Fluorescence fingerprints of oral bacteria

The rapid detection and identification of microorganisms is one of the most important factors in many cases of ill health. The purpose of this study was to determine the fluorescence characteristics of seven oral bacteria using emission spectra with the aim of distinguishing between the bacteria, and to compare fluorescence imaging methods for the direct assessment of oral bacteria. Fluorescence images of each bacterium were obtained under a 405‐nm light source using a two‐filter system. The emissions of all samples were measured with a fluorescence spectrometer. The complete fluorescence data set collected for each sample employed a three‐dimensional data cube. The differences in the autofluorescence characteristics of the seven oral bacteria were determined by principal components analysis (PCA). The fluorescence images of the oral bacteria varied with the genus and the filter system. The three‐dimensional excitation‐emission matrix fluorescence spectra exhibited distinctive fluorescence features associated with intracellular fluorophores. The seven bacteria could be clearly differentiated on the PCA score plot. The findings of this study indicate that oral bacteria can be identified based on their autofluorescence characteristics. Fluorescence spectroscopy coupled with PCA can be used to detect and classify oral bacteria.      

Spectral characteristics of caries autofluorescence obtained from different locations and caries severities

Dental caries usually occurs at interproximal and occlusal surfaces. The purpose of the present study was to determine if characteristic spectral factors extracted from autofluorescence (AF) spectra are informative regarding caries detection and the determination of caries stage as compared with DIAGNOdent results. AF spectra were obtained from caries lesions of different severities at two locations using a 405 nm laser. Three spectral factors, that is, spectral slope at 550 to 600 nm, spectral area under the curve at 500 to 590 nm and two‐peak ratio between 625 and 667 nm, were extracted. The values of three spectral factors linearly decreased as caries progressed. According to micro‐CT images, conventional visual and tactile inspections of lesions under or overestimated (25%‐65%) caries states, and brown or thickly stained layer on interproximal or occlusal surfaces, respectively, caused misclassifications of caries stage. Of the spectral factors examined, spectral slope and area under curve for interproximal and occlusal surfaces, respectively, were found to be significantly related to caries stage and showed least data overlap. For interproximal and occlusal surfaces, DIAGNOdent readings of different stages overlapped considerably though their mean values were significantly different regardless of stage.      

人体鼻咽组织的时间分辨自体荧光光谱研究

实验研究了在397 nm半导体脉冲激光激发下,人体离体鼻咽正常和癌变组织在600 nm荧光发射波长处的时间分辨自体荧光光谱特性。利用双指数衰减方程对时间分辨自体荧光光谱进行拟合后,获得相应的荧光强度随时间的指数衰减方程以及荧光平均寿命。人体鼻咽癌变和正常组织在600 nm处的自体荧光平均寿命分别为(2.94±0.51)ns和(4.29±0.71)ns,两者之间存在显著的差异。应用时间分辨光谱技术的诊断灵敏度和特异性分别为75%和100%。初步表明了时间分辨自体荧光光谱在早期鼻咽癌诊断的应用价值,该方法可望与传统的稳态荧光光谱结合起来,进一步提高早期鼻咽癌荧光诊断的准确率。     

Detection of mitochondrial dysfunction in vitro by laser‐induced autofluorescence

Mitochondrion plays a significant role in a variety of biological functions. Because of their diverse character and location in the cellular systems, mitochondria commonly get exposed to various extrinsic and intrinsic cellular stresses. The present study reports a novel approach to detection of mitochondrial dysfunction based on tryptophan autofluorescence of its proteins in mouse liver, using laser‐induced fluorescence (LIF) as a tool. Mitochondria, isolated from the mouse liver, were initially tested for purity and integrity using lactate dehydrogenase and succinate dehydrogenase (SDH) assays. Mitochondrial stress was induced by treating the isolated mitochondria with heavy metals at 10 and 0.01 mM for sodium arsenite and mercuric chloride, respectively. Upon treatment with the heavy metal, tryptophan autofluorescence quenching was recorded at 281 nm excitation. The functional integrity of the mitochondria treated with heavy metals was evaluated by measuring SDH and cytochrome c oxidase activities at various concentrations of mitochondria, which showed impaired activity as compared to control upto a concentration of 6.25 μg. A significant shift was also observed in the autofluorescence of proteins upto the level below 1 μg, suggesting their conformational change and hence altered structural integrity of mitochondria. Circular dichroism spectroscopy data of the mitochondrial proteins treated with heavy metals further validates their conformational change as compared to untreated control. The present study clearly shows that the LIF can be a novel detection tool to detect altered structural integrity of cellular mitochondria upon stress, and it also possesses the potentiality to combine with other interdisciplinary modalities.     

