Stray light and noise in confocal microscopy

The stray light and signal levels in a confocal microscope have been measured. These quantities determine the minimum reflectivity which can be observed with the system, as well as characterizing its noise performance and assisting with choice of appropriate pinhole size.     

Evaluation of algorithms for ratio imaging in fluorescence microscopy

Ratio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special-purpose image processors. Since lookup tables store integers in integer addresses, using a lookup table will generate rounding errors. The magnitude of the error will depend on the transformation performed and on the number of levels used in the lookup table. We examined ratio imaging by lookup table and computed the errors generated by both inversion and log subtraction methods. Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.     

Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and F?rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.     

Use of UV excitation in confocal laser scanning fluorescence microscopy

By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections.     

Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy

In the present study a new luminescent dye 3‐N‐(2‐pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3‐aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.     

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA– protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measure ments were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level ~10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.     

Robust pore size analysis of filamentous networks from three-dimensional confocal microscopy

We describe a robust method for determining morphological properties of filamentous biopolymer networks, such as collagen or other connective tissue matrices, from confocal microscopy image stacks. Morphological properties including pore size distributions and percolation thresholds are important for transport processes, e.g., particle diffusion or cell migration through the extracellular matrix. The method is applied to fluorescently labeled fiber networks prepared from rat-tail tendon and calf-skin collagen, at concentrations of 1.2, 1.6, and 2.4 mg/ml. The collagen fibers form an entangled and branched network. The medial axes, or skeletons, representing the collagen fibers are extracted from the image stack by threshold intensity segmentation and distance-ordered homotopic thinning. The size of the fluid pores as defined by the radii of largest spheres that fit into the cavities between the collagen fibers is derived from Euclidean distance maps and maximal covering radius transforms of the fluid phase. The size of the largest sphere that can traverse the fluid phase between the collagen fibers across the entire probe, called the percolation threshold, was computed for both horizontal and vertical directions. We demonstrate that by representing the fibers as the medial axis the derived morphological network properties are both robust against changes of the value of the segmentation threshold intensity and robust to problems associated with the point-spread function of the imaging system. We also provide empirical support for a recent claim that the percolation threshold of a fiber network is close to the fiber diameter for which the Euler index of the networks becomes zero.     

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.     

A dual fluorescence technique for visualization ofStaphylococcus epidermidis biofilm using scanning confocal laser microscopy

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (PVC) disks immobilized in a modified Robbins device using a seed and feed model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained onin situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.     

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.     

Quantitative imaging of intact cells and tissues by multi-dimensional confocal fluorescence microscopy

Confocal fluorescence microscopy enables visualisation and quantitation of fluorescent probes at high resolution deep within intact tissues, with minimal disturbance both of cell–cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix. We illustrate the principles of multiple-parameter 3-D (x,y,z) imaging using reconstruction of nuclear channels in mammalian cells. Repeated sampling in time generates 4-D (x,y,z,t) images which can be used to follow dynamic changes, such as blue-light-dependent chloroplast re-orientation, in intact tissues. Quantitative measurements from multi-dimensional images require calibration of the spatial dimensions of the image and the fluorescence intensity response. This must be determined throughout the volume, which must be sampled to correct for geometric distortion as well as photometric errors arising from the complete optical system, including the specimen. The effects of specimen calibration are illustrated for morphological analysis of stomatal closing responses to abscisic acid in Commelina from 4-D images. Calibrated 4-D imaging allows direct volume measurements and we have followed volume regulation of chondrocytes in cartilage explants during osmotic perturbation. In intact cartilage, unlike in isolated cells, the chondrocytes exhibit volume regulatory mechanisms. In other cases, the fluorescence intensity of the probe may be related to a physiological parameter of interest and changes in its distribution within the cell. Optical sectioning permits discrimination of signal in separate compartments within the cell and can be used to follow transport events between different organelles. We illustrate 3-D (x,y,t) measurements of vacuolar glutathione conjugate pump activity in intact roots of Arabidopsis by following the sequestration of a fluorescent conjugate between glutathione and monochlorobimane. Dynamic measurements of protein localisation are now possible following the introduction of chimeric fusion proteins with green fluorescent protein (GFP) from Aequoria victoria. We have analysed the disposition of heterochromatin in nuclei of living Schizosaccharomyces pombe cells expressing a chimeric construct between Swi6 and GFP. Heterochromatin dynamics can be followed throughout mitosis in 4-D (x,y,z,t) images. Statistical analysis of the fluorescence histograms from each nucleus over time provides quantitative support for aggregation and dispersion of Swi6-GFP clusters during mitosis, rather than dissociation of Swi6 from the heterochromatin. A wide range of single-wavelength and ratio probes are available for imaging different ion activities. We compare 3-D (x,y,t) measurements of ion activities made using single-wavelength (Fluo-3 for calcium) and ratio (BCECF for pH) measurements, using stomatal responses in Vicia faba to peptides from the auxin-binding protein of maize and tip growth in pollen tubes of Lilium longiflorum as examples. Ratioing techniques have many advantages for quantitative fluorescence measurements and we conclude with a discussion of techniques to develop ratioing of single-wavelength probes against alternative references, such as DNA, protein or cell wall material.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.     

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.     

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.     

Signal to noise analysis of multiple color fluorescence imaging microscopy

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.     

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning following by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.     

Comparing telomere length of sister chromatids in human lymphocytes using three-dimensional confocal microscopy

BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.