A New Histochemical Double-Stain Method Using Three-Dimensional Analysis with Confocal laser Scanning Microscopy

We describe a new technique for immunohistochemical and enzyme-histochemi-cal double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D_28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for ma⁺-ATPase in the rat kidney. The lead precipitation method for Ca²⁺-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35-calbindin-D_28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three-dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca²⁺-ATPase, were distributed deep in the section. The most intense signals from the silver partkles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution.     

Searching by parts: Towards fine-grained image retrieval respecting species correlation

Most of the existing works on fine-grained image categorization and retrieval focus on finding similar images from the same species and often give little importance to inter-species similarities. However, these similarities may carry species correlations such as the same ancestors or similar habits, which are helpful in taxonomy and understanding biological traits. In this paper, we devise a new fine-grained retrieval task that searches for similar instances from different species based on body parts. To this end, we propose a two-step strategy. In the first step, we search for visually similar parts to a query image using a deep convolutional neural network (CNN). To improve the quality of the retrieved candidates, structural cues are introduced into the CNN using a novel part-pooling layer, in which the receptive field of each part is adjusted automatically. In the second step, we re-rank the retrieved candidates to improve the species diversity. We achieve this by formulating a novel ranking function that balances between the similarity of the candidates to the queried parts, while decreasing the similarity to the query species. We provide experiments on the benchmark CUB200 dataset and Columbia Dogs dataset, and demonstrate clear benefits of our schemes.     

DISTRIBUTION AND SYSTEMATICS OF THE FRESHWATER GENUS TUOMEYA (RHODOPHYTA,BATRACHOSPERMACEAE)1

Twenty-seven populations of Tuomeya, including the types of Tuomeya fluviatilis Harvey and Baileya americana Kützing, were analyzed from the entire known range of the genus: northern Newfoundland to northern Florida and east of the Mississippi River. Key morphological features were examined in detail since it has been recently proposed that Tuomeya should be reduced to a section of the genus Batrachospermum. Our observations confirmed the presence of several characteristics unique to Tuomeya: gametophyte development from a basal mass of undifferentiated cells rather than a chantransia stage, pseudoparenchymatous growth, and carpogonia with obliquely to perpendicularly attached trichogynes. Based on these findings, we conclude that the genus Tuomeya should be retained. Using multivariate morphometrics, two groupings were found that differed significantly in plant length (X = 14.1 and 24.1 mm, respectively). However, since there was no other morphometric, environmental, or geographic basis for separation of the groupings, only one species is recognized, T. americana (Kützing) Papenfuss. Populations tend to occur in large streams (>2.8 m wide) with low ion content (≤ 100 μS · cm^?1).     

人体超微弱发光图像中的信号检验

用近期研制的具有单光子探测能力的超高灵敏度成像系统获得了人体体表生物超微弱发光的强度数据。为分析图像中的信号检验,二项分布被用于人手不同时间累积发光图像中的信号显著性检验,证实人手存在超微弱生物发光,手指的发光强度在３００～５５０ｐｈｏｔｏｎｓ／ｓ范围内,全手的发光强度在８５０～１２００ｐｈｏｔｏｎｓ／ｓ范围内。     

正常胎盘的超声图像定量分析研究

我们用ＤＦＹ－Ｉ型超声图像定量分析诊断仪,测定组织结构超声图像的分贝,灰阶值,本文检测分析了３９例正常台盘测值。     

SEXUAL DIFFERENTIATION OF GRIFFITHSIA (CERAMIALES, RHODOPHYTA): NUCLEAR PLOIDY LEVEL OF MIXED-PHASE PLANTS IN G. JAPONICA1

Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae .     

Image analysis, neural networks, and the taxonomic impediment to biodiversity studies

The taxonomic impediment to biodiversity studies may be influenced radically by the application of new technology, in particular, desktop image analysers and neural networks. The former offer an opportunity to automate objective feature measurement processes, and the latter provide powerful pattern recognition and data analysis tools which are able to 'learn' patterns in multivariate data. The coupling of these technologies may provide a realistic opportunity for the automation of routine species identifications. The potential benefits and limitations of these technologies, along with the development of automated identification systems are reviewed.     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.