Replicate-based noise corrected correlation for accurate measurements of colocalization.

It is widely recognized that the accuracy of colocalization measurements is dependent upon the quality of the source images. We demonstrate that, as the image quality increases, the measured colocalization, using the Pearson and Spearman rank correlation coefficients, approaches the true colocalization asymptotically. This means that in practice it is difficult to obtain images of sufficient quality for accurate measurements. We introduce the replicate-based noise corrected correlation (RBNCC) which aligns the measured colocalization with the true colocalization: a noise measurement is made for each fluorophore from a pair of replicate images, the two noise measurements are combined to generate a correction factor which is applied to the measured colocalization between the two fluorophores. In consequence, accurate measurements can be obtained even with noisy images, making RBNCC especially attractive for live imaging. Even with images of apparently good quality we found an average discrepancy of about 20% between the measured and corrected colocalization. A case is made for using the Spearman rank coefficient instead of the Pearson coefficient to measure colocalization.

Reducing image distortions due to temperature-related microscope stage drift.

Distortions in confocal 3D image data sets were related to movements of the microscope stage that matched fluctuations in laboratory temperature. Movement was apparent in all three axes. Adding a draught-proof enclosure, covering the stage, objective lenses and supporting structures, minimized these short-term fluctuations. However, the stage still tracked slow changes in laboratory temperature and was sensitive to heat sources mounted on the microscope. Suggestions are made about microscope design.

Association of calmodulin with cytoskeletal structures at different stages of HeLa cell division, visualized by a calmodulin-EGFP fusion protein.

The fusion protein of calmodulin (CaM) with the enhanced green fluorescent protein EGFP has been expressed in a stably transfected HeLa cell line in order to visualise the localisation of calmodulin during the cell cycle on a continuous basis in live cells, and for immunofluorescence colocalisation with cytoskeletal structures. High-resolution images of CaM-EGFP in the mitotic apparatus show the characteristic strongly convoluted structure of the centrosome. CaM-EGFP also apparently associates with both polar and mitotic microtubules, and with a specific intracentrosomal structure. During cytokinesis, CaM-EGFP is also found decorating selected oriented filaments in close proximity to microtubules in the midbody region. In interphase cells, it is seen with filamentous and punctuate localisation at the nuclear envelope. The intensity and continuity of the CaM-EGFP images suggest that a significant fraction of the cellular calmodulin remains attached to cytoplasmic structures during the cell cycle.

Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts.

The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

Imaging of Ca2+ transients in endothelial cells of single perfused capillaries: correlation of peak [Ca2+]i with sites of macromolecule leakage.

OBJECTIVE: To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca2+]i, does not increase uniformly within microvessel endothelial cells. METHODS: We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca2+]i using a cooled CCD camera. RESULTS: Control [Ca2+]i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca2+]i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca2+]i (274 +/- 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca2+]i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increase in [Ca2+]i were largest. CONCLUSIONS: These data indicate that variation in [Ca2+]i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.

Extravascular distribution of low-density lipoprotein and dextran in a high-permeability state.

Previous studies from this laboratory indicated that in the early phase of a high-permeability state, low-density lipoprotein (LDL) crosses the microvascular barrier by porous pathways. To determine the extravascular distribution of LDL (3,500,000 MW) and a smaller reference macromolecule [20,000 MW dextran (D20)] image processing techniques were employed, and extravascular accumulation of solutes from different microvessels was compared to quantitative fluorescence microscopy. Frog mesenteric venular microvessels (n = 8) were cannulated and perfused with fluorescent-labeled LDL and D20 and extravascular distribution of both solutes was imaged at control and after permeability was increased with the calcium ionophore ionomycin (5 microM). At the peak increase in apparent permeability (2-4 min after ionophore), the processed images of the microvessels demonstrated that the extravascular distribution of fluorescent-labeled solute was not uniform and that D20 accumulated outside the microvessel wall in some areas where LDL did not accumulate. The patterns of extravascular accumulation of LDL and D20 in a high-permeability state imply a distribution of effective microvascular pore sizes and/or a distribution of resistances to solute flow in the pore or in the tissue surrounding the microvessel.

Measurement of cytoplasmic calcium in single microvessels with increased permeability.

We investigated the hypothesis that an increase in cytoplasmic calcium ion concentration, [Ca2+]i, is one of the mechanisms responsible for increased microvessel permeability. We loaded the cells forming the walls of individually perfused microvessels in frog mesentery with fura-2 and measured [Ca2+]i in the control state and after adding the Ca2+ ionophore ionomycin to the perfusate. [Ca2+]i in the control state was 65 +/- 6 nM and increased to an initial peak of 285 +/- 29 nM after 1-3 min. After 4, 6, and 10 min, [Ca2+]i was 199 +/- 18, 163 +/- 16, and 129 +/- 9 nM, respectively. [Ca2+]i fell back to 77 +/- 7 nM after ionophore was removed. In similar experiments, hydraulic conductivity (Lp) increased to a peak of 9.5 times control after 1-3 min, then fell to 2.0 times control after 6 min. Lp remained elevated at this level for as long as ionophore was present in the perfusate. [Ca2+]i modulates the initial and sustained phases of the permeability increase. Both processes depend on external Ca2+ influx. Our experiments provide the first direct measurement of [Ca2+]i during a change in the permeability of an intact microvessel.