Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov—Smirnov test. The difference between the two patterns was at a P < 0?01 significance level.     

基于PCA和SVM算法的肝癌细胞显微后向散射光谱分类

为了实现对肝癌的早期实时和在体探测,基于前期搭建的光纤共聚焦后向散射(FCBS)光谱仪获取肝癌细胞的显微后向散射光谱,分别使用主成分分析(PCA)和支持向量机(SVM)两种算法,对获得的正常肝细胞株(L02)、低转移潜能肝癌细胞株(MHCC97-L)和高转移潜能肝癌细胞株(HCCLM3)三种细胞的后向散射光谱进行分类。使用PCA对获得的三种细胞光谱数据进行降维分析,得到的前两个主成分综合了全部信息的95.4%,由主成分1和主成分2的得分图可以观察到,三种细胞在直观上有明显的区分;对同一数据集选取69例对象通过SVM机器学习算法训练分类模型,随机抽取50例作为训练集,19例作为预测集,最终分类的准确度达到了94.7%。实验结果表明:使用光纤共聚焦后向散射(FCBS)光谱仪获取的细胞显微后向散射光谱可以分别通过PCA和SVM对不同转移潜能的肝癌细胞进行自动分类,这将为研究活检提供必要的检测手段。     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Monitoring of intracellular localization of Hepatitis B virus P22 protein using Laser Scanning Confocal Microscopy and Airyscan

The aim of this study was to assess nucleo‐cytoplasmic protein localization to better understand the exact intracellular localization of viral proteins involved with infections. Having determined the general protein localization of hepatitis B virus P22 precore protein, the aim was to more specifically resolve its intracellular organization. This was done using both laser scanning microscopy and Airyscan techniques. Using a 63× objective, the resolution obtained with Airyscan was increased by 1.5‐fold as compared to confocal microscopy (p value <.00001).     

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-μm vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.     

Three-dimensional optical microscopy using tilted views

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x–y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the ‘missing data’ and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0?25 μm in x and y, and 0?4 μm in z.     

Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli

Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC. The method is applied to two sets of 204 and 177 images that show the 70S ribosome of Escherichia coli, in the range of overlap views as defined by A. Verschoor and co-workers, and to two sets of 480 and 496 images of the 50S subunit of E. coli depleted of L7/L12 proteins in the well-defined crown view. Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates. These classes appear to be related to different orientations on the specimen grid (in the case of the 70S particle) and to different conformational states (50S subunit).     

Fuzzy sets-based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles

Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens. The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data. A number of partitional clustering algorithms and an extensive set of cluster-validity functionals (some already reported and some newly developed) have been applied to a test-data set and to two real-data sets of images. One of the real-data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E. coli 70S monosome in the range of overlap views. These two latter sets had been previously studied by another clustering methodology. The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.     

Confocal microscopy of the in-situ crystalline lens

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.     

Time-resolved confocal analysis of antibody penetration into living,solid tumor spheroids

The in vivo function of a biologically active molecule is governed in part by the dynamics of its distribution within its target tissue. To enhance our ability to probe living cells, we have endeavored to improve live confocal microscopy methods and to develop analytical methods that simplify the handling of the resulting complex data sets. To do this we attached a recently developed micro-incubation system to the stage of a Leica confocal laser scanning microscope and were able to maintain physiologic culture conditions over several hours. Axial stability was achieved by modifying the room air conditioning. Laser illumination was low enough to retain cell viability through several hours of continuous scanning. With this setup, planar, time-resolved data sets (xyt) were produced by continuously rescanning a single xy plane at the rate of one scan/min. As an alternative, volumetric data sets (xyz) were acquired by stepping the scanned plane through the z axis. In both types of data sets, a semi-quantitative determination of the concentration of a fluorescent reporter molecule (e.g., FITC) over a gray level range of 0--255 was recorded along with the positional information. Thus, concentration (as intensity of fluorescence, or i) gave a fourth variable by either scan method, resulting in high-density xyti or xyzi data sets. The biological model we used to examine these methods was the penetration of a FITC-labeled, anti-carcinoma monoclonal antibody into cultured spheroids of tumor cells bearing the antibody-binding epitope. In one case, the distribution of antibody-FITC conjugate was compared with that of a long wavelength membrane dye. DiIC₁₈(5). Several different software analyses were compared, including examining xyt data sets as “volumes.” We observed that by increasing the displayed resolution of one variable, the demonstrable resolution of the other variables was reduced. For example, with high temporal resolution, either quantitative or positional resolution had to be sacrificed. Thus, we needed to perform several different analyses of a single data set to compare all of the variables properly. In these experiments, the dynamic aspects of the changes in antibody-FITC distribution were examined. Along with comparison of antibody-FITC penetration with that of DiI, these data suggest an as yet unexplained biological transport of antibody into a tumor spheroid, which is not consistent with mere passive diffusion through the fluid of extracellular clefts. Using this model system, we have performed and analyzed highly time-resolved confocal microscopy on living specimens maintained under physiologic conditions.     

