Functional imaging of single synapses in brain slices.
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The strength of synaptic connections in the brain is not fixed, but can be modulated by numerous mechanisms. Traditionally, electrophysiology has been used to characterize connections between neurons. Electrophysiology typically reports the activity of populations of synapses, while most mechanisms of plasticity are thought to operate at the level of single synapses. Recently, two-photon laser scanning microscopy has enabled us to perform optical quantal analysis of individual synapses in intact brain tissue. Here we introduce the basic principle of the two-photon microscope and discuss its main differences compared to the confocal microscope. Using calcium imaging in dendritic spines as an example, we explain the advantages of simultaneous dual-dye imaging for quantitative calcium measurements and address two common problems, dye saturation and background fluorescence subtraction. (+info)
Miniaturization of fluorescence microscopes using fibre optics.
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In both medical research and diagnostics characterization of biological tissue on the cellular level relies on high-resolution optical microscopy. In most cases, however, tissue is excised for microscopic investigation, in part because conventional microscopes are bulky instruments. Imaging of cells in the intact living organism has been difficult. Over the last decade several groups have developed miniature confocal microscopes that use fibre optics to deliver light to the specimen and to measure either reflected or excited fluorescence light. In addition, two-photon excitation recently has been employed in a small portable 'fibrescope'. A potential clinical application of these microscope probes is their endoscopic use for optical biopsy of inner organs or guidance of conventional biopsy. As a mobile research tool they may permit imaging of neuronal activity in the brain of awake, behaving animals. Here, we review technological approaches to build miniaturized fluorescence microscopes and discuss their potential applications. (+info)
Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.
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Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell. (+info)
Early steps of supported bilayer formation probed by single vesicle fluorescence assays.
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We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. (+info)
GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice.
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To examine the behavior of tumor cells in tumors developing directly from mammary tissue in transgenic models, we have evaluated transgenic mice expressing green fluorescent protein (GFP). Using the mouse mammary virus promoter (MMTV) to directly drive expression of GFP, we find low levels of fluorescence in the mammary and salivary glands of transgenic animals. Using MMTV-Cre or WAP-Cre in combination with the Cre-activatable CAG-CAT-EGFP construct, we find stronger expression of GFP that is still tissue specific. These animals provide a range of expression of GFP that is suitable for analysis of transgenic mammary tumors and metastases in vivo at the single cell level of resolution. (+info)
Two-photon thermal bleaching of single fluorescent molecules.
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We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations. (+info)
Advantages and risks of multiphoton microscopy in physiology.
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Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out-of-focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction-limited volume combined with multiphoton or confocal imaging. (+info)
Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.
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In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning. (+info)