Extended morphological processing: a practical method for automatic spot detection of biological markers from microscopic images.

BACKGROUND: A reliable extraction technique for resolving multiple spots in light or electron microscopic images is essential in investigations of the spatial distribution and dynamics of specific proteins inside cells and tissues. Currently, automatic spot extraction and characterization in complex microscopic images poses many challenges to conventional image processing methods. RESULTS: A new method to extract closely located, small target spots from biological images is proposed. This method starts with a simple but practical operation based on the extended morphological top-hat transformation to subtract an uneven background. The core of our novel approach is the following: first, the original image is rotated in an arbitrary direction and each rotated image is opened with a single straight line-segment structuring element. Second, the opened images are unified and then subtracted from the original image. To evaluate these procedures, model images of simulated spots with closely located targets were created and the efficacy of our method was compared to that of conventional morphological filtering methods. The results showed the better performance of our method. The spots of real microscope images can be quantified to confirm that the method is applicable in a given practice. CONCLUSIONS: Our method achieved effective spot extraction under various image conditions, including aggregated target spots, poor signal-to-noise ratio, and large variations in the background intensity. Furthermore, it has no restrictions with respect to the shape of the extracted spots. The features of our method allow its broad application in biological and biomedical image information analysis

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation

Heterogeneous distribution of doublecortin-expressing cells surrounding the rostral migratory stream in the juvenile mouse

In the postnatal mammalian brain, neural stem cells of the ventricular-subventricular zone continue to generate doublecortin (Dcx)-expressing immature neurons. Throughout life, these immature neurons migrate to the olfactory bulb through the rostral migratory stream (RMS). In this study, we investigated the distribution of these putative immature neurons using enhanced green fluorescent protein (EGFP) expression in the area surrounding the RMS of the juvenile Dcx-EGFP mice. Through the combined use of an optical clearing reagent (a 2,2 '-thiodiethanol solution) and two-photon microscopy, we visualized three-dimensionally the EGFP-positive cells in the entire RMS and its surroundings. The resulting wide-field and high-definition images along with computational image processing methods developed in this study were used to comprehensively determine the position of the EGFP-positive cells. Our findings revealed that the EGFP-positive cells were heterogeneously distributed in the area surrounding the RMS. In addition, the orientation patterns of the leading process of these cells, which displayed the morphology of migrating immature neurons, differed depending on their location. These novel results provide highly precise morphological information for immature neurons and suggest that a portion of immature neurons may be detached from the RMS and migrate in various directions

Different Activity Patterns in Retinal Ganglion Cells of TRPM1 and mGluR6 Knockout Mice

TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamily, is the visual transduction channel downstream of metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells (BCs). Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB). In both TRPM1 and mGluR6 KO mouse retinas, OFF but not ON BCs respond to light stimulation. Here we report an unexpected difference between TRPM1 knockout (KO) and mGluR6 KO mouse retinas. We used a multielectrode array (MEA) to record spiking in retinal ganglion cells (RGCs). We found spontaneous oscillations in TRPM1 KO retinas, but not in mGluR6 KO retinas. We performed a structural analysis on the synaptic terminals of rod ON BCs. Intriguingly, rod ON BC terminals were significantly smaller in TRPM1 KO retinas than in mGluR6 KO retinas. These data suggest that a deficiency of TRPM1, but not of mGluR6, in rod ON bipolar cells may affect synaptic terminal maturation. We speculate that impaired signaling between rod BCs and AII amacrine cells (ACs) leads to spontaneous oscillations. TRPM1 and mGluR6 are both essential components in the signaling pathway from photoreceptors to ON BC dendrites, yet they differ in their effects on the BC terminal and postsynaptic circuitry