Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation

We theoretically demonstrate that enhanced penetration depth in three-dimensional multiphoton microscopy can be achieved using concentric two-color two-photon (C2C2P) fluorescence excitation in which the two excitation beams are separated in space before reaching their common focal spot. Monte Carlo simulation shows that, in comparison with the one-color two-photon excitation scheme, the C2C2P fluorescence microscopy provides a significantly greater penetration depth for imaging into a highly scattering medium. (C) 2008 Optical Society of America.

Methodology for reconstructing early zebrafish development from in vivo multiphoton microscopy

Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis.

Single molecule microscopy in 3D cell cultures and tissues

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

Optical microscopy methods for understanding learning and memory

Structural dynamics of dendritic spines is one of the key correlative measures of synaptic plasticity for encoding short-term and long-term memory. Optical studies of structural changes in brain tissue using confocal microscopy face difficulties of scattering. This results in low signal-to-noise ratio and thus limiting the imaging depth to few tens of microns. Multiphoton microscopy (MpM) overcomes this limitation by using low-energy photons to cause localized excitation and achieve high resolution in all three dimensions. Multiple low-energy photons with longer wavelengths minimize scattering and allow access to deeper brain regions at several hundred microns. In this article, we provide a basic understanding of the physical phenomena that give MpM an edge over conventional microscopy. Further, we highlight a few of the key studies in the field of learning and memory which would not have been possible without the advent of MpM.

Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas

Mortality in breast cancer is linked to metastasis and recurrence yet there is no acceptable biological model for cancer relapse. We hypothesise that there might exist primary tumour cells capable of escaping surgery by migration and resisting radiotherapy and chemotherapy to cause cancer recurrence. We investigated this possibility in invasive ductal carcinoma (IDC) tissue and observed the presence of solitary primary tumour cells (SPCs) in the dense collagen stroma that encapsulates intratumoural cells (ICs). In IDC tissue sections, collagen was detected with either Masson's Trichrome or by second harmonics imaging. Cytokeratin-19 (CK-19) and vimentin (VIM) antibodies were, respectively, used to identify epithelial-derived tumour cells and to indicate epithelial to mesenchymal transition (EMT). Confocal/multiphoton microscopy showed that ICs from acini were mainly CK-19 +ve and were encapsulated by dense stromal collagen. Within the stroma, SPCs were detected by their staining for both CK-19 and VIM (confirming EMT). ICs and SPCs were subsequently isolated by laser capture microdissection followed by multiplex tandem-PCR studies. SPCs were found to be enriched for pro-migratory and anti-proliferative genes relative to ICs. In vitro experiments using collagen matrices at 20 mg/cm 3, similar in density to tumour matrices, demonstrated that SPC-like cells were highly migratory but dormant, phenotypes that recapitulated the genotypes of SPCs in clinical tissue. These data suggest that SPCs located at the breast cancer perimeter are invasive and dormant such that they may exceed surgical margins and resist local and adjuvant therapies. This study has important connotations for a role of SPCs in local recurrence.

Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons.

PURPOSE: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells. MATERIALS AND METHODS: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy. RESULTS: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive. CONCLUSIONS: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans EmbryosV⃞

γ-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that γ-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans γ-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::γ-tubulin or GFP::β-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of γ-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of γ-tubulin. γ-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that γ-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.

Numerical analysis of gas and micro-particle interactions in a hand-held shock-tube device

A unique hand-held gene gun is employed for ballistically delivering biomolecules to key cells in the skin and mucosa in the treatment of the major diseases. One of these types of devices, called the Contoured Shock Tube (CST), delivers powdered micro-particles to the skin with a narrow and highly controllable velocity distribution and a nominally uniform spatial distribution. In this paper, we apply a numerical approach to gain new insights in to the behavior of the CST prototype device. The drag correlations proposed by Henderson (1976), Igra and Takayama (1993) and Kurian and Das (1997) were applied to predict the micro-particle transport in a numerically simulated gas flow. Simulated pressure histories agree well with the corresponding static and Pitot pressure measurements, validating the CFD approach. The calculated velocity distributions show a good agreement, with the best prediction from Igra & Takayama correlation (maximum discrepancy of 5%). Key features of the gas dynamics and gas-particle interaction are discussed. Statistic analyses show a tight free-jet particle velocity distribution is achieved (570 +/- 14.7 m/s) for polystyrene particles (39 +/- 1 mu m), representative of a drug payload.

In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath

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Note: Dynamic point spread function for single and multiphoton fluorescence microscopy

We propose and demonstrate a dynamic point spread function (PSF) for single and multiphoton fluorescence microscopy. The goal is to generate a PSF whose shape and size can be maneuvered from highly localized to elongated one, thereby allowing shallow-to-depth excitation capability during active imaging. The PSF is obtained by utilizing specially designed spatial filter and dynamically altering the filter parameters. We predict potential applications in nanobioimaging and fluorescence microscopy.

Note: Dynamic point spread function for single and multiphoton fluorescence microscopy

We propose and demonstrate a dynamic point spread function (PSF) for single and multiphoton fluorescence microscopy. The goal is to generate a PSF whose shape and size can be maneuvered from highly localized to elongated one, thereby allowing shallow-to-depth excitation capability during active imaging. The PSF is obtained by utilizing specially designed spatial filter and dynamically altering the filter parameters. We predict potential applications in nanobioimaging and fluorescence microscopy.

Spatial filter based 3D resolution improvement and polarization properties of multiphoton multiple-excitation-spot-optical microscopy

Three-dimensional (3D) resolution improvement in multi-photon multiple-excitation-spot-optical microscopy is proposed. Specially designed spatial filter is employed for improving the overall 3D resolution of the imaging system. An improvement up to a factor of 14.5 and sub-femto liter volume excitation is achieved. The system shows substantial sidelobe reduction (<4%) due to the non-linear intensity dependence of multiphoton process. Polarization effect on x-oriented and freely rotating dipoles shows dramatic change in the field distribution at the focal-plane. The resulting point-spread function has the ability to produce several strongly localized polarization dependent field patterns which may find applications in optical engineering and bioimaging.

Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy

Fast three-dimensional (3D) imaging requires parallel optical slicing of a specimen with an efficient detection scheme. The generation of multiple localized dot-like excitation structures solves the problem of simultaneous slicing multiple specimen layers, but an efficient detection scheme is necessary. Confocal theta detection (detection at 90 degrees to the optical axis) provides a suitable detection platform that is capable of cross-talk-free fluorescence detection from each nanodot (axial dimension approximate to 150 nm). Additionally, this technique has the unique feature of imaging a specimen at a large working distance with super-resolution capabilities. Polarization studies show distinct field structures for fixed and fluid samples, indicating a non-negligible field-dipole interaction. The realization of the proposed imaging technique will advance and diversify multiphoton fluorescence microscopy for numerous applications in nanobioimaging and optical engineering.