A tilting device for three-dimensional microscopy: Application to in situ imaging of interphase cell nuclei

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x-y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three-dimensional (3-D) organization of chromosomes in human interphase cell nuclei, the 3-D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3-D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer-controlled stepping motor for movement in the z-axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x-y direction. The technique was applied to 3-D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3-D data sets were registered at viewing angles of 0°, 90° and 180°; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2p geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.     

Spectral characterization of Dictyostelium autofluorescence

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from approximately 500 to 650 nm with a maximum at approximately 510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, pre-bleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium.     

Confocal imaging and second-order stereological analysis of a liquid foam

Foam structures are found in diverse fields of study; the structure of fire-fighting foam, upholstery foams and even the head on a pint of beer all share detailed similarities in their microstructure and dynamics. Despite impressive developments in the theory of two-dimensional foams the challenge in future will be to analyse and model the dynamics of three-dimensional foams. However, the myriad of gas/liquid interfaces in an aqueous foam make direct imaging of their structure difficult. In this study we circumvent this problem by using fluorescence confocal microscopy to acquire three-dimensional images of the structure of a coarsening aqueous foam. A stable aqueous foam was created by mixing commercial shaving foam with ethanol and a small amount of fluorescein solution. The foam was imaged in fluorescence mode such that the liquid fraction of the foam, containing the fluorescent dye, could be seen in optical sections. These images could be acquired in three-dimensional stacks of optical sections up to a depth of about 200 μm. Single images were also acquired as a time series. The time series of single optical section images clearly show the dynamics of the foam. Early images show a structure made mainly of spherical bubbles; later images show the polyhedral structure of the foam which coarsens as a function of time. The polyhedral nature of the foams is shown particularly clearly in stereo pair images of the three-dimensional image sets. The three-dimensional images of the foams were also analysed using second-order stereology (statistical summaries of spatial distribution). The x, y and z coordinates of the foam vertices were extracted from the images and used to compute the nearest neighbour (G-function) and reduced second moment (K-function) statistics. These statistics allow quantification of the range of length scales found in the foams. These results form part of an ongoing study of the coarsening of aqueous foams.     

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy‐to‐apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17‐day‐old laboratory Wistar rat embryo. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome,Picrosirius red and confocal microscopy techniques

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin‐Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image‐Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E‐confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland‐Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E‐confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E‐confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.     

Analysis of computational EELS modelling results for MgO-based systems

The ab-initio density functional theory (DFT) code CASTEP was used to model oxygen K edges in various magnesium oxide systems. Firstly, for the bulk material the process of geometry optimisation was carried out. Predicted oxygen K edges were found for a single cell with experimental lattice parameters, and parameters obtained after geometry optimisation, both with single electron core-holes in place. After geometry optimisation, a different predicted result was obtained, although it was qualitatively similar to the result for experimental lattice parameters in some respects. For example, approximately the same sets of peaks are observed, though in different energy positions, and with different relative peak intensities within those sets. Ultimately for the single cell results the experimental lattice parameters generated the predicted result that was in the closest agreement with experiment. It was further observed that a large supercell result (based on the experimental lattice parameters, utilising a core-hole) led to a slightly improved comparison with experiment as compared to the corresponding single cell result, although the latter result, and indeed a ground state calculation also give reasonable agreement with experiment. To rationalise these observations it was necessary to investigate the density of states (DOS) for the MgO cell and its constituent atoms, and it was observed that the conduction bands were of predominantly magnesium character. Furthermore, the core-hole’s introduction had relatively little overall effect on the p DOS prediction for oxygen, though there is a significant localised change close to the Fermi level. This work also considers interface and surface results. The principal aim of the study was to explore the interface of Fe (0 0 1)/MgO (0 0 1), crucial in certain classes of magnetic tunnel junctions (MTJs), which have significant technological applications. An initial step was to consider a MgO (0 0 1) surface. It was verified that a surface could be constructed such that within that surface a theoretical result could be found that matched the bulk result. It was then valid to use this surface as part of an interface with iron. Theoretical results obtained at that interface compare well with experimental results from an epitaxially grown MTJ, and various conclusions are drawn with regard to the nature of the interface.     

