Two-stage focus-hold system for rapid ultra-sensitive read-out of large-area biochips

We report here the development of a method for holding the focal plane in a fluorescence-based biochip scanner. The fast read-out of large (multiple cm²) glass slides as used in modern chip technology imposes severe constraints on the focal system. The limited focal depth of high-NA objectives together with the demand for single-molecule sensitivity challenges traditional focus-hold systems. Various long- and short-term effects disturb the often multiple hour-long data-acquisitioning process and cause blurred or unusable image data. Traditional focus-hold systems were often limited in terms of range, reaction time, sensitivity or demanded a large number of additional components. Our system uses the back-reflected illumination beam always present in total internal reflection fluorescence microscopy to generate an error proportional electrical signal, which in turn drives an actuator correcting the objective–sample distance. The latter consists of a fast but range-limited piezo drive attached to the objective and a slower motor coupled to the microscope's z-drive. With this combination, fast reaction times and virtually unlimited correction distances are possible. We show the applicability by scanning DNA microarrays on 27 × 18-mm² glass slides with single-molecule sensitivity over the whole array. Single-fluorescence dyes are imaged as diffraction-limited spots.     

Immobilization and stretching of 5′‐pyrene‐terminated DNA on carbon film deposited on electron microscope grid

The immobilization and stretching of randomly coiled DNA molecules on hydrophobic carbon film is a challenging microscopic technique, which possess various applications, especially for genome sequencing. In this report the pyrenyl nucleus is used as an anchor moiety to acquire higher affinity of double stranded DNA to the graphite surface. DNA and pyrene are joined through a linker composed of four aliphatic methylene groups. For the preparation of pyrene‐terminated DNA a multifunctional phosphoramidite monomer compound was designed. It contains pyrenylbutoxy group as an anchor moiety for π‐stacking attachment to the carbon film, 2‐cyanoethyloxy, and diisopropylamino as coupling groups for conjugation to activated oligonucleotide chain or DNA molecule. This monomer derivative was suitable for incorporation into automated solid‐phase DNA synthesis and was attached to the 5′ terminus of the DNA chain through a phosphodiester linkage. The successful immobilization and stretching of pyrene‐terminated DNA was demonstrated by conventional 100 kV transmission electron microscope. The microscopic analysis confirmed the stretched shape of the negatively charged nucleic acid pieces on the hydrophobic carbon film. Microsc. Res. Tech. 78:994–1000, 2015. © 2015 Wiley Periodicals, Inc.     

Fluorescent porphyrin counterstaining of chromatin DNA in conjunction with immunofluorescence methods using FITC-labelled antibodies

This work describes the use of two cationic porphyrins for fluorescent DNA counterstaining of HeLa cells also stained by means of indirect immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Staining HeLa cells with meso-tetra(4-N-methylpyridyl)porphine (T4MPyP) and meso-tetra(  p -N-trimethylanilinium)porphine (TMAP), both used at 5 × 10⁻⁶  m , gives rise to a deep red emission of chromatin from interphase nuclei and mitotic chromosomes when the cells are excited with blue (490 nm) light. The red-fluorescing chromatin contrasted very well with the yellowish-green emission from FITC-immunofluorescent staining. No significant difference in chromatin fluorescence found with either T4MPyP or TMAP was detected. Counterstaining with the porphyrins could be carried out as a separate step after immunolabelling or, more simply, by their inclusion in the mounting medium. Spectral analysis demonstrated that the fluorescent emission maximum of T4MPyP was at 665 nm and that the intensity of the fluorescent emission showed a considerable increase in the presence of DNA     

In situ fluorescence microscopy of bacteriophage aggregates

Virus aggregation is analyzed because of its potential use for both classifying viruses and understanding virus ecology and evolution. Virus aggregation is, however, problematic because aggregation causes loss of virions during processing for microscopy of any type. Thus, here we detect virus aggregation by fluorescence microscopy of wet-mounted dissections of dilute gel-supported plaques ( in situ fluorescence microscopy) of a test virus, the long-tail aggregating Bacillus thuringiensis bacteriophage, 0305φ8–36. Background fluorescence is reduced to nonproblematic levels by using the dye, DAPI (4',6-diamidino-2-phenylindole), to stain viral nucleic acid. In situ fluorescence microscopy reveals two in situ phases, one weakly fluorescent. Most bacteriophages partition to the weakly fluorescent phase. Aggregates of bacteriophage 0305φ8–36 are detected during fluorescence microscopy via the following. (1) Coordinated motion is found for visibly separate particles in solution; the motion is either thermally generated, fluid drift-induced or mechanical pressure-induced. (2) Aggregate dissociation is observed. Some of the larger aggregates are elastic and entangled with material of the weakly fluorescent phase. The larger aggregates sometimes sink at 1-g within specimens. In situ fluorescence microscopy rapidly distinguishes 0305φ8–36 from nonaggregating bacteriophages.     

Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy

Filamentous bacteriophage has been proposed as a vehicle that can carry and deliver therapeutics into mammalian cells for disease treatment, thus a protocol for imaging phage‐cell interaction is essential. Because high signal intensity is necessary to study the mechanism of interaction between filamentous bacteriophage and mammalian cells, it is important to optimize the procedure for fluorescence labeling of phage in order to understand such interaction. Here, we describe a procedure that gives intense fluorescence labeling and can show interactions between fd‐tet bacteriophage selected from phage libraries and mammalian cells (SKBR‐3 and MCF‐10A). The indirect labeling of phage with dye‐conjugated antibody and cytoskeleton associated proteins was significantly enhanced in the presence of a cross‐linking reagent called dithiobissuccinimidylpropionate (DSP) as shown by qualitative and quantitative fluorescence microscopy. The use of DSP resulted in high signal intensity in fluorescence imaging of phage‐cell complex. The DSP cross‐linker is believed to preserve soluble unbound proteins for fluorescence imaging. The interaction between the phage and mammalian cells was further confirmed by scanning electron microscopy. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Single-particle visualization of assembly: I. Dimerization in a planar zone

Single-particle fluorescence microscopy of association/dissociation is required for analysis of biological assembly reactions. Toward achieving this goal, Wang et al. (J. Microsc., 2004, 213 , 101–109) used molten agarose to concentrate thermally diffusing particles in a thin zone of solution next to the surface of a coverglass (plane of concentration). The present study details the first real-time, single-particle analysis of the association/dissociation of thermally diffusing particles in the plane of concentration. The test particles were procapsids of bacteriophage λ (radius = 31 nm). Quantification of thermal motion was developed and used to determine whether co-diffusing particles were bound to each other. The data are explained by (1) the presence of a molten agarose-generated barrier that is 93–155 nm from the coverglass surface, and (2) non-random orientation of procapsid dimers in the plane of concentration.     

Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone

To bypass limitations of ensemble averaging biochemical analysis, microscopy‐based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488‐stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re‐swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.     

A mixed microscopic method for differentiating seven species of “Bixie”‐related Chinese Materia Medica

Confusion in the species associated with the name of “Bixie” in Chinese Materia Medica began centuries ago. In recent decades, diverse medicinal plants from the genera Dioscorea and Smilax, and even minor species from the genus Heterosmilax, have been documented under the name Bixie or a very similar name as folk medicines in different areas of China. However, the traditional efficacies and chemical profiles of these herbs are not exactly the same and even vary wildly. Comprehensive authentication of multiple Bixie herbs has not yet been attempted. To differentiate and ensure the correct use of these Bixie‐related herbs, in this study, seven sorts of representative Bixie herbs (Dioscorea collettii, D. zingiberensis, D. nipponica, D. septemloba, Smilax china, S. glabra, and Heterosmilax japonica) were characterized based on the microscopic examination of their powders and cryotomed transverse sections. This is not only the first attempt to distinguish Bixie herbs by a comprehensive microscopic techniques, including common light microscopy, fluorescence microscopy, and polarized light microscopy, but also it is the first research to observe characteristics of transections of crude drugs under polarized lighting for the purpose of authentication. Polarized light has been found to provide a number of unique characteristics. The results indicate that starch granules, stone cells, vascular bundles, and other significant tissue features can be used to authenticate “Bixie” herbs. The method was proven to be quick, handy, specific, and simple. It should be widely applicable to other herbal materials. Microsc. Res. Tech. 77:57–70, 2014. © 2013 Wiley Periodicals, Inc.     

A new system for laser-UVA-microirradiation of living cells

Local nuclear irradiation of living cells has been used to gain insight into the dynamic changes that cell nuclei undergo in response to DNA damage. In particular, the effects of DNA double‐strand breaks (DSBs), a major threat to the genomic integrity of cells, have been studied by local nuclear irradiation with ionizing radiation. This method has the disadvantage that it requires expensive equipment to generate a sufficiently high density of focused or collimated ionizing radiation. After appropriate sensitization of the cellular DNA, nuclear microirradiation with UVA can also produce DSBs. In this communication we present a semi‐automatic system for laser‐UVA‐microirradiation based on a commercial laser scanning microscope. The system allows the convenient selection and precise irradiation of living cells, and could provide the basis for a more widespread availability of microirradiation facilities for DNA‐repair research.     

DNA测序电泳荧光信号的小波去噪分析

在DNA荧光测序中,噪声影响分析的准确度和检出限。相比其他滤波方法,小波分析具有良好的时频域分辨特性。在小波去噪处理中,正确选择合适的小波基函数和去噪阈值直接关系到信号去噪处理的质量。通过对毛细管电泳(CE)荧光信号的仿真分析,结果表明:选择sym7小波基函数、分解层数(lev=5)与使用软阈值,可以有效去除CE荧光光谱信号的噪声,提高分析准确度。将其用于处理实际的DNA电泳荧光信号,效果良好。     

The molecular machines of DNA repair: scanning force microscopy analysis of their architecture

The application of scanning force microscope (SFM, also called atomic force microscope or AFM) imaging to study the architecture of proteins and their functional assemblies on DNA has provided new and exciting information on the mechanism of vital cellular processes. Rapid progress in molecular biology has resulted in the identification and isolation of proteins and protein complexes that function in specific DNA transactions. These proteins and protein complexes can now be analysed at the single molecule level, whereby the functional assemblies are often described as nanomachines. Understanding how they work requires understanding their structure and functional arrangement in three dimensions. The SFM is uniquely suited to provide three‐dimensional structural information on biomolecules at nanometre resolution. In this review we focus on recent applications of SFM to reveal detailed information on the architecture and mechanism of action of protein machinery involved in safeguarding genome stability through DNA repair processes.     

Atomic force microscopy study of DNA deposited on poly l-ornithine-coated mica

Analyses of individual biomolecules, like DNA, or DNA–protein complexes, via atomic force microscopy, require ‘gentle’ methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly‐l ‐ornithine‐coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B‐form helical rise (0.34 nm bp^?1). The conformations of DNA that result are reminiscent of three‐dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Cooperativity and intermediate structures of single-stranded DNA binding-assisted RecA-single-stranded DNA complex formation studied by atomic force microscopy

The formation of a complex between RecA protein and single-stranded (ss) DNA was studied systematically by atomic force microscopy (AFM) by varying incubation time and the molecular ratio of RecA protein to single-stranded DNA binding (SSB) protein. New intermediate structures, such as small circular, tangled, and protruded structures in the absence of SSB and sharply turned structures in the presence of SSB, were clearly identified at the early stage of complex formation. These structures have probably resulted from competitive binding of RecA and SSB to DNA. After long incubation, only fully covered RecA-ssDNA and totally RecA-free SSB-ssDNA complexes were present regardless of RecA concentrations. Together with intermediate structures which consisted of only two parts, that is, ssDNA covered by SSB and by RecA proteins, the observation suggested strong neighbor cooperative binding of RecA to ssDNA assisted by SSB.     

Electron microscopic visualization of complementary labeled DNA with platinum‐containing guanine derivative

The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast‐enhanced heavy element. The stretched single‐chain DNA was obtained by modifying double‐stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single‐stranded DNA pieces on the support film with platinum containing guanine derivative to form base‐specific hydrogen bond; and (iii) producing a magnified image of the base‐specific labeled DNA. Stretched single‐stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum‐containing guanine derivative serves as a high‐dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom‐by‐atom analysis and it is promising way toward future DNA‐sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. Microsc. Res. Tech. 79:280–284, 2016. © 2016 Wiley Periodicals, Inc.     

Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores

We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.