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Showing papers in "Journal of Microscopy in 2000"


Journal ArticleDOI
TL;DR: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide‐field fluorescence microscope with strikingly increased clarity compared to both conventional and confocal microscopes.
Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

3,274 citations


Journal ArticleDOI
TL;DR: Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin.
Abstract: Summary Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm 22 to GW cm 22 , which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depthresolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a onephoton or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm 22 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible.

1,243 citations


Journal ArticleDOI

432 citations


Journal ArticleDOI
TL;DR: A speculative model of the vesicle trafficking network within growing hyphae is presented, consistent with dye internalization by endocytosis.
Abstract: Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

397 citations


Journal ArticleDOI
TL;DR: The use of the technique is illustrated in examination of a layered polymer film, a micrometeorite section, and an interplanetary dust particle section.
Abstract: Summary A method is described whereby a sequence of X-ray images at closely spaced photon energies is acquired using a scanning transmission X-ray microscope, and aligned. Near-edge absorption spectra can then be obtained both from large, irregular regions, and from regions as small as the spatial resolution of the microscope (about 40 nm in the examples shown here). The use of the technique is illustrated in examination of a layered polymer film, a micrometeorite section, and an interplanetary dust particle section.

364 citations


Journal ArticleDOI
TL;DR: Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip‐growing cells reveal the general principles that govern the relationship between the cytOSkeleton and cell growth.
Abstract: Tip-growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip-growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip-growing cells is among the fastest in biological systems. Tip-growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip-growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip-growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.

208 citations


Journal ArticleDOI
TL;DR: Axial scanned images of fluorescently labelled polystyrene beads using an oil immersion lens show restored sectioning ability at a depth of 28 µm in an aqueous specimen.
Abstract: We demonstrate aberration correction in two-photon microscopy. Specimen-induced aberrations were measured with a modal wavefront sensor, implemented using a ferro-electric liquid crystal spatial light modulator (FLCSLM). Wavefront correction was performed using the same FLCSLM. Axial scanned (xz) images of fluorescently labelled polystyrene beads using an oil immersion lens show restored sectioning ability at a depth of 28 mm in an aqueous specimen.

176 citations


Journal ArticleDOI
TL;DR: The advantages of the image correlation approach for measurement of correlation functions with high signal‐to‐noise in relatively short time periods are demonstrated and situations when these methods represent improvements over non‐imaging fluorescence correlation spectroscopy are discussed.
Abstract: We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments Using two-photon ICS, we measured diffusion coefficients as large as 10(-8) cm2 s(-1) that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions We also show the sensitivity of the method for measuring microscopic flow using analogous test samples We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells

165 citations


Journal ArticleDOI
TL;DR: This work demonstrates sub‐100 nm resolution water‐window soft X‐ray full‐field transmission microscopy with a compact system based on a 100 Hz table‐top regenerative debris‐free droplet‐target laser‐plasmaX‐ray source in combination with normal‐incidence multilayer condenser optics for sample illumination.
Abstract: We demonstrate sub-100 nm resolution water-window soft X-ray full-field transmission microscopy with a compact system. The microscope operates at lambda = 3.37 nm and is based on a 100 Hz table-top regenerative debris-free droplet-target laser-plasma X-ray source in combination with normal-incidence multilayer condenser optics for sample illumination. High-spatial-resolution imaging is performed with a 7.3% efficiency nickel zone plate and a 1024 x 1024 pixel CCD detector. Images of dry test samples are recorded with exposure times of a few minutes and show features smaller than 60 nm.

148 citations


Journal ArticleDOI
TL;DR: A novel method for retrospective shading correction is proposed that is efficient for a variety of differently structured images and as such may have applications in and beyond the field of microscopical imaging.
Abstract: Shading is a prominent phenomenon in microscopy, manifesting itself via spurious intensity variations not present in the original scene. The elimination of shading effects is frequently necessary for subsequent image processing tasks, especially if quantitative analysis is the final goal. While most of the shading effects may be minimized by setting up the image acquisition conditions carefully and capturing additional calibration images, object-dependent shading calls for retrospective correction. In this paper a novel method for retrospective shading correction is proposed. Firstly, the image formation process and the corresponding shading effects are described by a linear image formation model, which consists of an additive and a multiplicative parametric component. Secondly, shading correction is performed by the inverse of the image formation model, whose shading components are estimated retrospectively by minimizing the entropy of the acquired images. A number of tests, performed on artificial and real microscopical images, show that this approach is efficient for a variety of differently structured images and as such may have applications in and beyond the field of microscopical imaging.

