Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging

Fluorescence microscopy for the characterization of structural integrity

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity

Likelihood inference for particle location in fluorescence microscopy

We introduce a procedure to automatically count and locate the fluorescent particles in a microscopy image. Our procedure employs an approximate likelihood estimator derived from a Poisson random field model for photon emission. Estimates of standard errors are generated for each image along with the parameter estimates, and the number of particles in the image is determined using an information criterion and likelihood ratio tests. Realistic simulations show that our procedure is robust and that it leads to accurate estimates, both of parameters and of standard errors. This approach improves on previous ad hoc least squares procedures by giving a more explicit stochastic model for certain fluorescence images and by employing a consistent framework for analysis.Comment: Published in at http://dx.doi.org/10.1214/09-AOAS299 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Tip-Enhanced Fluorescence Microscopy at 10 Nanometer Resolution

We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness

Elastohydrodynamic study of actin filaments using fluorescence microscopy

We probed the bending of actin subject to external forcing and viscous drag. Single actin filaments were moved perpendicular to their long axis in an oscillatory way by means of an optically tweezed latex bead attached to one end of the filaments. Shapes of these polymers were observed by epifluorescence microscopy. They were found to be in agreement with predictions of semiflexible polymer theory and slender-body hydrodynamics. A persistence length of $7.4 \pm 0.2 \mu$m could be extracted.Comment: RevTex, 4 pages, 5 eps figs, submitted to PR