Confocal image

There are also laser-scanning confocal microscopes rapidly overtaking the TSM as a means of confocal microscopy. There is also a computer software program that produces a "confocal" image by recogni2ing the shapes of out-of-focus detail, ie, halos, and subtracting these from the in-focus image. 

Figure 4 shows confocal images of the staining pattern for DDC (Fig. 4A), serotonin (Fig. 4B), and TH (Fig. 4C) in the segmental ventral ganglion of the CNS from third instar larvae. Panels B and C are the same CNS double stained with serotonin and TH. The DDC-expressing cells can be categorized into a set of paired ventral lateral serotonin cells (Fig. 4A,B labeled VL in 4A), and two morphologically distinct types of dopamine cells, the medial dopamine cells (Fig. 4A,C labeled M) and the dorsal-lateral dopamine cells (Fig. 4A,C labeled DL). Figure 4 demonstrates clearly that individual DDC cells synthesize either serotonin or dopamine, but not both. One additional set of cells shows TH immunoreactivity in the ventral ganglion. These six large vacuolated cells are located more laterally than any other DDC cells (Fig. 4C, unlabeled short arrows). It is likely that the immunoreactivity in these cells results from a non-specific cross-reaction, since these cells are not...

Fig. 20.1. Confocal images of whole mounts of the ovijector region of A suum stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to show muscle and with an anti-RFamide antiserum coupled to fluorescein isothiocyanate (FITC) to show FaRPergic nerves. (A) Main ventral nerve cord encircles opening of ovijector where it meets the body wall and is immunopositive for FaRPs. (B) Flat-fixed preparation of the ovijector showing circular muscles and tracts of parallel FaRPergic nerves (arrows). (C) Detail of the circular muscle of ovijector and associated nerves (arrows). (D) A FaRPergic cell body is localized in the ventral nerve cord at junction with ovijector and provides innervation to ovijector muscle.

Note that parameters ft and 5 depend on signal amplifications in the utilized detectors and on the elements in the optical path (optical filter, spectral detection bands) only, while a and y are additionally influenced by relative excitation intensity. This is usually a fixed constant in wide-field microscopy but in confocal imaging laser line intensities are adjusted independently. Furthermore, note that the a factor equals 5 multiplied by y (see Appendix for further detail). 

Even in the nominal absence of laser fluctuations or other imagedegrading aberrations, the number of photons that hit the detector during the data collection period of the image (i.e., the exposure time for a CCD image or the pixel dwell time for a confocal image) will contain considerable noise. The photon count x follows a Poisson distribution (Fig. 7.7A) with mean value fi as... 

It can be shown that the standard deviation (SD) of this distribution is also just yfji. In other words, if one would repeatedly measure the same pixel that on average collects 100 photons during a single dwell time (a normal value for a rather bright confocal image ) one would record less than 100-2x /l00 = 80 photons or more than 100 + 2x /l00 = 120 photons just by coincidence in 5% of the measurements. This uncertainty is expressed as the... 

The factor g may account for integration time and electron multiplication in CCD imaging, or for the PMT gain in confocal imaging. 

Note that in confocal imaging, the PMT will generally be the same physical detector as... 

Note that when S and D are collected simultaneously (typically for confocal imaging) 0 and S are independent of relative laser line intensities. 

Salih, A., Jones, A., Bass, D. and Cox, G. (1997). Confocal imaging of exine for grass pollen analysis. Grana 36 215-224. 

Observations Figure 2 shows confocal images of 48 h tomato roots exposed to S. deppei aqueous leachate and then stained with DCFDA. A higher fluorescence is observed in treated-root hairs. 

Fig. 2 Confocal imaging. L. esculentum roots after 48 h of treatment, and with 25 pM DCF for 10 min. Control roots (A and C), S. deppei treated-roots (B and D). Panels C and D are higher magnification.

FIG. 1. A confocal image from a freshly isolated uterine myocyte from pregnant rat. The SR was loaded with mag-fluo-4 to show its structure and distribution. The stack of images was acquired using a Perkin Elmer Ultra VIEW LCI confocal imaging system. Images were combined to visualize the 3D distribution of the SR (A.V. Shmigol S. Wray, unpublished data). 

