Specific Visualization of Glioma Cells in Living Low-Grade Tumor Tissue

BACKGROUND: The current therapy of malignant gliomas is based on surgical resection, radio-chemotherapy and chemotherapy. Recent retrospective case-series have highlighted the significance of the extent of resection as a prognostic factor predicting the course of the disease. Complete resection in low-grade gliomas that show no MRI-enhanced images are especially difficult. The aim in this study was to develop a robust, specific, new fluorescent probe for glioma cells that is easy to apply to live tumor biopsies and could identify tumor cells from normal brain cells at all levels of magnification. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation we employed brightly fluorescent, photostable quantum dots (QDs) to specifically target epidermal growth factor receptor (EGFR) that is upregulated in many gliomas. Living glioma and normal cells or tissue biopsies were incubated with QDs coupled to EGF and/or monoclonal antibodies against EGFR for 30 minutes, washed and imaged. The data include results from cell-culture, animal model and ex vivo human tumor biopsies of both low-grade and high-grade gliomas and show high probe specificity. Tumor cells could be visualized from the macroscopic to single cell level with contrast ratios as high as 1000: 1 compared to normal brain tissue. CONCLUSIONS/SIGNIFICANCE: The ability of the targeted probes to clearly distinguish tumor cells in low-grade tumor biopsies, where no enhanced MRI image was obtained, demonstrates the great potential of the method. We propose that future application of specifically targeted fluorescent particles during surgery could allow intraoperative guidance for the removal of residual tumor cells from the resection cavity and thus increase patient survival

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

CD133 (Prominin) Negative Human Neural Stem Cells Are Clonogenic and Tripotent

CD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined

Spectrally resolved fluorescence lifetime imaging microscopy

We report a system for collecting spectrally resolved fluorescent lifetime images. Frequency domain fluorescence lifetime detection was combined with two-dimensional spectral imaging in a programmable array microscope. The spectroscopic fluorescence lifetime imaging microscopy (sFLIM) system has a resolution of similar to50 (lambda/Deltalambda) in the current arrangement and a wavelength range of similar to430-750 nm. With the sFLIM system, we recorded the lifetime spectra of rhodamine 6G, rhodamine B, and the DNA intercalation dye propidium iodide (PI) in cuvettes and an EGFP-fusion of the histone 2A variant D protein in Drosophila salivary gland explants in the presence and absence of PI. In the absence of PI, the EGFP-fusion exhibited a lifetime of 2.7 ns with little variation in wavelength. The lifetime of PI alone ranged from similar to1 ns in buffer to similar to18 ns when intercalated in the nuclei of intact cells. The combination of EGFP and PI in the Drosophila salivary gland explants exhibited strong fluorescence resonance energy transfer (FRET), a result consistent with the known nucleosomal structure of eukaryotic chromatin

Three-dimensional spectral imaging by Hadamard transform spectroscopy in a programmable array microscope

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains