Four-laser scanning confocal system for microarray analysis

We have constructed a confocal scanner suitable for routine microarray analysis from commercially available parts. We have outlined the details that should be considered when designing such an instrument and listed some of the specific components comprising the system [the full list of system components is available on CD from the corresponding author (D.J.G.) at no charge]. Here, we describe the methods used to test the linearity and sensitivity of the instrument. Performance was evaluated with two commonly used dyes, fluorescein and Cy5. While the instrument had a linear correlation between the dye concentration and fluorescence intensity, the observed deviation from a slope of 1.0 underscores the importance of running multipoint calibration experiments to obtain accurate dye quantitation over the full dynamic range of the scanner. This method has utility in testing commercial instruments in addition to the scanner described here. An array with over 300 spots dyed with Cy3 was scanned with our instrument and a high-end commercial instrument. The agreement between the two instruments was very good over a 1000-fold intensity range. Our scanner is a cost-effective alternative to more costly commercial scanners with similar capabilities.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of confocal microscopy system performance

BACKGROUND: The confocal laser scanning microscope (CLSM) has been used by scientists to visualize three-dimensional (3D) biological samples. Although this system involves lasers, electronics, optics, and microscopes, there are few published tests that can be used to assess the performance of this equipment. Usually the CLSM is assessed by subjectively evaluating a biological/histological test slide for image quality. Although there is a use for the test slide, there are many other components in the CLSM that need to be assessed. It would be useful if tests existed that produced reference values for machine performance. The aim of this research was to develop quality assurance tests to ensure that the CLSM was stable while delivering reproducible intensity measurements with excellent image quality. METHODS: Our ultimate research objective was to quantify fluorescence using a CLSM. To achieve this goal, it is essential that the CLSM be stable while delivering known parameters of performance. Using Leica TCS-SP1 and TCS-4D systems, a number of tests have been devised to evaluate equipment performance. Tests measuring dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, laser stability, photomultiplier tube (PMT) reliability, and system noise were either incorporated from the literature or derived in our laboratory to measure performance. These tests are also applicable to other manufacturer's systems with minor modifications. RESULTS: A preliminary report from our laboratory has addressed a number of the QA issues necessary to achieve CLSM performance. This report extends our initial work on the evaluation of CLSM system performance. Tests that were described previously have been modified and new tests involved in laser stability and sensitivity are described. The QA tests on the CLSM measured laser power, PMT function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance, and laser stability. Laser power stability varied between 3% and 30% due to various factors, which may include incompatibility of the fiber-optic polarization with laser polarization, thermal instability of the acoustical optical transmission filter (AOTF), and laser noise. The sensitivity of the system was measured using a 10-microm Spherotech bead and the PMTs were assessed with the CV concept (image noise). The maximum sensitivity obtainable on our TCS-SP1 system measured on the 10-microm Spherotech beads was approximately 4% for 488 nm, 2.5% for 568 nm, 20% for 647 nm, and 19% for 365 nm laser light. The values serve as a comparison to test machine sensitivity from the same or different manufacturers. CONCLUSIONS: QA tests are described on the CLSM to assess performance and ensure that reproducing data are obtained. It is suggested strongly that these tests be used in place of a biological/histological sample to evaluate system performance. The tests are more specific and can recognize instrument functionality and problems better than a biological/histological sample. Utilization of this testing approach will eliminate the subjective assessment of the CLSM and may allow the data from different machines to be compared. These tests are essential if one is interested in making intensity measurements on experimental samples as well as obtaining the best signal detection and image resolution from a CLSM. Published 2001 Wiley-Liss, Inc.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis.

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 micrograms/ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 microgram/ml (P less than 0.01) but the staining intensity at 0 microgram/ml was not different from that at 3 micrograms/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo- mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

Background

Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy.

Methods

Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table ​(Table1)1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop.

