Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics.

BACKGROUND:The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. METHODS:Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. RESULTS:In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 cells prove the method to be as generally applicable as the classical BrdUrd/Hoechst quenching technique, but without need for expensive ultraviolet laser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-3. CONCLUSIONS:Cell cycle studies of mammalian cells can be done by dual-laser flow cytometry without the need for ultraviolet lasers by using the BrdUrd-dependent fluorescence enhancement of TO-PRO-3. Total DNA content can be measured simultaneously using PI.     

Evaluation of c-erbB-2 overexpression and Her-2/neu gene copy number heterogeneity in Barrett's adenocarcinoma.

Amplification of the Her-2/neu gene is accompanied by overexpression of its cell surface receptor product, c-erbB-2 protein. To investigate the degree of intratumoural heterogeneity we applied immunohistochemistry in primary Barrett's adenocarcinoma (BCA) (n = 6) and dysplasia adjacent to the carcinoma (n = 4). In addition, fluorescence in situ hybridisation (FISH) was performed in primary BCA (n = 5) and dysplastic areas (n = 4). For an objective evaluation digital image analysis and laser scanning microscopy were used. Five of six BCA showed a marked intratumoral heterogeneous staining pattern ranging from areas in which the tumour cells were negative or faintly positive to tumour areas with a strong staining of the entire membrane. Among the two dysplastic areas also a heterogeneous staining pattern was observed. FISH analysis revealed marked heterogeneity of intratumoral gene copy number changes in all BCA showing populations with different fractions of cells with polysomy, low level amplification and high level amplification. One dysplasia showed a minor population with Her-2/neu signal clusters. In conclusion, we observed marked intratumoural heterogeneity of c-erbB-2 protein overexpression and Her-2/neu gene copy number in the majority of the primary BCA analyzed. Digital image analysis and laser scanning microscopy were helpful in quantifying the variations in protein expression and DNA copy number in individual tumour cells. The observed heterogeneity could hamper the exact diagnostic determination of the c-erbB-2 status in small biopsies and possibly influence the effectiveness of a potential c-erbB-2 targeting therapy. Figures on http://www.esacp.org/acp/2000/20-1/walch.htm+ ++.     

A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy.

The aim of this study was to develop a procedure to remove the TO-PRO-3 fluorescent dye from tissue sections and restain with TO-PRO-3, still allowing calculation of DNA content and distribution by confocal laser scanning microscopy (CLSM). This would allow repeated measurements on the same tissue sections and prevents loss of tissue material from valuable clinical samples. Thick sections (14 microm) were cut from a paraffin block of adrenal tissue and stained using TO-PRO-3. Image stacks were acquired by CLSM. Thereafter, three destaining approaches were tested based on incubation, at different temperatures and durations, in the medium that is normally used to dissolve TO-PRO-3. The same areas were imaged again to measure residual fluorescence and were subsequently restained and imaged again. The intensity of the images acquired after initial staining and restaining were compared. A number of 3-D (texture) features computed after segmentation of nuclei were compared as well. The best destaining result was obtained by incubation of sections at 37 degrees C in preheated medium twice for 20 min. On average, the 3-D feature values were comparable with those after initial staining. With the described protocol it is possible to remove TO-PRO-3 fluorescence from tissue sections that can successfully be restained with minimal influence on fluorescence intensity and nuclear chromatin distribution.     

用YO-PRO-1和PI联合染色定量检测细胞凋亡

经不同浓度staurosporine处理诱导凋亡的G7细胞样品,分别用YO-PRO-1/PI和AV/PI进行荧光染色,借助流式细胞仪检测凋亡情况,将两种检测方法得到的结果进行统计学分析显示,二者有显著的相关性(r=0.9659,P<0.01),且没有显著性差异(P<0.05);另外,上述凋亡细胞样品经YO-PRO-1/PI染色后在荧光显微镜下计数凋亡细胞比例的结果与AV/PI流式细胞仪的检测结果也有显著的相关性(r=0.9903,P<0.01),且没有显著性差异(P<0.05)。以上这些结果表明,用YO-PRO-1/PI对细胞进行染色、借助流式细胞仪和荧光显微镜均能准确地检测细胞凋亡,可替代AV/PI流式细胞仪方法用于细胞凋亡的检测。     

Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy

We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes.

Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.     

Predicting the efficacy of trastuzumab-based therapy in breast cancer: current standards and future strategies

Breast cancer is the most common female malignancy in many industrialized countries. Approximately one fourth of all women diagnosed with early breast cancer present with tumors that are characterized by erbB2 amplification. While the associated Her-2/neu receptor overexpression results in a high risk of relapse and poor prognosis, these tumors also represent a target for a selective monoclonal antibody therapy with trastuzumab (Herceptin). The combination of trastuzumab with chemotherapy has led to a considerable reduction of recurrences and to a significant reduction in breast cancer mortality both in the adjuvant and metastatic setting. Unfortunately, despite Her-2/neu overexpression, not all patients equally benefit from trastuzumab treatment, and almost all women with metastatic breast cancer eventually progress during antibody therapy. Moreover, trastuzumab is burdened with cardiotoxicity, thus increasing the risk of symptomatic congestive heart failure. In addition, the marginal costs for a 1 year therapy of trastuzumab-based therapy, which is currently considered to be the most effective treatment regimen in the adjuvant setting, may amount for up to US$ 40.000. Testing for erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH), respectively, and staining for Her-2/neu receptor overexpression by immunohistochemistry (IHC) represent the current standard for determining patient eligibility for trastuzumab-based therapy. However, while the negative predictive value of these assays for predicting the absence of benefit from trastuzumab-based therapy is sufficiently high, their positive predictive value remains insufficient, i.e. only a proportion of patients selected by these tests substantially benefit from trastuzumab-containing regimen. Accordingly, over the last years a number of biomarkers have been evaluated in their potential to predict response to trastuzumab-based therapies. These include markers auf activation of Her-2/neu (e.g., tyrosine phosphorylated Her-2/neu in tissue and cleaved Her-2/neu extracellular domain in serum) and its dimerization partners (e.g., EGFR), respectively, but also components of Her-2/neu-induced downstream signaling pathways that are crucial for the growth inhibitory effects of trastuzumab (e.g., PTEN and PI3K). Other parameters, such as topoisomerase-II alpha and c-myc co-amplifications, have also been identified as potentially useful predictors of response to trastuzumab-based chemotherapy regimen. While the benefit of these predictive biomarkers in the metastatic setting is currently explored, their usefulness in the adjuvant setting is still largely unknown. It is, however, undisputable that, within the group of Her-2/neu overexpressing tumors, further response predictors are needed in order to minimize trastuzumab-associated side effects, and to reduce the considerable societal costs that are associated with trastuzumab-based treatment regimen.     

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.     

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Quercetin-induced ubiquitination and down-regulation of Her-2/neu

Her-2/neu (ErbB2) is a transmembrane tyrosine kinase and acts as a co-receptor for the other EGFR family members. It is well known that high expression of Her-2/neu is associated with a poor prognosis in breast cancer. Quercetin, a flavonoid present in many vegetables and fruits, has been studied extensively as a chemoprevention agent in several cancer models. In this study, we observed that quercetin decreased the level of Her-2/neu protein in time- and dose-dependent manners and also inhibited the downstream survival PI3K-Akt signaling pathway in Her-2/neu-overexpressing breast cancer SK-Br3 cells. We also observed that quercetin induced polyubiquitination of Her-2/neu. When the proteasome pathway was blocked by MG-132 during quercetin treatment, accumulation of the NP-40 insoluble form of Her-2/neu occurred. Interestingly, data from immunocomplex studies revealed that quercetin promoted interaction between Her-2/neu and Hsp90 which is a molecular chaperone involved in stabilization of Her-2/neu. In this condition, inhibition of Hsp90 activity by a specific inhibitor, geldanamycin (GA), or intracellular ATP depletion caused dissociation of Hsp90 from Her-2/neu and promoted ubiquitination and down-regulation of Her-2/neu protein. In addition, the carboxyl terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, played a crucial role in the quercetin-induced ubiquitination of Her-2/neu. Inhibition of tyrosine kinase activity of Her-2/neu by quercetin could indicate an lateration in the Her-2/neu structure which promotes CHIP recruitments and down-regulation of Her-2/neu. We believe that by using quercetin, new therapeutic strategies can be developed to treat Her-2/neu overexpressing cancers.     

