Due to the internal nature of mammalian development, much of the research performed is of a static nature and depends on interpolation between stages of development. This approach cannot explore the dynamic interactions that are essential for normal development. While roller culture overcomes the problem of inaccessibility of the embryo, the constant motion of the medium and embryos makes it impossible to observe and record development. We have developed a static mammalian culture system for imaging development of the mouse embryo. Using this technique, it is possible to sustain normal development for periods of 18-24 h. The success of the culture was evaluated based on the rate of embryo turning, heart rate, somite addition, and several gross morphological features. When this technique is combined with fluorescent markers, it is possible to follow the development of specific tissues or the movement of cells. To highlight some of the strengths of this approach, we present time-lapse movies of embryonic turning, somite addition, closure of the neural tube, and fluorescent imaging of blood circulation in the yolk sac and embryo.

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

Abstract. Multiphoton microscopy is becoming a popular mode of live and fixed cell imaging. This mode of imaging offers several advantages due to the fact that fluorochrome excitation is a nonlinear event resulting in excitation only at the plane of focus. Multiphoton excitation is enhanced by the use of ultrafast lasers emitting in the near IR, offering better depth penetration coupled with efficient excitation. Because these lasers, such as titanium:sapphire lasers, offer tunable output it is possible to use them to collect multiphoton excitation spectra. We use the software-tunable Coherent Chameleon laser coupled to the Zeiss LSM 510 META NLO to acquire x-y images of biological samples at multiple excitation wavelengths, creating excitation lambda stacks. The mean intensity of pixels within the image plotted versus excitation wavelength reveals the excitation spectra. Excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in much the same way that emission fingerprinting can be used to generate crosstalk free channels from emission lambda stacks using the META detector. We show how this technique can be used to eliminate autofluorescence and to produce crosstalk-free images of dyes with very close overlap in their emission spectra that cannot be separated using emission fingerprinting. Moreover, excitation fingerprinting can be performed using nondescanned detectors (NDDs), offering more flexibility for eliminating autofluorescence or crosstalk between fluorochromes when imaging deep within the sample. Thus, excitation fingerprinting complements and extends the functions offered by the META detector and emission fingerprinting. We correct biases in the laser and microscope transmission to acquire realistic multiphoton excitation spectra for fluorochromes within cells using the microscope, which enables the optimization of the excitation wavelength for single and multilabel experiments and provides a means for studying the influence of the biological environment on nonlinear excitation.

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