Table 1

Patient Demographic Data

Results obtained with a sensitive confocal scanning system designed for epifluorescence

A wide variety of specimens has been examined with our apparatus, a commerical version of which is being manufactured by Bio-Rad/Lasersharp. The advantages expected of a confocal system have been realised in practice, the most striking advantage being the exclusion of glare from out-of-focus structures. This has made it possible to image cytological details in unflattened cells and intact tissues that were previously inaccessible to the light microscope.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 μg/ ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 μg/ml (P < 0.01) but the staining intensity at 0 μg/ml was not different from that at 3 μg/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo⁻ mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

Combined molecular ecological and confocal laser scanning microscopic analysis of peat bog methanogen populations

Confocal laser scanning microscopy, using fluorescently labelled oligonucleotide probes targeting the 16S rRNA of different physiological groups of methanogens, was used to identify which methanogenic genera were present and to describe their in situ spatial locations in samples taken at different depths from blanket peat bog cores. Total bacterial DNA was also extracted and purified from the samples and used as template for amplification of 16S rRNA and regions of methyl CoM reductase-encoding genes using the polymerase chain reaction, as well as for oligonucleotide hybridisation experiments. These techniques, used in concert, demonstrated that methanogens of several physiological groups were present in highest numbers in the mid regions of 25 cm deep peat cores. Some discrepancies were apparent in the findings of the microscopic and molecular methods, though these may be partially accounted for by the different sensitivities of the techniques employed. The combined approaches used in this study gave an insight into the diversity and distribution of methanogens in peat environments not possible using molecular ecological methods alone.     

High-performance liquid chromatographic system for separating sugar phosphates and other intermediary metabolites

A simple method for separating intermediates of carbohydrate metabolism, including the difficult-to-resolve sugar phosphates, using anion-exchange high-performance liquid chromatography is described. A gradient of decreasing borate concentration (10 to 0 mM) and increasing ionic strength (0 to 150 mM NH₄Cl) separates phosphorylated sugars based on the strength of the ester complex that they form with borate anion. Lyophillized samples are reconstituted in a low ionic strength aqueous medium (5 mM triethanolamine-HCl, pH 8.1) and chromatographed on a commercially available anion-exchange column (Hamilton PRP-X100). The process requires only 3 h and permits nanomole detection of the phosphorylated intermediates of the glycolytic and pentose shunt pathways.     

High-performance liquid chromatography of unmodified rosin and its applications in contact dermatology

Rosin is a well recognised skin sensitiser and is also amongst the most common causes of occupational asthma. Due to its complex chemical composition, it is difficult to isolate its many components and this has hindered progress in the identification of the specific respiratory and contact allergens it contains. This paper reports the application of high-performance liquid chromatography and other analytical techniques to the isolation and identification of contact allergens in complex mixtures such as rosin. HPLC methods were developed in order to isolate as many rosin components as possible and these were then patch tested on rosin sensitive individuals. The structure of the most dermatologically active component was then determined using mass spectrometry, nuclear magnetic resonance and infrared techniques. An HPLC method has also been developed which will enable the identification of rosin in commercial products, providing a valuable tool for determining the cause of rosin contact allergy. Furthermore, mass spectral data for the common abieitic-type resin acids are compiled which were used to confirm the identification of the HPLC resin acid peaks and have not been reported previously.     

Optical and digital microscopic imaging techniques and applications in pathology

The conventional optical microscope has been the primary tool in assisting pathological examinations. The modern digital pathology combines the power of microscopy, electronic detection, and computerized analysis. It enables cellular-, molecular-, and genetic-imaging at high efficiency and accuracy to facilitate clinical screening and diagnosis. This paper first reviews the fundamental concepts of microscopic imaging and introduces the technical features and associated clinical applications of optical microscopes, electron microscopes, scanning tunnel microscopes, and fluorescence microscopes. The interface of microscopy with digital image acquisition methods is discussed. The recent developments and future perspectives of contemporary microscopic imaging techniques such as three-dimensional and in vivo imaging are analyzed for their clinical potentials.     

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca²⁺ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.     

Software controlling algorithms for the system performance optimization of confocal laser scanning microscope

The relative slow scanning speed of a galvanometer commonly used in a confocal laser scanning microscopy system can dramatically limit the system performance in scanning speed and image quality, if the data collection is simply synchronized with the galvanometric scanning. Several algorithms for the optimization of the galvanometric CLSM system performance are discussed in this work, with various hardware controlling techniques for the image distortion correction such as pixel delay and interlace line switching; increasing signal-to-noise ratio with data binning; or enhancing the imaging speed with region of interest imaging. Moreover, the pixel number can be effectively increased with Acquire-On-Fly scan, which can be used for the imaging of a large field-of-view with a high resolution.     