Video‐assisted parathyroid gland mapping with autofocusing

Preservation of the parathyroid gland (PTG) in neck endocrine surgery is important for regulating the amount of calcium in the blood and within the bones. Localization of the PTG has been attempted using various methods such as ultrasound, sestamibi, computerized tomography, magnetic resonance imaging and indocyanine green fluorescence imaging. These methods cannot be used during surgery, have high sensitivity or have PTG specificity. However, autofluorescence technique has shown high sensitivity and does not require exogenous contrast. In this study, a new optical system was designed and developed into a clinical system. The system enabled easier and faster focusing on the surgical area and high‐resolution video imaging while maintaining a clear image. The system was located above the head of the surgeon. The surgeon was able to see the real‐time autofluorescent image on the monitor next to the operating table at any time to locate the PTG. The PTG buried in the adipose tissue and connective tissue was located easily and accurately. The clinical trial conducted in this study consisted of 56 parathyroid cases in 26 patients. For the statistical results, the sensitivity and accuracy in this redesigned autofluorescent imaging system were 98.1% and 96.4%, respectively.      

A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue

Immunoblotting is used to determine many important characteristics of proteins. After electrophoretic separation, proteins are transferred to a membrane and reacted with a specific antibody. The antibody-protein complex is then visualized by radiographic, chromogenic, chemiluminescent, or more recently described fluorescence detection methods. Fluorescence-based detection offers some advantages over other approaches, including increased sensitivity, improved quantifiable range, and the ability to detect multiple antigens on the same blot. However, this technique is unavailable for analysis of green plant tissues by standard extraction methods because of contaminating autofluorescent pigments. We have compared 3 methods for extracting protein from plant tissue for use with infrared fluorescence-based immunoblot analysis. We report a trichloroacetic acid-acetone method that effectively eliminates autofluorescence while retaining the immunogenicity of a target protein.     

A rapid hypersensitive response associated with resistance in the willow Salix viminalis against the gall midge Dasineura marginemtorquens

Great genotypic variation in resistance against the gall midge Dasineura marginemtorquens Bremi (Diptera: Cecidomyiidae) exists within its willow host Salix viminalis L. (Salicaceae). In some resistant genotypes larvae die within 40 h after attempting to initiate galls. The present study tested the hypothesis that the hypersensitive response (HR) is involved in incompatible interactions between D. marginemtorquens and S. viminalis. By means of UV/blue light and visible light microscopy, we verified a rapidly (within 12 h after egg hatch) spreading cell death of an HR-type due to larval attack in resistant willow genotypes. Twelve h after egg hatch, the cell death had spread up to six cell layers in resistant S. viminalis genotypes whereas in susceptible genotypes only up to two cell layers were affected. In the groups of dead cells on the resistant genotypes accumulation of phenolics was observed within 24 h after egg hatch. The rapidity of the cell death induction, the early local accumulation of phenolic compounds, and the strong association of the cell death with larval mortality suggest that the formation of dead cells in resistant willow genotypes should be interpreted as a true HR.     

THE STORAGE GLUCAN OF PHAEOCYSTIS GLOBOSA (PRYMNESIOPHYCEAE) CELLS1

A non-colony-forming axenic strain of Phaeocystis globosa (Harlot) Lagerheim was shown to produce a water-soluble β-d -glucan. This glucan consisted of about 20 glucose units, mainly (l→3)-linked, with branching at position 6. Therefore, it can be classified as a chrysolaminaran. Glucan production occurred mainly during the stationary growth phase and resulted in concentrations as high as 76 pg glucose per cell. When cultures were deprived of light the glucans were consumed, which supports their possible role as compounds used for temporary storage of energy.