Automated Quantitative Bone Analysis in In Vivo X-ray Micro-Computed Tomography.

Measurement and analysis of bone morphometry in 3D micro-computed tomography volumes using automated image processing and analysis improve the accuracy, consistency, reproducibility, and speed of preclinical osteological research studies. Automating segmentation and separation of individual bones in 3D micro-computed tomography volumes of murine models presents significant challenges considering partial volume effects and joints with thin spacing, i.e.

Flat field correction for high-throughput imaging of fluorescent samples.

Vignetting of microscopic images impacts both the visual impression of the images and any image analysis applied to it. Especially in high-throughput screening high demands are made on an automated image analysis. In our work we focused on fluorescent samples and found that two profiles (background and foreground) for each imaging channel need to be estimated to achieve a sufficiently flat image after correction.

Calculation of photon-count number distributions via master equations.

Fitting of photon-count number histograms is a way of analysis of fluorescence intensity fluctuations, a successor to fluorescence correlation spectroscopy. First versions of the theory for calculating photon-count number distributions have assumed constant emission intensity by a molecule during a counting time interval. For a long time a question has remained unanswered: to what extent is this assumption violated in experiments? Here we present a theory of photon-count number distributions that takes account of intensity fluctuations during a counting time interval.

Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction.

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay.

Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy: trypsin proteinase reaction.

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels.