SummaryTest systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm −2 and about 300 J cm −2 for native cells (corresponding to about 250−3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi-or total internal reflection illumination) and decreased to values between 50 J cm −2 and less than 1 J cm −2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high-resolution wide field or laser scanning microscopy of single cells, typically 10−20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in superresolution microscopy based on single molecule detection.
In the past, the majority of antitumor compound-screening approaches had been performed in two-dimensional (2D) cell cultures. Although easy to standardize, this method provides results of limited significance because cells are surrounded by an artificial microenvironment, are not exposed to hypoxia gradients, and lack cell-cell contacts. These nonphysiological conditions directly affect relevant parameters such as the resistance to anticancer drugs. Multicellular tumor spheroids more closely resemble the in vivo situation in avascularized tumors. To monitor cellular reactions within this three-dimensional model system, we stably transfected a spheroid-forming glioblastoma cell line with Grx1-roGFP2, a green fluorescent protein (GFP)-based glutathione-specific redox sensor that detects alterations in the glutathione redox potential. Functionality and temporal dynamics of the sensor were verified with redox-active substances in 2D cell culture. Based on structured illumination microscopy using nonphototoxic light doses, ratio imaging was then applied to monitor the response of the glutathione system to exogenous hydrogen peroxide in optical sections of a tumor spheroid. Our approach provides a proof of concept for biosensor-based imaging in 3D cell cultures.
In the context of various approaches to super-resolution microscopy, structured illumination microscopy (SIM) offers several advantages: it needs rather low light doses (with a low risk of phototoxicity or photobleaching), is comparably fast and flexible concerning the use of microscopes, objective lenses and cameras, and has potential for 3D imaging. This paper describes an experimental setup for SIM with first diffraction orders of a spectral light modulator (SLM) creating an interference pattern in two dimensions. We kept this system rather compact with a comparably large illuminated object field, validated it with nano-beads and applied it further to living cells for imaging the cytoskeleton, mitochondria or cell nuclei with a resolution slightly above 100 nm. Its advantages, challenges and limitations—concerning cameras, acquisition time, depth of imaging, light exposure, and combining it with further super-resolving methods—are discussed.
Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z -stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.
An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide-field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 μm diameter, in agreement with visual observation. Smaller sub-structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2-dimensional cell cultures are confirmed by 3-dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label-free detection of apoptosis and may represent a first step towards label-free in vivo diagnostics.
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