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.     

Confocal laser scanning light microscopy of the extra-thecal epithelia of undecalcified scleractinian corals

The extra-thecal epithelia of cryofixed undecalcified, freeze-substituted polyps of the scleractinian corals Galaxea fascicularis and Tubastrea faulkneri and axial and basal polyps of Acropora formosa have been examined, in anhydrously prepared thick slices, by confocal laser scanning light microscopy. The avoidance of chemical fixation and decalcification makes it possible to determine whether previously seen structures are real or artefactual products of swelling, shrinkage and distortion. All of the epithelia of all the corals examined are characterised by well defined intercellular spaces. Mucocytes are present in all cell layers in Galaxea and Tubastrea but are not present in any cell layers in the axial polyp of Acropora although they are abundant in the oral ectoderm of the basal polyps in this coral. Zooxanthellae are absent in Tubastrea, the epithelia of the exert septa of Galaxea and the axial polyp of Acropora. The calicoblastic ectoderm is generally composed of thin squamous cells with large intercellular spaces. At rapidly calcifying regions such as the tips of the exert septa of Galaxea, the calicoblastic cells are elongated with extensive arborisation of the basal regions of the cells. They are separated by large intercellular spaces and contain numerous fluorescent granules. The apical regions of these cells appear to be closely applied to the surface of the skeleton. There is no evidence of a space between the apical region of the calicoblastic cells and the skeleton.     

番茄种子经激光和咖啡因处理后其当代生物效应的研究

咖啡因具有抑制一些酶的活性,可减弱或消除生物体自身对激光引起的生物效应的修复作用。试验证明咖啡因对番茄种子萌发有强烈的抑制作用,咖啡因和激光复合处理生物效应显著,可降低番茄种子的发芽,并促进番茄果实增大和生长前期速率加快。     

Damage to living trees contributes to almost half of the biomass losses in tropical forests

Accurate estimates of forest biomass stocks and fluxes are needed to quantify global carbon budgets and assess the response of forests to climate change. However, most forest inventories consider tree mortality as the only aboveground biomass (AGB) loss without accounting for losses via damage to living trees: branchfall, trunk breakage, and wood decay. Here, we use ~151,000 annual records of tree survival and structural completeness to compare AGB loss via damage to living trees to total AGB loss (mortality + damage) in seven tropical forests widely distributed across environmental conditions. We find that 42% (3.62 Mg ha⁻¹ year⁻¹; 95% confidence interval [CI] 2.36–5.25) of total AGB loss (8.72 Mg ha⁻¹ year⁻¹; CI 5.57–12.86) is due to damage to living trees. Total AGB loss was highly variable among forests, but these differences were mainly caused by site variability in damage-related AGB losses rather than by mortality-related AGB losses. We show that conventional forest inventories overestimate stand-level AGB stocks by 4% (1%–17% range across forests) because assume structurally complete trees, underestimate total AGB loss by 29% (6%–57% range across forests) due to overlooked damage-related AGB losses, and overestimate AGB loss via mortality by 22% (7%–80% range across forests) because of the assumption that trees are undamaged before dying. Our results indicate that forest carbon fluxes are higher than previously thought. Damage on living trees is an underappreciated component of the forest carbon cycle that is likely to become even more important as the frequency and severity of forest disturbances increase.     

A dual fluorescence technique for visualization ofStaphylococcus epidermidis biofilm using scanning confocal laser microscopy

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (PVC) disks immobilized in a modified Robbins device using a seed and feed model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained onin situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.     

Body Position,Age and Mass Effect of Adiposity on Adipose Tissue Red Cell Flux in Morbid Obesity

Objective: To measure red cell flux of adipose tissue in morbidly obese patients' pannus in the upright and supine position to determine factors which would render the lower pannus susceptible to ischemic necrosis. Design: A cohort study of morbidly obese subjects without ischemic necrosis. Setting: University teaching hospital. Patients: Twenty-three consecutive morbidly obese patients referred for gastroplasty. Measurements: Red cell flux, measured as RMS voltage by a laser Doppler velocimeter. An optical fiber with a tip diameter of 250μ was inserted into the upper and lower pannus and output recorded in the upright and supine positions. Other variables recorded were age, BMI, blood pressure and serum lipids. Results: Adipose tissue red cell flux demonstrates considerable spatial and temporal heterogeneity from subject to subject and in various locations in the pannus. No differences in red cell flux were detected in response to change in position. However, regression analysis demonstrated that the gradient between the upper and lower abdomen in the supine position was increasingly positive with age and in the upright position it was increasingly positive with increasing weight or BMI. Conclusions: These data suggest that red cell flux is heterogeneously distributed in the abdominal pannus and is not greatly influenced by body position. However, with increasing age and adiposity there is a gradient for decreased red cell flux to the lower portion of the pannus. This may be a factor in rendering this part of the pannus prone to ischemic fat necrosis.     

准分子激光治疗近视眼

本文对准分子激光PRK治疗近视眼息者503例的早期临床结果进行分析,近视范围为－2．0至－15．0D,根据术前屈光度不同分为三组,术后随访三个月．结果证实准分子激光是一种安全、有效、预测性较好的治疗近视眼方法．全部病人提高了裸眼视力,绝大部分保持了最佳矫正视力,且没有产生危及视力的并发症．     

低能量He—Ne激光血管内照射治疗银屑病21例报告

21例寻常型银屑病患者,经用He－Ne激光血管内照射,功率3．5－5mw,每日照射一次,每次1小时,10次一疗程,同时伴用vitc2givq．d及鼻吸氧,二疗程间休息4－7天,经5－35次治疗,近期疗效：近期痊愈5例（23．81％）,显效6例（28．57％）,好转10例（47．62％）,总有效率100％,复发1例（4．76％）。     

Biophoton emission

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moroever, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

氦氖激光对绵羊精清中酶活性效应的研究

本文研究结果表明低剂量的氦氖激光可以提高绵羊精清中GOT和LDH酶的活性,并对其机制作了初步的探讨。