41,500 research outputs found
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
Towards many colors in FISH on 3D-preserved interphase nuclei
The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + widefield microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. Copyright (c) 2006 S. Karger AG, Basel
Using Machine-Learning to Optimize phase contrast in a Low-Cost Cellphone Microscope
Cellphones equipped with high-quality cameras and powerful CPUs as well as
GPUs are widespread. This opens new prospects to use such existing
computational and imaging resources to perform medical diagnosis in developing
countries at a very low cost.
Many relevant samples, like biological cells or waterborn parasites, are
almost fully transparent. As they do not exhibit absorption, but alter the
light's phase only, they are almost invisible in brightfield microscopy.
Expensive equipment and procedures for microscopic contrasting or sample
staining often are not available.
By applying machine-learning techniques, such as a convolutional neural
network (CNN), it is possible to learn a relationship between samples to be
examined and its optimal light source shapes, in order to increase e.g. phase
contrast, from a given dataset to enable real-time applications. For the
experimental setup, we developed a 3D-printed smartphone microscope for less
than 100 \$ using off-the-shelf components only such as a low-cost video
projector. The fully automated system assures true Koehler illumination with an
LCD as the condenser aperture and a reversed smartphone lens as the microscope
objective. We show that the effect of a varied light source shape, using the
pre-trained CNN, does not only improve the phase contrast, but also the
impression of an improvement in optical resolution without adding any special
optics, as demonstrated by measurements
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