Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila.

In a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently-stained 12.5-day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.

Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser.

We have studied the wavelength dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose-staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540-800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution-based measurements.

Increased signals from short-wavelength-excited fluorescent molecules using sub-Ti:Sapphire wavelengths.

We report the use of an all-solid-state ultrashort pulsed source specifically for two-photon microscopy at wavelengths shorter than those of the conventional Ti:Sapphire laser. Our approach involves sum-frequency mixing of the output from an optical parametric oscillator (λ= 1400-1640 nm) synchronously pumped by a Yb-doped fibre laser (λ= 1064 nm), with the residual pump radiation. This generated an fs-pulsed output tunable in the red spectral region (λ= 620-636 nm, ~150 mW, 405 fs, 80 MHz, M(2) ~ 1.3). We demonstrate the performance of our ultrashort pulsed system using fluorescently labelled and autofluorescent tissue, and compare with conventional Ti:Sapphire excitation. We observe a more than 3-fold increase in fluorescence signal intensity using our visible laser source in comparison with the Ti:Sapphire laser for two-photon excitation at equal illumination peak powers of 1.16 kW or less.

A compact instrument for adjusting laser beams to be accurately coincident and coaxial and its use in biomedical imaging using wave-mixed laser sources.

Biomedical imaging applications that involve nonlinear optical processes such as sum-frequency generation (SFG) and four-wave mixing require that the pulses are synchronized in time and the beams are coaxial to better than 400 µrad. For this reason, folding mirrors are normally used to extend the beam path over a few meters so that detectors can be put into the beams to check their overlap at the start of a long path and also at the end of it. We have made a portable instrument with a footprint of only 22 cm × 11 cm × 16 cm that uses a short focal length lens and a telephoto combination for viewing the near-field and far-field simultaneously. Our instrument is simple to build and use, and we show its application in coherent anti-Stokes Raman scattering microscopy and SFG-based two-photon fluorescence microscopy.

Use of a white light supercontinuum laser for confocal interference-reflection microscopy.

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460-700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser.

Digitisation of electron microscope films: six useful tests applied to three film scanners.

A series of simple tests have been used to measure the performance of flat-bed film scanners suitable for digitisation of electron micrographs. Two of the film scanners evaluated are commercially available and one has been constructed in the laboratory paying special attention to the needs of the electron microscopist. The tests may be useful for others.

How the confocal laser scanning microscope entered biological research.

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.

Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics.

In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long-established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain-free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy.

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

Three-dimensional cryoelectron microscopy of 16-protofilament microtubules: structure, polarity, and interaction with motor proteins.

We present a three-dimensional (3D) map, reconstructed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtubules (MTs) in ice. We compare it with the tubulin in six 3D maps of MTs decorated with motor domains, three from frozen MTs decorated with kinesin or ncd in the tightly bound AMP-PNP state, and three from negatively stained MTs decorated with kinesin in different nucleotide states. The comparison confirms that kinesin and ncd bind to identical sites and interact with both monomers of a tubulin dimer. Maps of specimens in negative stain and in ice are similar except that the protein in the top half of a motor domain appears denser in negative stain. The interactions have only a small effect on tubulin structure; the outward appearance is unchanged, but there seems to be a small internal rearrangement. The relative polarity of undecorated and decorated MTs is evident from their 3D structures. This agrees with the absolute polarities indicated by the orientations of motors in decorated specimens and by polar superposition patterns calculated for undecorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria.

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study.

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines.

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.

The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy.

Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements.

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.

Molecular analysis of neurofibrillary degeneration in Alzheimer's disease. An immunohistochemical study.

Antibodies directed against three regions of tau, ubiquitin, and B-amyloid were used in a histologic study of neurofibrillary degeneration in Alzheimer's disease to distinguish two populations of neurofibrillary tangles. Intracellular tangles were immunolabeled exclusively by two antibodies raised against antigens in the fuzzy coat of the paired helical filament (PHF). Extracellular tangles were distinguished by selective immunolabeling with a monoclonal antibody raised against antigens in the PHF core. This was associated with removal of the fuzzy coat and exposure of PHF-core epitopes. In the transition from intracellular to extracellular compartments in vivo, tangles appeared to undergo changes similar to protease digestion in vitro. The transition was associated with the appearance of amyloid immunoreactivity. These findings suggest that tangle degradation occurs in a series of distinct stages, including ubiquitination of some unknown molecule, a change in tau immunoreactivity, and partial proteolysis of tangle-bound tau in extracellular tangles.

Replication occurs at discrete foci spaced throughout nuclei replicating in vitro.

Demembranated Xenopus sperm nuclei were induced to replicate synchronously in a low-speed supernatant (LSS) of Xenopus eggs by preincubation in a high-speed supernatant (HSS). DNA replication was observed by incorporation of [alpha-32P]dATP, BrdUTP or biotin-dUTP. Biotin-dUTP incorporation, visualised with fluorescent streptavidin, reveals a striking pattern of replication foci throughout replicating nuclei. We show that this represents a precursor to the bright uniform fluorescence seen later. Confocal microscopic analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100-300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells. Foci are evenly distributed throughout the nuclei and are not concentrated at or near the nuclear envelope. Complete replication of each nucleus occurs in an average time of only one hour in this system. Hence we calculate that there must be at least 300-1000 replication forks together in each cluster. Furthermore, pulse labelling at later times in the period of replication reveals a similar pattern of foci indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of DNA replication.

Results obtained with a sensitive confocal scanning system designed for epifluorescence.

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

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.