

Calibration↗

PubMed2004

Localization accuracy in single-molecule microscopy.

One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.

Biopolymers↗

PubMed2004

Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions.

The imaging of cellular dynamics in three dimensions using a standard microscope is severely limited due to the fact that only one focal plane can be imaged at a given point in time. Here we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. This is achieved by a modification of the emission pathway of a standard microscope. The efficacy of the design is shown by imaging bead samples and an FcRn-green fluorescent protein expressing tubule that leaves a sorting endosome and subsequently exocytoses at the plasma membrane.

Cell Line↗