Jim Swoger

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.

The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis.

Tailoring the axial shape of the point spread function using the Toraldo concept

A novel procedure for shaping the axial component of the point spread function of nonparaxial focusing systems by use of phase-only pupil filters is presented. The procedure is based on the Toraldo technique for tailoring focused fields. The resulting pupil filters consist of a number of concentric annular zones with constant real transmittance. The number of zones and their widths can be adapted according to the shape requirements. Our method is applied to design filters that produce axial superresolution in confocal scanning systems.

Multiple imaging axis microscopy improves resolution for thick-sample applications

The multiple imaging axis microscope (MIAM) is a wide-field optical microscope that observes a sample simultaneously from multiple directions without requiring the sample to be rotated or tilted. The prototype is capable of high-resolution imaging of the interior of a 300-microm-diameter sample consisting of fluorescent microbeads suspended in an agarose gel. Compared with a single-axis system, the MIAM can achieve a reduction of the axial point-spread function elongation by a factor of 5.8 and a 3.5-fold improvement in volume resolution by simple linear image combination techniques.

Optical scanning holography as a technique for high-resolution three-dimensional biological microscopy

The applicability of optical scanning holography (OSH) to the field of microscopic imaging for biological applications is assessed. A generalized mathematical description of OSH that takes into account polarization effects, high numerical apertures, and generalized illumination wave fronts is presented. This description is used to show that the proposed single-beam scanning technique relaxes the restrictions under which OSH functions correctly compared with the conventional double-beam scanning method. It is also shown that, although in general OSH is restricted to thin samples, this condition can be relaxed in nonrefracting fluorescence samples, which are of importance in biological microscopy.

Decrease in Cell Volume Generates Contractile Forces Driving Dorsal Closure

Biological tissues must generate forces to shape organs and achieve proper development. Such forces often result from the contraction of an apical acto-myosin meshwork. Here we describe an alternative mechanism for tissue contraction, based on individual cell volume change. We show that during Drosophila dorsal closure (DC), a wound healing-related process, the contraction of the amnioserosa (AS) is associated with a major reduction of the volume of its cells, triggered by caspase activation at the onset of the apoptotic program of AS cells. Cell volume decrease results in a contractile force that promotes tissue shrinkage. Estimating mechanical tensions with laser dissection and using 3D biophysical modeling, we show that the cell volume decrease acts together with the contraction of the actin cable surrounding the tissue to govern DC kinetics. Our study identifies a mechanism by which tissues generate forces and movements by modulating individual cell volume during development.

4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies

A study demonstrating an imaging framework that permits the determination of cell lineages during organogenesis of the posterior lateral line in zebrafish is presented. The combination of Selective Plane Illumination Microscopy and specific fluorescent markers allows retrospective tracking of hair cell progenitors, and hence the derivation of their lineages within the primodium. It is shown that, because of its superior signal-to-noise ratio and lower photo-damaged properties, SPIM can provide significantly higher-quality images than Spinning Disk Confocal technology. This allows accurate 4D lineage tracing for the hair cells over tens of hours of primordium migration and neuromast development.

Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Resolution in optical microscopy.

Publisher Summary This chapter describes resolution in optical microscopy. Optical microscopes are fundamentally limited in the resolution they can achieve. The resolution depends on the wavelength of the light (both incident and detected), on the numerical aperture (NA) of the optical arrangement, and on the specimen to be observed or the experiment to be performed. Live specimens are also dynamic and sensitive to photobleaching and thermal damage, which imposes a limit on the duration for which they can be observed and on the power of the incident light. Fluorescence is excited throughout its illumination cone, but only fluorescence emitted from the focal point is imaged through the confocal pinhole to the detector. A useful tool for comparing the performance of optical microscopes is the point-spread function (PSF). The PSF can be defined in two complementary. The intensity PSF (hereafter referred to simply as the PSF) can be measured by taking images of—for example, a subresolution bead as it is scanned through the focus of a microscope. Real specimens are, of course, rarely point sources.

Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.