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Cell-based assays are essential in both basic research and drug discovery. Three-dimensional cellular spheroids are more realistic models of tumors and healthy tissues compared to standard two-dimensional cultures. Employing spheroids improves the reliability and the physiological significance of cell-based assays. We present a detailed drug assay protocol performed with live cellular spheroids. We employ automated epifluorescence live microscopy to investigate the effects of drugs on the spheroids over several days. We describe the spheroid preparation, manipulation, live fluorescence imaging, and data processing. We quantify the autophagy-triggering effects of the drugs (-)-Gossypol and Rapamycin in glioma cell spheroids. The formation of the autophagosomes and the fusion of the autophagosomes with lysosomes in the treated spheroids are monitored over time and space with a mRFP:GFP:LC3 fusion protein.

Dynamic microscopy of living cells and organisms alone does not reveal the high level of complexity of cellular and subcellular organization. All observable processes rely on the activity of biochemical and biophysical processes and many occur at a physiological equilibrium. Experimentally, it is not trivial to apply a perturbation that targets a specific process without perturbing the overall equilibrium of a cell. Drugs and more recently RNAi certainly have general and undesired effects on cell physiology and metabolism. In particular, they affect the entire cell. Pulsed lasers allow to severe biological tissues with a precision in the range of hundreds of nanometers and to achieve ablation on the level of a single cell or a subcellular compartment. In this chapter, we present an efficient implementation of a picosecond UV-A pulsed laser-based nanosurgery system and review the different mechanisms of ablation that can be achieved at different levels of cellular organization. We discuss the performance of the ablation process in terms of the energy deposited onto the sample and compare our implementation to others recently employed for cellular and subcellular surgery. Above the energy threshold of ionization, we demonstrate how to achieve single-cell ablation through the induction of mechanical perturbation and cavitation in living organisms. Below this threshold, we induce cytoskeleton severing inside live cells. By combining nanosurgery with fast live-imaging fluorescence microscopy, we show how the apparent equilibrium of the cytoskeleton can be perturbed regionally inside a cell.

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.

Publisher Summary Microscopy, and in particular confocal microscopy and two-photon excitation, provide a three-dimensional resolution; therefore, a three-dimensional resolution criterion is necessary that documents that a resolution improvement along one axis is not imposed at the expense of a decrease along another axis. Some techniques using—for example, annular apertures—improve the lateral resolution but at the same time degrade the axial resolution. The confocal microscope reduces the illumination field and the detection field to the physical limit determined by diffraction and hence discriminates all light emitted outside the focal volume. In a 4Pi(A) confocal microscope, two opposing lenses use spherical waves to illuminate a focal spot coherently and produce a standing wave that modulates the intensity along the optical axis. Two minima are slightly more than λ/2n apart. By adjusting the phase and moving the object relative to the focal spot while recording the fluorescence intensity as a function of the position, images are generated. In the confocal theta microscope, the axial resolution can be improved by a factor of three, and low-NA systems may have axial resolutions that are better than those achieved with confocal high-NA systems.

7 pages, 3 figures, 1 table., Transverse and longitudinal momentum distributions of 9Li fragments from 11Li break-up reactions in C, Al and Pb targets have been measured at 280 MeV/u. The two-neutron removal cross-section was measured to be σ(−2n) = 0.26 ± 0.02 b for the carbon target, σ(−2n) = 0.47 ± 0.08 b for the aluminum target and σ(−2n) = 1.9 ± 0.4 b for the lead target. No significant difference is observed between the narrow widths (FWHM ≈ 47 MeV/c) of the transverse and longitudinal momentum distributions of the 9Li fragments. The physical implications of this are discussed., This work was supported by the German Federal Minister for Research and Technology (BMFT) under Contracts 06 DA 641 I, 06 BO 103 and 06 MZ 106 and by GSI via Hochschulzusammenarbeitsvereinbarungen under Contracts DA RICK, BO FRE, F ELE, Mz KRD and partly supported by the Polish Committee of Scientific Research under Contract PB 1158/P3/92/02 and CICYT under contract AEN92-078%CO2-02 (MJGB).

Publisher Summary Confocal scanning-laser microscopy combined with sophisticated image-processing techniques allow the cell biologist to explore the three-dimensional infrastructure of the cell, thus conferring a high degree of precision in localization experiments. This chapter discusses some of the inherent problems in preservation of biological specimens so that reliable data can be acquired with a confocal fluorescence microscope. The methodology is the result of an effort to study the genesis of epithelial polarity in the Madin-Darby canine kidney (MDCK) cell line. While specific methods have been developed to study these particular cells, the lessons learned about their preservation should be widely applicable. A description of the confocal principle introduces basic concepts of confocal fluorescence microscopy. One can then understand how images are recorded and how the information may be interpreted.