Three-dimensional reconstruction by confocal laser scanning microscopy in routine pathologic specimens of benign and malignant lesions of the human breast

 Confocal laser scanning microscopy (CLSM) has become an exciting new instrument because of its increased resolution over conventional wide-field microscopy and its high performance three-dimensional (3D) optical sectioning. Although CLSM has been used extensively in cell biology, few applications have been reported in routine clinical pathology. In this study, 3D reconstruction was performed on routine formalin-fixed, paraffin-embedded tissues of normal mammary duct, simple ductal hyperplasia, intraductal papillary hyperplasia, ductal carcinoma in situ, invasive carcinoma, and lymph node metastatic carcinomas of the human breast by using computer-assisted CLSM in conjunction with a 3D reconstruction software package (microVoxel). The selected specimens were sectioned at 30 μm, mounted on glass slides, and stained with the DNA fluorescent probe, YOYO-1 iodide. The nuclear DNA and chromatin texture were clearly demonstrated after pretreatment with RNAase and hydrolysis with 2 N HCl. High quality 3D images were obtained by processing the optical section stacks with volume render and surface display parameters in microVoxel. 3D morphologic characteristics of different breast lesions were examined in various orientations by angular image rotation. The clearly benign lesions (simple ductal hyperplasia and intraductal papillary hyperplasia) revealed similar 3D morphologic features, including: (1) smooth nuclear surface and homogeneous chromatin fluorescence intensity; (2) hyperplastic cell nuclei showing similar shape and volume; and (3) clear-cut margin of basement membrane defined by spindle-shaped myocytes of the ductal outer layer. In contrast, carcinomas displayed remarkably different features in 3D morphology, including: (1) irregular nuclear surface; (2) marked nuclear pleomorphism (irregular, angulated and indented shape of nuclear volume); (3) irregular and coarse chromatin texture; (4) chaotic arrangement of tumor cell nuclei; and (5) absence of myocytes, indicating no clear margin at the site of infiltration of cancer cells. In conclusion, nuclear structure, specifically demonstrated by CLSM of YOYO-1 iodide fluorescently stained cells, used in tandem with 3D volume morphologic reconstruction, may provide a useful research diagnostic tool in pathology. Accepted: 7 November 1996     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

Routine assessment of hormonal receptor and her-2/neu status underscores the need for more therapeutic targets in Kenyan women with breast cancer