141 citations


Journal ArticleDOI
TL;DR: The study found the interface biologically and chemically active over the 12‐week implantation period, as it indicates that the pseudowollastonite can be physically and chemically integrated into the structure of living bone tissue, and therefore could be suitable for repair or replacement ofliving bone.
Abstract: In vitro experiments show that pseudowollastonite (α-CaSiO3) is a highly bioactive material that forms a hydroxyapatite surface layer on exposure to simulated body fluid and also to human parotid saliva. This finding is very significant, as it indicates that the pseudowollastonite can be physically and chemically integrated into the structure of living bone tissue, and therefore could be suitable for repair or replacement of living bone. The physical and chemical nature of the remodelled interface between the pseudowollastonite implants and the surrounding bone has been studied after in vivo implantation of 20 pseudowollastonite cylinders into rat tibias. The interfaces formed after 3, 6, 8 and 12 weeks of implantation were examined histologically using an optical microscope and also by analytical scanning electron microscopy. SEM and X-ray elemental analysis showed that the new bone was growing in direct contact with the implants. Other examinations found that the bone was fully mineralized. The ionic exchange taking place at the implant interface with the body fluids was essential in the process of the implant integration through a dissolution-precipitation- transformation mechanism. The study found the interface biologically and chemically active over the 12-week implantation period. The rate of new bone formation decreased after the first 3 weeks and reached constant value over the following 9 weeks. The osteoblastic cells migrated towards the interface and colonized the surface at the contact areas with the cortical regions and also bone marrow.

Journal ArticleDOI
TL;DR: A cryo scanning transmission X‐ray microscope which uses soft X‐rays from the National Synchrotron Light Source is developed, capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 1010 Gray.
Abstract: We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 microm at temperatures of around 100 K We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 1010 Gray This makes possible studies where multiple images of the same specimen area are needed, such as tomography (Wang et al (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II Tomography J Microsc 197, 80-93) or spectroscopic analysis

Journal ArticleDOI
TL;DR: Multiple frequency fluorescence lifetime imaging microscopy was successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co‐expressed in live cells.
Abstract: The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co-expressed in live cells.

Journal ArticleDOI
TL;DR: In this article, spectral precision distance microscopy (SPDM) is used to estimate the bary centers (intensity weighted centroid analogous to the center of mass) of a given target in spectrally differential images.
Abstract: Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, eg confocal laser scanning microscopy (CLSM) However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts) In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a ‘resolution equivalent’, many times smaller than the conventional optical resolution Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution

Journal ArticleDOI
TL;DR: It is demonstrated that skeleton graph analysis is a powerful technique to describe trabecular bone microarchitecture in complex disordered porous media.
Abstract: This paper introduces a new three-dimensional analysis of complex disordered porous media. Skeleton graph analysis is described and applied to trabecular bone images obtained by high resolution magnetic resonance imaging. This technique was developed bearing in mind topological considerations. The correspondence between vertices and branches of the skeleton graph and trabeculae is used in order to get local information on trabecular bone microarchitecture. In addition to real topological parameters, local structural information about trabeculae, such as length and volume distributions, are obtained. This method is applied to two sets of samples: six osteoporosis and six osteoarthritis bone samples. We demonstrate that skeleton graph analysis is a powerful technique to describe trabecular bone microarchitecture.

Journal ArticleDOI
TL;DR: Using a cryo scanning transmission X‐ray microscope, tomographic data‐sets of frozen hydrated mouse 3T3 fibroblasts are obtained and 100 nm lateral and 250’nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution.
Abstract: Using a cryo scanning transmission X-ray microscope (Maser, et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc. 197, 68-79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.

Journal ArticleDOI
TL;DR: It is found that the Hilbert transform provides a rapid qualitative pre‐processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which is used in this study.
Abstract: Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.

