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Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup., (© 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.)

This paper investigates a postprocessing approach to correct spatial distortion in two-photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field-of-view, deep-tissue studies of vascular structures. Based on simple geometric modelling of the object-of-interest, a distortion function is directly estimated from the image volume by deconvolution analysis. Such distortion function is then applied to subvolumes of the image stack to adaptively adjust for spatially varying distortion and reduce the image blurring through blind deconvolution. The proposed technique was first evaluated in phantom imaging of fluorescent microspheres that are comparable in size to the underlying capillary vascular structures. The effectiveness of restoring three-dimensional (3D) spherical geometry of the microspheres using the estimated distortion function was compared with empirically measured point-spread function. Next, the proposed approach was applied to in vivo vascular imaging of mouse skeletal muscle to reduce the image distortion of the capillary structures. We show that the proposed method effectively improve the image quality and reduce spatially varying distortion that occurs in large field-of-view deep-tissue vascular dataset. The proposed method will help in qualitative interpretation and quantitative analysis of vascular structures from fluorescence microscopy images., (© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.)

The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level., (© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.)

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three-dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more., (© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.)

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms 'quasi-confocal images' can be derived from a given number of such phase-images. Here, we present an alternative structured illumination microscopy approach, which employs two-dimensional patterns instead of a one-dimensional one. While in one-dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two-dimensional approach it is shifted at a single, pattern-dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern-shift and -rotation. Moreover, our two-dimensional approach also yields a better signal-to-noise ratio in the evaluated image., (© 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.)

Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy., (© 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.)

We describe a microscopy design methodology and details of microscopes built to this 'open' design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam 'end-stations' at Oxford and Surrey Universities, showing the versatility and extendibility of this approach., (© 2013 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

We present endoscopic focal modulation microscopy (FMM) for minimally invasive imaging of deep tissue with high contrast, and then compare the results using endoscopic confocal microscopy (CM). Deep tissue imaging is achieved using a needle-like endoscopic probe based on gradient-index (GRIN) lenses. A tissue-like phantom with fluorescent micro-beads and rat kidney tubules were imaged through the endoscopic probe with FMM. FMM effectively rejected the background signals from the out-of-focus plane, thereby enhancing the image contrast and the optical sectioning ability. The combination of the GRIN endoscopic probe and FMM provides deep tissue imaging with better contrast than endoscopic CM., (© 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.)

Traditional confocal microscopy uses a physical aperture barrier to prevent out-of-focus light from reaching the detector. The physical nature of a conventional aperture limits control over the system confocality. We describe a new line scanning confocal microscope that eliminates a need for a physical aperture by employing a software-controllable rolling shutter on a CMOS camera. A confocal image is obtained by synchronizing motion of the rolling shutter and the laser line scanning over a sample. Confocal resolution of this microscope is adjustable in real time and independently established for each fluorescence channel by changing the rolling shutter width. This technology has been implemented in the IN Cell Analyzer 6000 system by GE Healthcare., (© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.)

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations., (© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.)

We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores' emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging., (© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.)

In this paper we report stimulated emission depletion (STED) and two-photon excitation (2PE) fluorescence microscopy with continuous wave (CW) laser beam using a new generation laser scanning confocal microscope equipped for STED-CW (TCS STED-CW, Leica Microsystems, Mannheim, Germany). We show the possibility to achieve CW-2PE with the very same beam used for STED-CW. This feature extends the performance of the microscope allowing multimodal imaging (CW-2PE, STED-CW, confocal)., (© 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.)

In conventional microscopes, fluorescence emission is separated from the backscattered illumination using the Stokes shift, whereby the emission occurs at a longer wavelength to the excitation. Such separation is usually achieved through a combination of wavelength filters that divide the spectrum into mutually exclusive excitation and emission bands. It is therefore impossible in these microscopes to access the full excitation/emission spectrum of the specimen in a single image. We report on a microscope that acquired fluorescence images using illumination across the spectral range 450-680 nm; the full emission spectrum was detected simultaneously across the same range. The microscope was also combined with structured illumination optical sectioning to give three-dimensionally resolved images with improved background rejection. Full spectrum fluorescence images of biological specimens are demonstrated. As this system is more versatile than the standard fluorescence microscope, it could be of benefit in many fluorescence imaging applications.

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 microm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.

This paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to real-time imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly-L-lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron-sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.

Capillary-based high-pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub-cellular-dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub-cellular-dimension spatial resolution in the imaging of fluorescent micro-spheres. Objectives with a range of numerical apertures (0.5-1.3) and working distances (0.1-7.4 mm) are considered. We show that spatial (or point-spread function, PSF) deconvolution improves image contrast in capillary-based images by comparing deconvolution results with those obtained from slide-mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat-geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high-pressure imaging fields.