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.     

Entropy of corneal nerve fibers distribution observed by laser scanning confocal microscopy: A noninvasive quantitative method to characterize the corneal innervation in Sjogren's syndrome patients

Sjogren's syndrome (SS) is a progressive autoimmune condition mainly affecting the salivary and lacrimal glands with an incidence of primary SS between 1/100 and 1/1,000. SS implies an alteration in the epithelium and subepithelium innervation, with consequent reduction of corneal sensitivity. It is necessary to have noninvasive quantitative methods to characterize the status of the corneal nerve fibers of the patients in order to choose and follow the best therapy. Entropy (information dimension) of the nerve corneal fibers distribution observed by confocal microscopy was evaluated in patients with primary SS (n = 30, 6 males, 24 females, 21–81 years), diagnosed by biopsy of salivary gland and blood tests and in sex‐ age‐matched healthy subjects (n = 12). Corneal nerve fiber density, Langerhans cell count, and cell density in the nerve plexus images were also evaluated. In selected patients salivary gland atrophy degree was also evaluated. Nerve corneal distribution observed by confocal microscopy is fractal. Entropy of the corneal nerve distribution statistically distinguishes between SS patients and healthy subjects: patients present a lower value of information dimension of the corneal nerve fibers distribution than healthy individuals (P < 0.001). Percentage of grouped cases classified by entropy according to the subjects (selected patients vs. healthy) showed a 100% sensitivity and 96% specificity, P < 0.0001 with a low value of coefficient of variation among the individuals (6–7 times lower than the other morphometric indexes). Entropy correlated with the severity of the disease (salivary gland atrophy degree, P < 0.01). Evaluation of entropy of the corneal nerve distribution observed by a laser confocal microscopy appears to quantitatively and noninvasively characterize an aspect of the SS patients in relation to the recognition of an impairment of their ocular surface, giving us for the first time a method to objectively and precisely characterize the corneal innervation status in the SS patients. Microsc. Res. Tech. 78:1069–1074, 2015. © 2015 Wiley Periodicals, Inc.     

The factors affecting surface roughness measurements of the machined flat and spherical surface structures – The geometry and the precision of the surface

The quantitative determination of surface roughness is of vital importance in the field of precision engineering. This paper presents an experimental study of the roughness analyses for the flat and spherical surfaces of machined metal in order to compare the roughness data taken from the cloud data produced by the stylus type profilometer and two optical-based measurement instruments, namely the infinite focus microscope and the confocal laser scanning microscope.In this experimental study, the roughness measurements for fifteen flat and six spherical surfaces were repeated six times using three different measurement instruments. Great care was paid to measure the same location for each measurement. For the comparison of the measurement techniques, the same measurement process was applied to the flat and spherical surfaces individually, and the configurations of the measurement instruments (filter type, cut-off, resolution etc.) were synchronized. R_a, two-dimensional (2D) roughness parameter and S_a, three-dimensional (3D) roughness parameter were also compared. The measurement results for the samples having spherical surfaces indicated a considerably high difference in values taken from the stylus profilometer and two optical-based measurement instruments in contrast to those for flat surfaces.     

Response of E. faecalis biofilms to different associations of auxiliary substances during root canal preparation: A confocal laser microscopy analysis

Objective: This in vitro study evaluated the effect of different endodontic auxiliary chemical substances over Enterococcus faecalis (Ef) biofilm through confocal laser scanning microscopy (CLSM). Methods: Forty‐five bovine incisors were infected with Ef for 21 days. Teeth were divided into five groups: group 1: 2.5% NaOCl + EDTA, group 2: 2% CHX gel + EDTA, group 3: 2% CHX liquid + EDTA, group 4: 2.5% NaOCl + 2% CHX gel + EDTA, group 5: 2.5% NaOCl + 2% CHX liquid + EDTA and a negative and a positive control group (NCG; PCG). The samples were stained with SYTO9 and propidium iodide and analyzed by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Scores were standardized according to the total bacterial load (TBL)—1: ≤25%, 2: >25 ≤50%, 3: >50 ≤75%, 4: >75% and debris—1: absence of debris; 2: presence of debris. Statistical analysis was carried out through the Kruskal–Wallis and the Fischer exact tests (P = 0.05). Results: No statistical differences were observed to CFU, debris and bacterial viability. Conclusion: None of the tested substances could completely eliminate Ef from the root canal space. Microsc. Res. Tech. 76:658–662, 2013. © 2013 Wiley Periodicals, Inc.