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PubMed2004

Triple-color coincidence analysis: one step further in following higher order molecular complex formation.

Confocal fluorescence spectroscopy is a versatile method for studying dynamics and interactions of biomolecules in their native environment with minimal interference with the observed system. Analyzing coincident fluctuations induced by single molecule movement in spectrally distinct detection channels, dual-color fluorescence cross-correlation, and coincidence analysis have proven most powerful for probing the formation or cleavage of molecular bonds in real time. The similarity of the optical setup with those used for laser scanning microscopy, as well as the non-invasiveness of the methods, make them easily adaptive for intracellular measurements, to observe the association and dissociation of biomolecules in situ. However, in contrast to standard fluorescence microscopy, where multiple fluorophores can be spectrally resolved, single molecule detection has so far been limited to dual-color detection systems due to the harsh requirements on detection sensitivity. In this study, we show that under certain experimental conditions, employing simultaneous two-photon excitation of three distinct dye species, their successful discrimination indeed becomes possible even on a single molecule level. This enables the direct observation of higher order molecular complex formation in the confocal volume. The theoretical concept of triple-color coincidence analysis is outlined in detail, along with an experimental demonstration of its principles utilizing a simple nucleic acid reaction system.

Equipment Design↗
PubMed2004

Characterization of a range of fura dyes with two-photon excitation.

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.

Animals↗
PubMed2003

High-efficiency transfection of individual neurons using modified electrophysiology techniques.

Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection of neurons requires high and reliable efficiency together with good cell survival. Here, we describe the modification of electrophysiology techniques for SCE leading to very simple and efficient (up to 80%) transfection of neurons in organotypic rat hippocampus and mouse cortex slice cultures. Electroporation-mediated transfection was visualized in real-time by two-photon microscopy at the cellular level using fluorescently labeled oligonucleotides and plasmid DNA. Small oligonucleotides enter the cell immediately during pulse application while large plasmids remain localized for more than 10 min at the cell membrane before they enter the cell by an, as yet, unknown process. SCE does not affect the electrophysiology of transgene-expressing cells. Expression of several neuronal green fluorescent protein-tagged proteins demonstrates that the method can be employed to analyze subcellular trafficking and targeting in single living neurons.

Animals↗
PubMed2005

[Emerging endoscopic techniques. The arrival of virtual histology].

Endoscopic technology has evolved in such a way that gastroenterologists can now visualize and store high-resolution images of the gastrointestinal tract. This has improved the approach to precancerous and cancerous lesions of the gastrointestinal tract and biliary tree. However, certain difficulties remain, especially in relation to diagnosis. In the last few years, multiple techniques have been developed that, using the properties of light, enable an instantaneous histologic diagnosis to be made while endoscopy is being performed. What has been called the "optical biopsy" allows highly exact information to be obtained, both from the morphological and functional point of view. Some of these techniques, such as chromoendoscopy and magnification, are already being performed in clinical practice while others are still under investigation. The aim of the present article is to review the underlying principles and applications of these emerging techniques.

Endoscopy, Gastrointestinal↗
PubMed2003

Two-photon fluorescence excitation of macroscopic areas on planar waveguides.

In this paper, we report the first successful demonstration, to our knowledge, of two-photon fluorescence excitation (TPFE) using planar thin-film waveguide structures of macroscopic excitation dimensions (square millimeters to square centimeters in size). The high intensity of excitation light required for TPFE is available not only at a single focus point but along the whole trace of the beam guided in the waveguide structure. Line profiles of the fluorescence excited by TPFE show excellent correlation with the geometry of the launched laser beams. A clear second-order dependence of the fluorescence intensity on the excitation intensity confirms the two-photon character of fluorescence generation. Spectra of the emission generated by one-photon excitation and by two-photon excitation show only minor differences.

Equipment Design↗
PubMed2003

A two-photon and second-harmonic microscope.

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.

Animals↗
PubMed2006

Two-photon fluorescence excitation and related techniques in biological microscopy.

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

Biology↗
PubMed2005

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development.

The present study was conducted to evaluate the function of cumulus cells during bovine IVF Oocytes within cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

Animals↗
PubMed2005

In vivo monitoring of fluorescent nanosphere delivery in anesthetized rats using an implantable fiber-optic microprobe.

