Microscopy brightfield

IHC stains as used for brightfield microscopy are sandwich procedures, consisting of sequential staining steps, each of which contributes amplification of the original signal. Specificity of the stain is provided by the primary antibody. The primary antibody also contributes to the sensitivity of the stain, but sensitivity is also the combination of each of the other steps of the stain protocol. It is critical that the steps used to amplify the signal in the stain process are not overdone, as these amplification steps can easily produce excess back-... 

In contrast to brightfield microscopy, which uses specimen features such as light absorption, fluorescence microscopy is based on the phenomenon in which absorption of light by fluorescent molecules called fluorescent dyes or fluorophores (known also as fluorochromes) is followed by the emission of light at longer... 

Fluorophores were introduced to fluorescence microscopy in the early twentieth century, but did not see widespread use until the early 1940s when Albert Coons developed a technique for labeling antibodies with fluorescent dyes, thus giving birth to the field of immunofluorescence (http //www.olympusconfocal.com/ theory/fluorophoresintro.html). By attaching different fluorophores to different antibodies, the distribution of two or more antigens can be determined simultaneously in the tissue section and, in contrast to brightfield microscopy, even in the same cells and in the same cell structures (see Chap. 8). 

Aqueous mounting media for Aqueous mounting media for Organic mounting media brightfield microscopy fluorescence microscopy... 

Counterstaining counterstain nuclei if necessary, e.g., with DAPI for fluorescence microscopy or with hematoxylin (see Sect. 7.4) for brightfield microscopy. 

Mounting mount sections in aqueous medium or balsam for brightfield microscopy or in anti-fade medium for fluorescence microscopy (see Sect. 3.2.2). Notes. A11 incubations are at room temperature unless otherwise noted. Nuclear dyes (DAPI, Hoechst 33342 and Propidium Iodide) supplied as lyophilized solids are usually reconstituted in methanol. The stock solutions (5 mg/ml) are stable for many years when stored frozen at <—20°C and... 

Mount sections in aqueous medium or balsam for brightfield microscopy or in anti-fade medium for fluorescence microscopy (see Sect. 3.5). 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

To obtain the fractal dimension of a network of particles, acquiring images of the microstructure is necessary. Many forms of microscopy can be used, including brightfield microscopy, confocal laser scanning microscopy, scanning electron microscopy, and in the case of fat crystal networks, polarized light microscopy. 

Figure 23-29 Optical image using brightfield microscopy showing a carbon fiber microelectrode adjacent to a bovine chromaffin cell from the adrenal medulla. The extracellular solution was 10 mM TRIS buffer containing 150 rnM NaCl, 2 mM CaCfi, 1.2 mM MgCfi, and 5 mM glucose. The black scale bar is 50 p.m. (From L. Buhler and R. M. Wightman, unpublished work. With permission.)...

Fig. 5. The components of the EZ-TAXIS system are shown in (a) with the individual components in their order of assembly from top to bottom shown in (b). (c) An example of HL-60 cells migrating toward chemoattractant in the EZ-TAXIS assay visualized with brightfield microscopy.

An 8-well chamber will yield four molds. Only bottom surface (side touching permanox chamber slide) of epoxy mold is sticky. Additionally, Lab-Tek 8 well cover glass 1.5 chamber slide (Nunc) may be used for epifluorescence and brightfield microscopy. However, the epoxy that hold the chamber walls to the coverslip is autofluorescent and thus less suitable for TIRF. 

FIGURE 48.4 (a) Model of microdialysis system. Schematicof the mass transfer. (b)The schematic of stacked microdialysis system. The depth and width of PDMS channels are 30 and 1500 pm. The depth and width of SU-8 channels are 15 and 400 pm. (c) Electrodes SU-8 and PDMS channels under brightfield microscopy, (d) Photo of a complete device. (Reproduced from Hsieh, Y.-C. and Zahn, J. D., Biosens. Bioelectron., 22, 2422-2428, 2007. With permission.)... 

Place mixture on a microscope slide, add a cover slip, and examine by brightfield microscopy. 

Fig. 2 Schematic diagram of laser optical tweezers (LOT) system. (Top panel) An infrared laser beam was steered to trap and move pm-size particles in the focal plane. At a sufficiently large laser intensity, the gradient force dominates over the scattering force. (Lower panel) Membrane tethers are extracted from live cells. Dielectric beads (0.5 pm in diameter) were conjugated with antibodies against integiins and attached to the cell membrane. LOT traps one bead and pulls it away from the cell, as indicated by the arrow. Thin membrane tethers extending from the beads to the cell body appear. Using conventional brightfield microscopy, the tether length and its diameter are measured which, in turn, provide an estimate of the plasma membrane tension.

Figure 4.2. Botrytis cinerea as viewed with brightfield microscopy at a magnification of 400x. Photograph provided with the kind permission of WineBugs LLC.

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