Journal ArticleDOI
TL;DR: This review attempts to describe the major immunocytochemical strategies that are in current use for transmission electron microscopy, to enable readers to select the most profitable technique (or compromise) available to reflect both their resources and their ability to address their biological questions with vigour.
Abstract: This review attempts to describe the major immunocytochemical strategies that are in current use for transmission electron microscopy. Emphasis is placed on suggesting the most appropriate application of each technique that is described. The review is not intended to identify the microscopist's ‘cure all’ technique for immunocytochemistry but rather to describe what techniques are available and what are their relative merits. Expensive equipment and technically complicated methods are not always necessary to collect robust data. I hope to present a review that will enable its readers to select the most profitable technique (or compromise) available, to reflect both their resources and their ability to address their biological questions with vigour.

Journal ArticleDOI
TL;DR: An extremely simple method by which optically sectioned fluorescence images may be obtained with conventional microscopes using laser illumination using a one‐dimensional grid pattern, together with a rotating ground glass diffuser.
Abstract: We describe an extremely simple method by which optically sectioned fluorescence images may be obtained with conventional microscopes using laser illumination. A one-dimensional grid pattern is introduced into the illumination system, together with a rotating ground glass diffuser. This causes an image of the grid pattern to be projected into the specimen. Images taken at three spatial positions of the grid are processed in a simple manner to provide optically sectioned images of fluorescent specimens.

Journal ArticleDOI
Neil1, Wilson1, Juškaitis1
TL;DR: The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.
Abstract: We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.

Journal ArticleDOI
TL;DR: An iterative thresholding algorithm for the segmentation of cells from noisy cell images that changes iteratively with both the previous segmentation and image local activity is introduced.
Abstract: We introduce an iterative thresholding algorithm for the segmentation of cells from noisy cell images. The thresholding image, which is initially a constant, changes iteratively with both the previous segmentation and image local activity. Experimental results for both synthesized and real cell images are provided to demonstrate the performance of the algorithm.

Journal ArticleDOI
TL;DR: Two fluorochromes, ER‐TrackerTM Blue‐White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY‐BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular‐vacuole system at the light microscope level in living cells.
Abstract: Two fluorochromes, ER-TrackerTM Blue-White DPX dye and the fluorescent brefeldin A (BFA) derivative, BODIPY-BFA, label the endoplasmic reticulum (ER) in hyphal tips of Pisolithus tinctorius and allow its differentiation from the tubular-vacuole system at the light microscope level in living cells. The ER-Tracker dye labels a reticulate network similar in distribution to ER as seen in electron micrographs of freeze-substituted hyphae. BODIPY-BFA stains a thicker axially aligned structure with an expanded region at the apex, which is similar to that seen when hyphae are stained with ER-Tracker dye in the presence of unconjugated BFA. This structure is considered to be ER modified by BFA, a view supported by ultrastructural observations of the effect of BFA on the fungal ER. Both fluorescent probes also stain punctate structures, which are most likely to be Golgi bodies. Neither probe labels the tubular-vacuole system.

Journal ArticleDOI
TL;DR: An oscillating diamond knife is invented with the aim to minimize compression artefacts in ultrathin sections, coincident with the stroke direction, and consists of a diamond knife on which a low voltage piezoelectric translator is mounted, which oscillates when the piezo is driven by an alternating voltage source.
Abstract: With the aim to minimize compression artefacts in ultrathin sections, coincident with the stroke direction, we have invented an oscillating diamond knife. Results and theoretical considerations explaining its function are discussed. During conventional ultrathin sectioning the resultant compression is in the order of 20–35% of section height. This holds true for sections of samples embedded into Lowicryl HM20 and of the polymer polystyrene, cut with a 45° diamond knife and floated on water. The oscillating knife reduces this compression almost completely. It consists of a diamond knife on which a low voltage piezoelectric translator (piezo) is mounted, which oscillates when the piezo is driven by an alternating voltage source. No additional cutting artefacts were observed in the micrographs when they were compared with sections produced without oscillating the knife.

