Stephen W. Paddock

Hairy and Emc negatively regulate morphogenetic furrow progression in the drosophila eye

The initial steps of pattern formation in the developing Drosophila eye involve the coordination of cell cycles, changes in cell shape, and the specification of the R8 photoreceptor cell. These events begin several cell rows ahead of the morphogenetic furrow and are positively regulated by secreted signaling proteins and the proneural HLH transcription factor atonal (ato). Two HLH regulatory proteins that function to suppress neuronal development in other tissues, extra macrochaetae (emc) and hairy (h), are expressed ahead of the morphogenetic furrow. While neither h nor emc is required for photoreceptor cell determination, in emc-h-clones the morphogenetic furrow and differentiated eye field advance up to eight ommatidial rows ahead of adjacent wild-type tissue. This indicates that morphogenetic furrow progression and neuronal differentiation are negatively regulated by a combination of anteriorly expressed HLH regulatory proteins.

Introduction to confocal microscopy and three-dimensional reconstruction.

Publisher Summary This chapter introduces the principle of confocal microscopy. The different types of confocal microscopes currently available and the various applications of confocal microscopy are discussed. Methods of specimen preparation for confocal microscopy are provided as guidelines and should be generally applicable to most cell types. The chapter also describes three-dimensional reconstruction, four-dimensional imaging, and the methodology for producing color prints and slides of confocal data. In conventional microscopy, much of the depth or volume of the specimen is uniformly and simultaneously illuminated in addition to the plane in which the objective lens is focused. This leads to out-of-focus blur from areas above and below the focal plane of interest. Out-of-focus light reduces contrast and decreases resolution, making it difficult to discern various cellular structures. In contrast, the illumination in a confocal microscope is not simultaneous, but sequential. The illumination is focused as a spot on one volume element of the specimen at a time.

Further developments of the laser scanning confocal microscope in biomedical research.

Abstract The laser scanning confocal microscope (LSCM) is a valuable research tool for imaging fluorescently labeled biological specimens. Rather than cutting sections of the tissue with a knife, it is now possible to produce relatively noninvasive “optical sections” using the LSCM as an imaging tool. This has made the imaging of living cells in situ more of a practical option. This minireview briefly describes some of the improvements made to the LSCM over the past 5 years and, in more detail, outlines many of the current biomedical applications of the LSCM, including single and multiple labeling of fixed and living specimens, physiological imaging, 3-dimensional imaging, and the use of the LSCM for lineage tracing and in correlative microscopy.

The Laser-Scanning Confocal Microscope in Biomedical Research

The laser-scanning confocal microscope (LSCM) produces improved light microscope images of both fixed and living cells and tissues. Moreover, the serial optical-sectioning power of the LSCM has made three dimensional reconstruction of light microscope images a practical option. The different confocal microscopes that have resulted in the current generation of the LSCM and the applications of the LSCM for biomedical research are briefly reviewed: further details can be found elsewhere (1–3). Historical Perspective Marvin Minskys Microscope. The confocal microscope was invented in 1955 by Marvin Minsky specifically for studying neural networks in the living brain (4, 5). All modern confocal microscopes are based on Minskys original idea, which was patented in 1957. Basically, illumination and detection are confined to a single diffraction-limited point in the specimen. The point is scanned across the specimen and light from the specimen is built into an image of a precise optical section of the specimen. The method of image formation in a confocal microscope is fundamentally different from that in a conventional wide field microscope, in which the entire specimen is bathed in light, usually from a mercury or xenon source, and the final image quality can be degraded by light scattered from out-of-focus structures. In Minskys original confocal microscope, the point source of light is produced by a pinhole placed in front of a zirconium arc source. The point of light is focused by an objective lens onto the specimen, and light that passes through the specimen is focused by a second objective lens onto a second pinhole, which has the same focus as the first pinhole, i.e., it is confocal with the first pinhole. Light that passes through the second pinhole is detected by a low noise photomultiplier.

Tools of the Trade Image Manipulation: Confocal images to go?

Biologists are no longer restricted to using a single algorithm in their manipulation and display of data acquired using confocal microscopy.