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Results for “Microscopy, Fluorescence, Multiphoton”

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PubMed2005

Deep tissue two-photon microscopy.

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

Animals↗

PubMed2006

An improved mRFP1 adds red to bimolecular fluorescence complementation.

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.

Gene Expression Profiling↗

PubMed2006

Imaging cellular network dynamics in three dimensions using fast 3D laser scanning.

Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 microm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.

Animals↗

PubMed2006

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin.

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron-glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.

Animals↗

PubMed2003

Analysis of molecular diffusion in ftsK cell-division mutants using laser surgery.

Escherichia coli cells that lack the carboxy-terminal part of FtsK fail to segregate their chromosomes properly during cytokinesis and tend to form chains. These chains are possibly formed as a result of DNA being trapped in the division planes or a failure to fuse the membrane during septum formation. If so, small molecules might diffuse between the apparent cell compartments. To investigate this theory, we developed an optical workstation that allows simultaneous imaging of and surgical operations on cellular objects in the sub-micrometre range. By surgical incisions of E. coli cell poles, diffusion of propidium iodide (PI) can be followed in real time. This analysis showed that PI was unable to diffuse from one cell equivalent to another in chain-forming ftsK mutants. Thus, the cytoplasm of the cell compartments in the chains seems to be fully separated.

Cell Division↗

PubMed2007

The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.

Albumins↗

PubMed2005

A lab-on-a-chip compatible bioaffinity assay method for human alpha-fetoprotein.

A new lab-on-a-chip compatible binding assay platform is introduced. The platform combines dry-chemistry bioaffinity reagents and the recently introduced ArcDia TPX binding assay technique. The technique employs polymer microspheres as a solid phase reaction carrier, fluorescently labeled antibody conjugates, and detection of fluorescence emission from the surface of individual microspheres by two-photon excitation fluorescence. Signal response of the technique is independent of the reaction volume, thus the technique is particularly well suited for detection of bioaffinity reactions from miniature volumes. Performance of the new assay platform is studied by means of an immunometric assay of human alpha-fetoprotein (hAFP) in 384-plate format, and the results are compared to those of a corresponding wet-chemistry assay method. The results show that the ArcDia TPX detection technique can be combined with dry-chemistry reagents without compromises in assay performance. The microchip field has so far been characterized with a lack of microchip-compatible detection platforms which would allow cost-effective microchip design and sensitive bioaffinity detection. The presented detection technique is expected to provide a solution for this shortage.

Humans↗

PubMed2006

A femtosecond time-resolved investigation of dual fluorescence from N6,N6-dimethyladenine.

The excited electronic state dynamics of N(6),N(6)-dimethyladenine (DMAde), a molecule known to emit dual fluorescence, has been studied in aqueous solution using femtosecond fluorescence up-conversion spectroscopy. Time profiles of the fluorescence of DMAde excited at lambda= 258 nm were measured at a series of wavelengths in the range 320 nm or= 500 nm), which appeared slightly delayed compared to the UV fluorescence, the long-lived fluorescence component (tau(3)) dominated, the second component (tau(2)) disappeared. The results are consistent with the assumption that DMAde is primarily excited to a short-lived local excited (LE) electronic state that fluoresces mostly in the UV and decays rapidly, on a approximately 0.5 ps timescale, to an intramolecular charge transfer (ICT) state that emits only at longer wavelengths in the visible spectrum. The fluorescence-time profiles and transient fluorescence spectra reconstructed from the time profiles provided further information on secondary relaxation processes within and between the excited states and their non-radiative relaxation to the electronic ground state.

Adenine↗

PubMed2002

Fibre-optic two-photon scanning fluorescence microscopy.

Two geometries of a novel two-photon fluorescence microscope incorporating single-mode fibre optics for the delivery of ultrashort-pulsed illumination to a remote sample are characterized. First, a 785 nm single-mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non-linear response of the fibre to intense ultrashort-pulsed light. Second, a 785 nm single-mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort-pulsed laser beam caused by the non-linear response of the fibre to ultrashort-pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort-pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low-cost, compact instrument. Sets of three-dimensional images of fluorescent polymer microspheres and biological material confirm these features.

Eukaryota↗

PubMed2003

Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.

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.

Biological Transport↗

PubMed2006

[Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!].

One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.

Cells↗

PubMed2004

Two-dimensional optical spectroscopy: two-color photon echoes of electronically coupled phthalocyanine dimers.

