Three-dimensional Fluorescence Lifetime Imaging with a Single Plane Illumination Microscope provides an improved signal to noise ratio.

We designed a widefield frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM)setup, which is based on a Single Plane Illumination Microscope (SPIM). A SPIM provides an inherent optical sectioning capability and reduces photobleaching compared to conventional widefield and confocal fluorescence microscopes. The lifetime precision of the FLIM was characterized with Rhodamine 6G solutions of different quencher concentrations [KI]. We demonstrate the high spatial resolution of the SPIM-FLIM combination in the intensity domain as well as in the lifetime domain with latex bead samples and multiple recordings of three-dimensional live Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the bleaching rate after 600 images have been recorded is below 5%.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

BACKGROUND AND PURPOSE: Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). METHODS: Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. RESULTS: Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. CONCLUSIONS: The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Multi-view image fusion improves resolution in three-dimensional microscopy.

A non-blind, shift-invariant image processing technique that fuses multi-view three-dimensional image data sets into a single, high quality three-dimensional image is presented. It is effective for 1) improving the resolution and isotropy in images of transparent specimens, and 2) improving the uniformity of the image quality of partially opaque samples. This is demonstrated with fluorescent samples such as Drosophila melanogaster and Medaka embryos and pollen grains imaged by Selective Plane Illumination Microscopy (SPIM). The application of the algorithm to SPIM data yields high-resolution images of organ structure and gene expression, in some cases at a sub-cellular level, throughout specimens ranging from several microns up to a millimeter in size.

Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM).

Advances in the life sciences rely on the ability to observe dynamic processes in live systems and in environments that mimic in-vivo situations. Therefore, new methodological developments have to provide environments that resemble physiologically and clinically relevant conditions as closely as possible. In this work, plasma-induced laser nanosurgery for three-dimensional sample manipulation and sample perturbation is combined with optically sectioning light-sheet based fluorescence microscopy (SPIM) and applied to three-dimensional biological model systems. This means: a) working with a biological system that is not confined to essentially two dimensions like cell cultures on cover glasses, b) gaining intrinsic optical sectioning capabilities by an efficient three-dimensional fluorescence imaging system, and c) using arbitrarily-shaped three-dimensional ablation-patterns by a plasma-induced laser ablation system that prevent damage to surrounding tissues. Spatial levels in our biological applications range from sub-microns during delicate ablation of single microtubules over the confined disruption of cell membranes in an MDCK-cyst to the macroscopic cutting of a millimeter-sized Zebrafish caudal fin with arbitrary three-dimensional ablation patterns. Dynamic processes like laser-induced hemocyte migration can be studied with our SPIM-microscalpel in intact, live embryos.

ojoplano-mediated basal constriction is essential for optic cup morphogenesis.

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage.

Light-sheet-based fluorescence microscopy (LSFM) is a fluorescence technique that combines optical sectioning, the key capability of confocal and two-photon fluorescence microscopes with multiple-view imaging, which is used in optical tomography. In contrast to conventional wide-field and confocal fluorescence microscopes, a light sheet illuminates only the focal plane of the detection objective lens from the side. Excitation is, thus, restricted to the fluorophores in the volume near the focal plane. This provides optical sectioning and allows the use of regular cameras in the detection process. Compared to confocal fluorescence microscopy, LSFM reduces photo bleaching and photo toxicity by up to three orders of magnitude. In LSFM, the specimen is embedded in a transparent block of hydrogel and positioned relative to the stationary light sheet using precise motorized translation and rotation stages. This feature is used to image any plane in a specimen. Additionally, multiple views obtained along different angles can be combined into a single data set with an improved resolution. LSFMs are very well suited for imaging large live specimens over long periods of time. However, they also perform well with very small specimens such as single yeast cells. This perspective introduces the principles of LSFM, explains the challenges of specimen preparation, and introduces the basics of a microscopy that takes advantage of multiple views.

Lateral modulation boosts image quality in single plane illumination fluorescence microscopy.

A new microscope combines optical sectioning by fluorophore excitation using a single light sheet with structured illumination. Several images with laterally intensity-modulated light sheets are recorded from scattering fluorescent specimens. By applying a simple data processing scheme, the nonmodulated volumes are identified. The blurred features become dark, and the resultant images are improved in terms of contrast and resolution. Hence, the instrument is capable of discriminating against contributions to the image that are induced by the optical properties of the specimen. The new microscope's capabilities are demonstrated by imaging the internals of the head of an adult Drosophila melanogaster (fruit fly) expressing green fluorescent protein-labeled polycomb proteins.

High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy.

We report that single (or selective) plane illumination microscopy (SPIM), combined with a new deconvolution algorithm, provides a three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy in large samples. We demonstrate this by imaging large living multicellular specimens obtained in a three-dimensional cell culture. The ability to rapidly image large samples at high resolution with minimal photodamage provides new opportunities especially for the study of subcellular processes in large living specimens.

Dynamic organization of the actin cytoskeleton during meiosis and spore formation in budding yeast.

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non-polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post-meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate-rich compartments, which may be chitosome analogous structures.