Fluorophore unmixing based on bleaching and recovery kinetics using MCR-ALS.

Fluorescence microscopy is a key technology in the life sciences, though its performance is constrained by the number of labels that can be recorded. We propose to use the kinetics of fluorophore photodestruction and subsequent fluorescence recovery to distinguish multiple spectrally-overlapping emitters in fixed cells, thus enhancing the information that can be obtained from a single measurement. We show that the data can be directly processed using multivariate curve resolution - alternating least squares (MCR-ALS) to deliver distinct images for each fluorophore in their local environment, and apply this methodology to membrane imaging using DiBAC4(3) and concanavalin A - Alexa Fluor 488 as the fluorophores. We find that the DiBAC4(3) displays two distinct degradation/recovery kinetics that correspond to two different label distributions, allowing us to simultaneously distinguish three different fluorescence distributions from two spectrally overlapping fluorophores. We expect that our approach will scale to other dynamically-binding dyes, leading to similarly increased multiplexing capability.

Lack of in vitro evidence for storage of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1,25(OH)2D3 binding protein in skeletal matrix.

A few studies have reported on the measurement of 1, 25-dihydroxycholecalciferol (1,25(OH)2D3) in bone, using chloroform/methanol extraction and radioreceptor assay. As the significance of bone 1,25(OH)2D3 content was not defined in any of these reports, the objective of the current investigation was to determine whether 1,25(OH)2D3 may be stored in skeletal matrix. Bone powder samples from the iliac crest were extracted in ethylacetate/cyclohexane and 1,25(OH)2D3 isolated from the extract by means of Sephadex LH-20 and high pressure liquid chromatographic separation and subsequently measured by radioimmunoassay (RIA). Within the detection range of the RIA, no 1,25(OH)2D3 could be measured, suggesting that 1,25(OH)2D3 is not stored in skeletal matrix. Vitamin D bone concentrations previously measured may therefore have reflected plasma contamination. Consistent with this hypothesis, only traces of skeletal 1,25(OH)2D3 binding protein were measured when compared with serum values. Although 1,25(OH)2D3 may act as a potential local determinant of bone remodeling, there is no evidence supporting a delayed paracrine function by matrix-derived 1, 25(OH)2D3.

Alterations of the mineralization profile and osteocalcin concentrations in osteoarthritic cortical iliac crest bone.

The relation between bone mineralization and osteocalcin content was investigated in iliac crest cortical bone obtained at necropsy in young females and in two groups of elderly women with and without osteoarthritis of the hands evaluated by X-ray. Using density fractionation technique, the bone was separated into fractions of increasing density from 1.72 to 2.30 g/ml. The mineralization profile revealed a significant shift to higher densities in the osteoarthritis cases compared with young adults (P less than 0.005) and age-sex-matched controls (P less than 0.001). The ash, calcium, and phosphorus content of the bone increased with increasing density of the fractions whereas collagen content, measured as hydroxyproline, decreased. The osteocalcin concentration of each fraction was determined in the supernatants obtained after EDTA-extraction in the presence of protease inhibitors. In the young control and osteoarthritis group, the osteocalcin content in the lowest density fractions was higher compared with the older non-osteoarthritic group. Osteocalcin content of the high density fractions, representing highly mineralized osteons, was the same in the three groups studied. These findings support the hypothesis that quality differences in bone may explain the inverse relationship between osteoarthritis and osteoporosis.