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

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a 4-fold shift towards higher values in concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, which is almost exclusively localized in the nucleus under high and..

Calibrating evanescent-wave penetration depths for biological TIRF microscopy

Roughly half of a cells proteins are located at or near the plasma membrane. In this restricted space the cell senses its environment, signals to its neighbors and ex-changes cargo through exo- and endocytotic mechanisms. Ligands bind to receptors, ions flow across channel pores, and transmitters and metabolites are transported against con-centration gradients. Receptors, ion channels, pumps and transporters are the molecular substrates of these biological processes and they constitute important targets for drug discovery. Total internal reflection fluorescence microscopy suppresses background from cell deeper layers and provides contrast for selectively imaging dynamic processes near the basal membrane of live-cells. The optical sectioning of total internal reflection fluorescence is based on the excitation confinement of the evanescent wave generated at the glass-cell interface. How deep the excitation light actually penetrates the sample is difficult to know, making the quantitative interpretation of total internal reflection fluorescence data problematic. Nevertheless, many applications like super-resolution microscopy, colocalization, fluorescence recovery after photobleaching, near-membrane fluorescence recovery after photobleaching, uncaging or photo-activation-switching, as well as single-particle tracking require the quantitative interpretation of evanescent-wave excited images. Here, we review existing techniques for characterizing evanescent fields and we provide a roadmap for comparing total internal reflection fluorescence data across images, experiments, and laboratories.Comment: 18 text pages, 7 figures and one supplemental figur

Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load

Confocal microscopy

Chapter focusing on confocal microscopy. A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy

J Fluorescence

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research