OBJECTIVE: To report the expression of estrogen receptors, progesterone receptors and human epidermal growth factor receptor (Her-2/neu) in 158 Kenyan women with breast cancer and correlation with other prognostic indicators in this high-risk group. This study stressed the importance of routine assessment of the steroid receptors and Her-2/neu as a mode of therapeutic selection of patients for antihormonal or targeting monoclonal antibody (Herceptin) therapy, directed at the juxtamembrane domain of Her-2/neu protein in the developing countries such as Kenya. STUDY DESIGN: The study population consisted of 158 female patients with histologically confirmed breast carcinoma seen at the pathology department of The Nairobi Hospital. An immunohistochemical (IHC) study of ER, PR and Her-2/neu was conducted, followed by fluorescent in situ hybridization (FISH) validation for Her-2/neu gene amplification in cases initially scored as positive 2+ with IHC. Mastectomy samples registered at the pathology department of The Nairobi Hospital were used for this study. The study was approved by the institution's ethical review committee and informed consent obtainedfrom the concerned patients. RESULTS: In the studied cohort, positivity for both hormonal receptors and Her-2/neu was noted in 10 (6.33%) cases and negativity in 44 (27.85%) cases. Conversely, Her-2/neu negativity was noted in 32 (20.25%) cases with both steroid receptors positive and Her-2/neu positivity with both steroid receptors negative in 20 (12.66%) cases. Overall, no predictive factor was found in the Her-2/neu amplified 31/153 (20.26%) cases completely assessed with IHC and FISH. Grade III invasive ductal carcinomas, however, had a high prevalence of Her-2/neu overexpression. Association of both menopausal status (p = 0.044) and progesterone receptor status (p = 0.004) with high grade tumors were found to be statistically significant at 95% CI (p < 0.5). Consistent with other studies, Her-2/neu overexpression in this cohort was 20.26%. CONCLUSION: Her-2/neu positivity may activate ER expression through signaling kinases, and the combined target of mitogenic estrogen plus the monoclonal antibody therapy against Her-2/neu-overexpressing tumors expand chances of survival for patients in developing countries such as Kenya. The cost factor for these tests, selection for appropriate combined therapies and lack of awareness were noted as limiting factors for access to basic health care service and resulted in advanced tumor grade at time of patient presentation.     

Evaluation of a Continuous Quantification Method of Apoptosis and Necrosis in Tissue Cultures

In tissue-engineering and other life sciences, there is a growing need for real-time, non-destructive information on apoptosis and necrosis in 2D and 3D tissue cultures. Previously, propidium iodide was applied as a fluorescent marker for monitoring necrosis. In the current study this technique was extended with a fluorescent apoptosis marker, YO-PRO-1, to discriminate between both stages of cell death. The main goal was to evaluate the performance of YO-PRO-1 and propidium iodide during monitoring periods of up to 3 days. Apoptosis was induced in C2C12 cultures and the numbers of YP-positive and PI-positive nuclei were counted in time. The performance of the dual staining was evaluated with a metabolic measure and a probe intensity study. Cell metabolism was unaffected during the first 24 h of testing. In conclusion, the YP/PI dual staining method was found to be a powerful tool in obtaining real-time spatial information on viability in cell and tissue culture without culture disruption.     

The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial cells in soil samples without interference from background fluorescence

Visualization of microorganisms in soils and sediments using fluorescent dyes is a common method in microbial ecology studies, but is often hampered by strong nonspecific background fluorescence that can mask genuine cellular signals. The cyanine nucleic acid binding dyes TO-PRO-3 and TOTO-3 iodide enabled a clear detection of microbial cells in a mineral soil, while nonspecific background was greatly reduced compared with commonly used dyes. When used as counterstains for fluorescence in situ hybridization (FISH), both cyanine dyes allowed identification of microbial cells despite strong background from nonspecifically bound probes. TO-PRO-3 and TOTO-3 are easy to use and represent superior alternatives for detecting microorganisms in soil environments.     

Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy

Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.     

Quality assessment of confocal microscopy slide based systems: performance.

BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.     

Application of YO-PRO-1 as an Epifluorescent Dye for in situ Detection of Small Amount DNA in Plant Cells

i.e. plastid and mitochondrial DNA in the plant cells such as the sperm cell of Jasminum nudiflorum, the generative cell of Pharbitis lim-bata, the cultured cell of Nicotiana tabacum and the root cell of Vicia faba with epifluorescence microscopy and laser confocal microscopy using YO-PRO-1 as a fluorescent dye. The excitation for YO-PRO-1 was blue light in epifluorescence microscopy and 488 nm Kr/Ar ion laser in confocal microscopy. Dimorphic epifluorescent spots that corresponded plastid DNA and mitochondrial DNA were distinctly detected in the cells of each species examined. In this report, we introduce YO-PRO-1 as a new epifluorescent dye for successful in situ detection of small amount DNA in plant live cells and cell sections with perticular emphasis on the importance of sample preparation. Received 10 November 1998/ Accepted in revised form 13 January 1999