Light and confocal microscopic observations of adult Schistosoma mansoni from mice fed on a high-fat diet

The morphological aspects of Schistosoma mansoni adult worms recovered from albino mice fed on a cholesterol-rich diet compared to mice fed on a standard chow were investigated. After feeding on their respective diets for over a period of 5 months, mice were subcutaneously infected with c. 50 S. mansoni cercariae/mouse. Blood samples were obtained 1 day prior to experimental infections and 63 days later, when mice were euthanized by jugular section (hypovolaemic shock). Total cholesterol (TC) levels were determined. Recovered worms were stained with hydrochloric carmine, and preserved as whole-mounts for examination by bright-field and laser confocal microscopy. The infected mice fed on high-fat chow showed higher levels of serum lipoproteins than the infected mice fed on standard chow, except for very-low-density lipoprotein cholesterol (VLDL-c) and triglycerides (TG). In this experiment, worms from mice fed on a high-fat chow showed a greater percentage of morphological differentiation regarding supernumerary testes, seminal vesicle, and seminal receptacle. In mice of this group, the rate of oocyte laying in the ovary was much higher than in control females. The present results suggest that cholesterol could be actively involved in the modulation of cell signalling and reproduction, because the lobes contained fully developed oocytes in variable amounts, different from control males. The data presented here are the first to report the role of a cholesterol-rich diet affecting the development of S. mansoni worms.     

Dendritic pattern development of the honeybee antennal lobe neurons: a laser scanning confocal microscopic study

The processing of odorant signals is performed, in the olfactory bulb of vertebrates or in the antennal lobe of insects, by different types of neurons which display specific morphological and functional features. The present work characterizes the morphogenesis of the main neuronal types which participate in olfactory discrimination in the adult honeybee (Apis mellifera). Neurons were stained intracellularly with Lucifer yellow at different stages of pupal development and in the adult, and imaged by laser scanning confocal microscopy. Attending to branching patterns, all pupal neurons could be attributed to morphological types previously established in the adult. Given the functional importance of intraglomerular dendritic arbors in the processing of olfactory information, the study focused on their development. The two main classes, dense and sparse intraglomerular arbors, display adultlike features as early as the second day of pupal development. However, morphometric measurements and confocal observations show that their general pattern undergoes continuous maturation processes until late pupal stages and after emergence of the adult. Among these, the results point out a pruning of dendritic branches in sparse arbors, but not in dense arbors. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 461–474, 1999     

应用激光共聚焦扫描技术对海马脑片神经元内钙的观察

用微量注射法将荧光剂Ｆｌｕｏ－３注入大鼠海马,对神经元进行在体荧光标记,可清晰地标记多个神经细胞。联合应用激光共聚焦扫描显微镜,观察大鼠海马脑片ＣＡ１锥体细胞在青霉素,谷氨酸模拟致痫及缺糖缺氧时胞内钙的变化。结果显示：无镁时,谷氨酸和青霉素可致海马ＣＡ１锥体细胞胞内钙的缓慢增加;离体缺糖缺氧时ＣＡ１锥体细胞胞内钙亦增多。     

Immunofluorescence confocal laser scanning microscopy and immuno-electron microscopic identification of keratins in human materno-foetal interaction zone

We show here that at least 5 keratin proteins are present in villous trophoblast and the same 5 in extravillous trophoblast. A further 14 tested were undetectable in these tissues. In contrast, 10 of the 19 keratins tested were present in amniotic epithelium. The marking of amniotic epithelium on the one hand, as distinct from villous and extravillous trophoblast on the other, can be achieved using 5 keratins (K4, 6, 13, 14 and 17) with a mixture of positive and negative discrimination that is expected, in combination, to be highly sensitive. All the specific keratins identified in trophoblast were apparently up-regulated on the pathway to extravillous trophoblast. Co-ordinated differentiation at the molecular expression level is indicated by this finding. The relevant keratins are K5, 7, 8, 18 and 19. Specific keratins have been identified that are down-regulated in villous trophoblast in pre-eclamptic pregnancy. This difference between healthy and pre-eclamptic chorionic villous trophoblast keratin expression was statistically significant in 4 out of the 5 keratins. This was not the case for the extravillous trophoblast at the immunofluorescence confocal level but significant differences were obtained using immunogold electron microscopy. We suggest that the villous trophoblast in pre-eclamptic placentae is cytoskeletally weaker with respect to the filaments made from these specific proteins and that this is one reason why, in pre-eclampsia, trophoblast is deported in greater quantity than in healthy placentae.