Two-color photon echo peak shift spectroscopy was used to study electronic coupling in a phthalocyanine homodimer. Two optical parametric amplifiers were used to produce pulses to excite the split lower states of LuPc2-. The existence of a two-color peak shift indicates the existence of correlation between these two dipole-allowed states. The nature of this correlation is discussed based on theoretical predictions of the interactions between exciton and charge resonance states.

Computer Simulation↗

PubMed2005

Direct measurement of relative and collective diffusion in a dilute binary colloidal suspension.

Experimental characterization of the dynamics of multicomponent fluids is a problem of general importance to the field of complex fluids. We demonstrate a new experimental approach, termed two-color Fourier imaging correlation spectroscopy, which allows direct measurement of the partial dynamic structure factors, S(11)(k,tau), S(22)(k,tau), and S(12)(k,tau), where 1, 2 label the component species of a binary colloidal suspension. Linear combinations of the partial dynamic structure factors yield the characteristic time-correlation functions of the binary fluid. These are the correlation functions of concentration fluctuations S(CC)(k,tau), number density fluctuations S(NN)(k,tau), and cross-correlations between number density and concentration fluctuations S(NC)(k,tau). Test measurements are performed on a dilute symmetric mixture of fluorescently labeled 0.5 and 1.0 microm polystyrene spheres. From these data, we determine generalized collective and relative diffusion coefficients, and compare them to the predictions for an ideal mixture of noninteracting particles.

Algorithms↗

PubMed2007

Large two-photon absorptivity of hemoglobin in the infrared range of 780-880 nm.

Porphyrin molecules have a highly conjugated cyclic structure and are theorized to have unusually large two-photon absorptivities (sigmaTPA), i.e., sigmaTPA approximately 10(2) GM. The authors tested this claim. Ultrafast two-photon absorption (TPA) spectroscopy was performed on solutions of hemoglobin, which contains a naturally occurring metaloporphyrin. They used a pump-probe technique to directly detect the change in transmission induced by TPA over the wavelength range of lambda0=780-880 nm. As controls, they measured the TPA of the dyes rhodamine 6G and B; their measurements both verify and extend previously reported values. In new results, hemoglobin was found to have a peak two-photon absorptivity of sigmaTPA approximately 150 GM at lambda0=825 nm, near a resonance of the Soret band. This value supports theoretical expectations. They also found a significant difference in the TPA of carboxyhemoglobin versus oxyhemoglobin, e.g., sigmaTPA=61 GM versus sigmaTPA=18 GM, respectively, at lambda0=850 nm, which shows that the ligand affects the electronic states involved in TPA.

Computer Simulation↗

PubMed2006

Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing.

The image quality of a two-photon microscope is often degraded by wavefront aberrations induced by the specimen. We demonstrate here that resolution and signal size in two-photon microcopy can be substantially improved, even in living biological specimens, by adaptive wavefront correction based on sensing the wavefront of coherence-gated backscattered light (coherence-gated wavefront sensing, CGWS) and wavefront control by a deformable mirror. A nearly diffraction-limited focus can be restored even for strong aberrations. CGWS-based wavefront correction should be applicable to samples with a wide range of scattering properties and it should be possible to perform real-time pixel-by-pixel correction even at fast scan speeds.

Animals↗

PubMed2006

Natural killer cell behavior in lymph nodes revealed by static and real-time imaging.

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.

Animals↗

PubMed2006

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).

Animals↗

PubMed2004

Identification of second harmonic optical effects from vaccine coated gold microparticles.

This study investigates the optical effects observed from uncoated and protein vaccine coated gold microparticles while imaging with two-photon excitation in the Mie scattering regime. When observed with time correlated single photon counting fluorescence lifetime microscopy, the emission from the gold microparticles appeared as an intense instrument-limited temporal response. The intensity of the emission showed a second-order dependence on the laser power and frequency doubling of the emitted light was observed for fundamental light between 890 and 970 nm. The optical effect was attributed to two-photon induced second harmonic generation. The vaccine coated gold microparticles had a much weaker second harmonic signal than the uncoated gold microparticles. Chemical analysis of the surface of the gold microparticles revealed that the vaccine coating decreases the surface charge thereby diminishing the observed second harmonic signal. These optical properties can be exploited to identify both the location of the protein vaccine coating as well as the gold microparticles in vitro and potentially to investigate the vaccine delivery kinetics in vivo.

Drug Delivery Systems↗