An implantable needle-type fiber-optic microprobe was constructed to monitor in vivo fluorescent substances in anesthetized rats. This fiber-optic microprobe was composed of coaxial optical fibers that were catheterized using a thin-wall tube of stainless steel (o.d. approximately 400 microm; i.d. approximately 300 microm). When the fiber-optic microprobe was placed in solutions containing various concentrations of fluorescent nanospheres (20 nm), either in the presence or in the absence of 10% Lipofundin acting as an optical phantom, we observed nanosphere concentration-dependent responses of the fluorescence intensity. The microprobe was then implanted into the livers and brains of anesthetized rats to monitor the in situ extravasation of preadministered fluorescent nanospheres from vasculature following the hepatic and cerebral ischemic insults. Both types of ischemic insults showed immediate increases in fluorescent intensities when 20-nm fluorescent nanosphere were administered, but neither ischemic insult induces such an increase when we administered 1000-nm fluorescent nanospheres. Additional experiments can be performed to further narrow the size range of the increase in blood vessel permeability following ischemic insult; such "size" information may be valuable when formulating drugs for optimal local delivery. Although a wide variety of fluorescent substances are used intensively for in vitro biological studies, the in vivo and in situ monitoring of these substances is studied much less often, probably because of difficulties in the efficient assembly of miniaturized fiber optics to detect the relatively weak fluorescence signal arising within such a turbid medium as tissue. To our knowledge, the use of our implantable fiber-optic microprobe is the first minimally invasive technique capable of investigating the "size window" of vascular permeability for the in vivo delivery of nanospheres in both ischemic livers and brains.

Anesthesia↗
PubMed2005

Lipid--protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy.

Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and shades providing the researcher not only with quantitative intensity information but also with spatial resolution and visual comprehension of two- or three-dimensional images. In this Account, we review the use of two-photon excitation microscopy and FCS in the study of lipid-protein interactions. We discuss these new methodologies and techniques, and we present examples of different complexity from qualitative to quantitative, from simple model systems to studies in living cells.

Cell Membrane↗
PubMedDate not supplied

In situ observation of molecular diffusion in solid supports using two-photon fluorescence microscopy.

We demonstrate that two-photon fluorescence microscopy is a useful tool for monitoring the diffusion of molecules through polymeric solid supports. As a proof of principle, TentaGel beads were bound to a cover slip, and diffusion of Rhodamine 6G through single beads was observed in real time. Diffusion experiments performed in 40 different beads indicate that there is a considerable degree of heterogeneity in diffusion rates from bead to bead.

Color↗
PubMed2005

Direct observation of organic contaminant uptake, storage, and metabolism within plant roots.

Two-photon excitation microscopy (TPEM) is used to visualize and track the uptake and movement of anthracene and phenanthrene from a contaminated growth medium into living unmodified roots of maize and wheat over a 56-day period. The degradation of anthracene was also directly observed within the cortex cells of both species. The power of this technique is that neither the plant nor the compound require altering (staining or sectioning) to visualize them, meaning they are in their natural form throughout the experiment. Initially both compounds bound to the epidermis along the zone of elongation, passing through the epidermal cells to reach the cortex within the root hair, and branching zones of the root. The PAHs entered the epidermis radially; however, once within the cortex cells this movement was dominated by slow lateral movement of both compounds toward the shoot. Highly focused "streams" of compound were observed to form over time; zones where phenanthrene concentrated extended up to 1500 microm in length over a 56-day period, for example, passing through several adjoining cells, and were detectable in cell walls and cell vacuoles. Radial movement was not observed to extend beyond the cortex cells to reach the vascular tissues of the plant. The longitudinal movement of both compounds was not observed to extend beyond the root base into the stem or vegetative parts of the plant. The lateral movement of both compounds within the cortex cells was dominated by movement within the cell walls, suggesting apoplastic flow through multiple cell walls, but with a low level of symplastic movement to transport compound into the cellular vacuoles. Degradation of anthracene to the partial breakdown products anthrone, anthraquinone, and hydroxyanthraquinone was observed directly in the zones of root elongation and branching. The technique and observations have important applications to the fields of agrochemistry and phytoremediation.

Anthracenes↗