Journal ArticleDOI
TL;DR: Calculations show that when CARS is performed in a microscopic volume, the phasematching restraint on tuning over the vibrational band is strongly relaxed, and the possible vignetting of the CARS emission is much reduced.
Abstract: Three-dimensional microscopy based on coherent anti-Stokes Raman scattering (CARS) is a powerful new imaging technique, in which the contrast arises from molecular vibrations. Based on a simple numerical model, it is shown how the CARS interaction volume depends on the focusing parameters and the type of phasematching used. Collinear phasematching yields an ellipsoidal interaction volume, with lateral dimensions that readily cause vignetting of the CARS signal emission at the collection microscope objective. A folded BoxCARS phasematching geometry, on the other hand, results in an almost cylindrical interaction volume — at the cost of a reduced resolution, for which the possible vignetting of the CARS emission is much reduced. In addition, this type of phasematching provides spatial separation of the signal from the input laser beams, permitting simple signal detection of low frequency vibrational modes. Calculations show that when CARS is performed in a microscopic volume, the phasematching restraint on tuning over the vibrational band is strongly relaxed. A first example of CARS imaging using a folded BoxCARS imaging geometry is shown.

Journal ArticleDOI
Geday1, Kaminsky1, Lewis1, Glazer1
TL;DR: It is shown how the phase δ, and thus optical retardance, can be extracted from combining measurements of |sin δ| at different wavelengths, and the algorithm derived in this paper is valid not only for birefringence studies, but can be applied to all studies of interfering light waves.
Abstract: Modulation techniques for measuring changes in optical birefringence, such as the rotating-polariser method (Wood & Glazer, 1980, J. Appl. Crystallogr. 13, 217), allow one to determine |sin δ|, δ = 2πLΔn/λ, Δn= double refraction, L = light path and λ = wavelength. However, they generally suffer from not providing absolute values of the optical retardance or are limited to relatively low retardance values. In addition, knowledge of the absolute phase is required when establishing the correct values of optical orientation information. In this paper, it is shown how the phase δ, and thus optical retardance, can be extracted from combining measurements of |sin δ| at different wavelengths. The new approach works on each single point of a 2-D picture without the need to correlate with neighbouring points. There is virtually no limit to the retardance, and the computational efforts are small compared with other methods (e.g. Ajovalasit et al. 1998, J. Strain Analysis 33, 75). When used with imaging techniques, such as the rotating polariser method of Glazer, Lewis & Kaminsky 1996 (Proc. R. Soc. London SeriesA452, 2751) this process has the potential to identify automatically optically anisotropic substances under the microscope. The algorithm derived in this paper is valid not only for birefringence studies, but can be applied to all studies of interfering light waves.

Journal ArticleDOI
TL;DR: Cytoplasmic glutathione concentrations in roots of intact Arabidopsis seedlings were found to be 2–3 mm in most cell types, suggesting that additional cell–cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.
Abstract: Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S-bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15-20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S-conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2-3 mM in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell-cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.

Journal ArticleDOI
TL;DR: A novel method of chromatic aberration measurement and software correction using sub‐resolution bead imaging and computer image analysis is described, which enables the determination of both longitudinal and lateral chromatic aberrations.
Abstract: Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10-20 nm laterally and 10-60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.

Journal ArticleDOI
TL;DR: Experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae are reviewed, showing that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations.
Abstract: Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Journal ArticleDOI
TL;DR: This paper reviews digital imaging technologies for TEM at different voltages, and contrasts the performance of digital imaging systems with that of TEM film.
Abstract: The digital revolution currently under way, as evidenced by the rapid development of the Internet and the world-wide-web technologies, is undoubtedly impacting the field of transmission electron microscopy (TEM). Digital imaging systems based on charge-coupled device (CCD) technologies, with pixel array size up to 2 k × 2 k at the present and increasing, are available for TEM applications and offer many attractions. Is it time to phase out film cameras on TEMs and close the darkrooms for good? This paper reviews digital imaging technologies for TEM at different voltages, and contrasts the performance of digital imaging systems with that of TEM film. The performance characteristics of CCD-based digital imaging systems, as well as methods for assessing them, are discussed. Other approaches to digital imaging are also briefly reviewed.

Journal ArticleDOI
TL;DR: The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemicsupercoiling is the predominant form adopted by plasmid DNA.
Abstract: Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide:base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic:toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.