Bolton TB, Gordienko DV 1998 Confocal imaging of calcium release events in single smooth muscle cells. Acta Physiol Scand 164 567—575... 

Wier WG, ter Keurs HE, Marban E, Gao WD, Balke CW 1997 Ca2+ sparks and waves in intact ventricular muscle resolved by confocal imaging. Circ Res 81 462-469... 

FIG. 3. Confocal images showing the location of the SR in live myocytes within an intact, small diameter (< 250 nm passive diameter), pressurized (70 mmHg) artery from the rat mesenteric artery arcade. The artery was loaded with Fluo-4 as the membrane-permeant acetoxymethyl ester. Some of this high-affinity, Ca2+ indicator dye is often sequestered in the SR (cf. Goldman et al 1990). The SR can then be readily visualized, especially when [Ca2+]CYx is low (as in the panels at 0 and 6.8 s), because the intra-SR dye is saturated with Ca2+, and fluoresces brightly. This artery was treated with 1.0 fim phenylephrine (PE), which caused the [Ca2+]CYT level to oscillate asynchronously in the cells seen in the centre of the panel. The cell outlines are clearly visible when [Ca2+]CYT tiscs, as in the panels at 3.4 and 10.2 s. Note that nearly all of the SR (the very bright areas, especially in the 0 and 3.4 s panels) lies parallel to, and immediately beneath the PL (from Miriel at al 1999, with permission). 

Transient spontaneous increases in intracellular ionized Ca2+ concentration, [Ca2+] , were first detected in smooth muscle as bursts of openings of Ca2+ activated K+ channels (Benham Bolton 1986). Several years later, laser scanning confocal imaging showed that similar spontaneous transient increases in... 

FIG. 2. Confocal image of an isolated smooth muscle cell from guinea-pig ileum which has been permeabilized with staphylococcal os-toxin and incubated with 150 /iM Fluo-3 acid which stains the SR. The SR is predominantly localized to the periphery of this type of smooth muscle as seen on the left hand side where the image plane is through the centre of the cell, whereas an extensive network is seen where the image plane is adjacent to the plasma membrane as seen in the right hand portion of the cell. 

Imbert D, Cullander C (1999) Buccal mucosa in vitro experiments I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability. J Control Release 58 39-50... 

When using isolated single cells, discrimination between binding and uptake is rather easily assessed by combining the confocal image with a usual transmission image. When the focus is set to the middle of the single cell,... 

Fig. 3. Comparisons of wide-field (A) and confocal fluorescence images (B, mesoglea level C, apical) of rhodamine phalloidin-stained F-actin in a whole-mount hydra tentacle. The hydra was fixed and stained as described in Chapter 18. The bar represents 25 pm. All images were collected with a Nikon (New York) Microphot FX microscope (x40 objective lens). Confocal images were collected with the microscope connected to a Bio-Rad (Hercules, CA) MRC600 laser-scanning confocal system.

The extraction of meaningful data from confocal images of living cells requires the establishment of fluorescent probe loading conditions for each specimen type. Consideration should be given to ... 

A number of methods are available for the characterization and examination of SAMs as well as for the observation of the reactions with the immobilized biomolecules. Only some of these methods are mentioned briefly here. These include surface plasmon resonance (SPR) [46], quartz crystal microbalance (QCM) [47,48], ellipsometry [12,49], contact angle measurement [50], infrared spectroscopy (FT-IR) [51,52], Raman spectroscopy [53], scanning tunneling microscopy (STM) [54], atomic force microscopy (AFM) [55,56], sum frequency spectroscopy. X-ray photoelectron spectroscopy (XPS) [57, 58], surface acoustic wave and acoustic plate mode devices, confocal imaging and optical microscopy, low-angle X-ray reflectometry, electrochemical methods [59] and Raster electron microscopy [60]. 

In cardiomyopathic hamsters Luque et al. [1994] stained for Cx43 using confocal microscopy and found that some of the cardiomyocytes stain normally but others stain diffusely, with a pixel intensity distribution of the confocal images showing a 90% increase in the number of pixels and a 60% decrease in pixel intensity in the cardiomyopathic hearts as compared to control hearts. Thus, Cx43 seemed to be present in the cells but did not become localized on the membranes as in normal cells. 

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