The release of histamine and other inflammatory mediators from mast cells and basophils is the primary event in a variety of acute allergic and inflammatory conditions. The mechanism of the release of granules from these cells and the degranulation process of the cells is still not clear. Fluorescence microscopy has played an essential role in cell biology by allowing visualization of specific molecular components of cells. Recently, confocal laser scanning microscopy and atomic force microscopy have been introduced into the field for studying the dynamic molecular organization. We review current views, including our recent imaging work on mast cell activation and degranulation, using these new microscopic methods.
We considered the image acquisition system and algorithm of dynamic thresholding method for automatically obtaining the appropriate binary image of beef rib-eye area. Our proposed image acquisition system, in which the physical object was covered with non-reflecting glass plate and cooking oil was filled in the space between them, was able to exclude irregular reflection spots. The dynamic thresholding method consisted of three steps. First, the threshold value of a split region was determined by ordinary thresholding method. Second, the threshold value of each pixel was estimated with linear function of threshold values at vertexes of the target region. Finally, binarization was carried out for each pixel based on its threshold value. This algorithm is described herein. We were able to obtain the appropriate binary image using the proposed image acquisition system and the dynamic thresholding method.
Osmotic cell volume perturbations were approximated by the changes of cell height by scanning HeLa cells loaded with di-8-anepps (a potential-sensitive fluorescent indicator) along the z-axis with a confocal microscope. Cells challenged with a 280 mosM buffer for 10 min did not induce significant changes in cell height. When cells were shocked with a 190 mosM buffer, a rapid cell swelling followed by a slow regulatory volume decrease (RVD) was observed. When the osmolarity of the buffer was adjusted to 170 or 150 mosM, rapid cell swelling was obtained again but cells could not regain their original volume. On the other hand, hypertonic (340 mosM) shock induced cell shrinkage and no regulatory volume increase was observed. Changes in the total area of an optical section 2 μm above the cover slip during 190 mosM hypotonic shock were also determined. Comparison of the changes in cell height to changes in area indicates that cell height was a more sensitive indicator to study cell swelling and RVD in a monolayer of HeLa cells. Results in our study also illustrate that challenge of cells with 190 mosM buffer induced a transient increase in the intracellular free Ca2+ level ([Ca2+]i). When BAPTA/AM-loaded cells were exposed to a Ca2+-free-EGTA 190 mosM buffer, cell swelling was elicited as usual but the rise of [Ca2+]i and the RVD response were abolished. Interestingly, suppression of there-uptake of Ca2+ by thapsigargin (2 μM), a microsomaI Ca2+-ATPase inhibitor, to sustain the increase in [Ca2+]i during the 190 mosM shock did not produce a ‘normal’ but rather a weak and delayed RVD. In contrast, challenge to the cells with the 190 mosM solution in the presence of ionomycin (5 μM) displayed a fast transient Ca2+ response and an accelerated RVD response. These observations therefore indicate that an increase in [Ca2+]i followed by a decrease is an important factor in the triggering of cell volume decrease in HeLa cells.
Actin polymerization occurs in the process of neutrophil migration toward chemotactic stimuli, and rapid increase of Ca2+ concentration is also observed after stimulation of chemotactic substances such as formyl peptides. We monitored the changes in intracellular actin concentration ([actin]i) and Ca2+ concentration ([Ca2+]i) simultaneously in living guinea pig neutrophils, to investigate the relationship between Ca2+ and actin in the mechanism of chemotaxis. To induce chemotaxis, we used N-formyl-methionylleucylphenylalanine (FMLP) and a unique chemotactic peptide [Lys7] I-CP which causes only chemotaxis but no additional responses, such as superoxide production. To monitor the changes in [actin]i and [Ca2+]i simultaneously, rabbit muscle actin was labeled with a fluorescent dye, iodoacetamido morpholino rhodamine (IMR), and injected into neutrophils together with fluo3, a fluorescent Ca2+ indicator, and monitored with confocal laser microscope. Rapid increases in [actin]i and [Ca2+]i were observed in the focal plane of FMLP-stimulated neutrophils, but not observed in [Lys7] I-CP-stimulated neutrophils. However, in both cases, injected actin mostly coexisted with higher concentration of Ca2+. These results suggest that the inerease in [Ca2+]i is not strongly related to chemotaxis, but is needed for actin polymerization in the process of chemotaxis.