Dyes, fluorescence microscopy

The ability to image lateral heterogeneity in Langmuir monolayers dates back to Zocher and Stiebel s 1930 study with divergent light illumination [166]. More recently the focus shifted toward the use of fluorescence microscopy of mono-layers containing a small amount of fluorescent dye [167]. Even in single-corn-... 

Hayazawa, N Inouye, Y. and Kawata, S. (1999) Evanescent field excitation and measurement of dye fluorescence using a high N.A. objective lens in a metallic probe near-field scanning optical microscopy J. Microsc., 194, 472-476. 

The synthesis and characterization of a somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, labeled with an indo-dicarbo- and an indotricarbocyanine dye at the V-terminal amino group were described in [34], The ability of these fluorescent contrast agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, wherein the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. The intracellular localization was visualized using NIR fluorescence microscopy. 

Bullen A, Saggau P (1999) High-speed, random-access fluorescence microscopy II. Fast quantitative measurement with voltage-sensitive dyes. Biophys J 76 2272-2287... 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

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). 

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... 

When fluorescent dyes are used in the experiments, autofluorescence (or natural fluorescence) of some tissue components can cause background problems and complicate the use of fluorescence microscopy. The simplest test is to mount... 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

Fluorescent pH indicators offer much better sensitivity than the classical dyes such as phenolphthalein, thymol blue, etc., based on color change. They are thus widely used in analytical chemistry, bioanalytical chemistry, cellular biology (for measuring intracellular pH), medicine (for monitoring pH and pCC>2 in blood pCC>2 is determined via the bicarbonate couple). Fluorescence microscopy can provide spatial information on pH. Moreover, remote sensing of pH is possible by means of fiber optic chemical sensors. 

Optical fluorescence microscopy is a powerful and sensitive method for obtaining information about the orientation of luminescent dye molecules in small crystals. In Figure 1.10, we show unpolarized and linearly polarized fluorescence of two perpendicularly lying zeolite L crystals loaded with DSC. 

Figure 1.13. (1) Electron microscopy picture of a zeolite L crystal with a length of 1.5 pm. (2-5) True color fluorescence microscopy pictures of dye loaded zeolite L crystals. (2-4) Fluorescence after excitation of only Py+ (2) after 5-min exchange with Py+, (3) after 2 h exchange with Py+, (4) after additional 2 h exchange with Ox+. (5) The same as 4 but after specific excitation of only Ox+. (See insert for color representation.)...

Figure 1.18 shows fluorescence microscopy images of a bipolar three-dye antenna material with POPOP in the middle, followed by Py+ and then by Ox+. The different color regions that can be observed in this simple experiment are impressive. The red color of the luminescence (1) disappears, when the crystal is observed trough a polarizer parallel to the crystal axis while the blue emission disappears when turning the polarizer by 90°. This material is very stable and is easy to handle. 

Two-photon fluorescence microscopy has also been used with good effect in the near-IR. For example, Ferguson et al.r24> at the University of Strathclyde have used 270 fsec pulses from a titanium sapphire (Ti sapphire) laser at 790 nm to observe visible fluorescence from dyes in zebra fish larvae and erythrocytes. The high depth and lateral definition afforded by the two-photon process and confocal microscopy are useful here. Also, the use of near-IR excitation minimizes photobleaching. 

The research group led by Dr. Djilali at the University of Victoria has developed an ex situ experimental technique using fluorescent microscopy to study the liquid water transport mechanisms inside diffusion layers and on their surfaces [239-243]. The diffusion layer is usually placed between two plates (the top plate may or may not have a channel) the liquid water, which is pumped through a syringe pump, flows from the bottom plate through the DL. Fluorescein dye is added to the water for detection with the microscope. 

The two cationic dyes, Py+ as a donor and Ox" as an acceptor, were found to be very versatile for demonstrating photonic antenna functionalities for light harvesting, transport, and capturing, as illustrated in Fig. 7. They can be incorporated into zeolite L by means of ion exchange, where they are present as monomers because of the restricted space. In this form they have a high fluorescence quantum yield and favourable spectral properties. The insertion of the dyes can be visualised by means of fluorescence microscopy. The fluorescence anisotropy of Ox -loaded zeolite L has recently been investigated in detail by conventional and by confocal microscopy techniques [15],... 

In Fig. 12 in Ref 25, fluorescence microscopy images of different dye-loaded zeolite L single crystals are shown. Each line consists of three pictures of the same sample, but with different polarizations of the fluorescence observed. In the first one, the total fluorescence of the crystals is shown, and in the others, the fluorescence with the polarization direction indicated by the arrows is displayed. The zeolite was loaded with the following dyes (A) Py+, (B) PyGY", (C) PyB +, (D) POPOP (see Table 1). Most crystals show a typical sandwich structure with fluorescent dyes at the crystal ends and a dark zone in the middle. This situation can be observed when the diffusion of the dyes in the channels has not yet reached its equilibrium situation. It illustrates nicely how the molecules penetrate the crystals via the two openings on each side of the one-dimensional channels. 

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