Spiral wave phenomena exist in many scales of nature and have attracted the attention of scientists from different fields. Although much work has been done on qualitative analysis of spiral waves, the mechanism of spiral waves' spontaneous formation and termination in living systems is still not clear. Here, by using total internal reflection fluorescence microscopy, we show the spiral waves of calcium signals in single rat cardiac myocytes and the simulation of the waves comprising calcium sparks. The mechanism of the formation and termination of spiral waves is attributable to the calcium release channels' refractory resulting from their stochastic release. We suggest that this mechanism can be adapted to other living systems.

A scanning-less single-photon counting system for FLIM and fluorescence anisotropy wide-field imaging is described and characterized in this paper. The two polarizations of the fluorescence are divided by a Glan prism and acquired at the same time by the Q(A) detector. Fluorescence decay profiles can be reconstructed for any desired area up to each pixel and used to calculate time-resolved fluorescence anisotropy decays.

We describe the construction and characterization of a laser-line-scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near-infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi-photon-induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron-multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed-loop controlled sample scanning stage and a piezo-driven objective actuator. The full widths at half maximum of the point-spread function of the system were estimated to be 0.39-0.40, 0.33 and 0.56-0.59 mum for the X (lateral axis along the line-scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub-cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re-absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub-cellular domains. The efficiency of the new line-scanning spectromicroscope was estimated in comparison with our own point-by-point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line-scanned region, as expected.

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

We describe a flexible, multifaceted optical setup that allows quantitative measurement and manipulation of biomolecules and nanoparticles in biomimetic and cellular systems. We have implemented integrated biophotonics techniques (i.e. differential interference contrast, wide-field fluorescence, prism- and objective-based total internal reflection excitation, single particle tracking, fluorescence correlation spectroscopy and dynamic holographic optical trapping) on a single platform. The adaptability of this versatile, custom-designed system allows us to simultaneously monitor cell morphology, while measuring lateral diffusion of biomolecules or controlling their cellular location or interaction partners.

We report the demonstration of time-correlated single-photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white-light supercontinuum excitation source. By using a 100 fs-pulsed Ti:Sapphire laser with a low-frequency chirp to pump a 30-cm long section of photonic crystal fibre, a ps-pulsed white-light supercontinuum was created. Optical bandpass interference filters were then applied to this broad-bandwidth source to select the 488-nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non-infected material. The white-light generation source provides a flexible method of producing variable-bandwidth visible and ps-pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.

We describe a novel automated technique for visualizing the three-dimensional distribution of fluorochrome-labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome-labelled microdamage within an 8-mm diameter cylinder of trabecular bone at a voxel size of 3 x 3 x 8 microm3. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three-dimensional visualization. The three-dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.

The relaxation of fluorescence from diffraction-limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo-conversion was measured in solutions of varied viscosity (eta), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point-spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D(ap). The model incorporates two novel features that make it of broad utility. First, the model includes the no-flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D(ap). Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D(ap) and eta(-1) were obtained for the two proteins: for PAGFP, D(aq)= 89 +/- 2.4 microm2 s(-1) (mean +/- 95% confidence interval), and for EGFP D(aq)= 91 +/- 1.8 microm2 s(-1). In CHO cells, the application of the model yielded D(ap)= 20 +/- 3 microm2 s(-1) (PAGFP) and 19 +/- 2 microm2 s(-1) (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked 'bound fraction' explanation of the deviation of the terminal fluorescence recovery from its pre-bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488-nm excitation, and the requirement for approximately 1/200th the cumulative light energy to produce data of comparable signal/noise.

High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.

The axial (z-) resolution of approximately 100 nm provided by 4Pi and I5M fluorescence microscopy relies on the coherent addition of spherical wavefronts of two opposing high aperture angle lenses. Both microscopes feature a point-spread function (PSF) with a sharp central spot that is accompanied by axially shifted sidelobes which leads to replication artefacts in the raw image data. In a 4Pi-microscope the sidelobes are less pronounced than in I5M and without relevant lateral (x,y) substructure, making their posterior removal in the image reliable and fast. On the other hand, high speeds of raw data acquisition are more easily gained by I5M. Moreover, I5M features a stronger signal as compared to the commonly employed two-photon excitation (2PE) 4Pi-imaging mode. We investigate here the capability of both techniques to image (aqueous) specimens without artefacts. To this end, we consider the optical transfer function (OTF) of the two microscopes in conjunction with the signal-to-noise-ratio (SNR) of the object to be imaged. The imaging of E. coli bacteria with an interconvertable setup enabled a direct comparison of the two imaging modes. As both systems rely on high aperture angles, water-immersion lenses of the largest numerical aperture available (NA = 1.2) were employed. The experimental results are corroborated by simulations assuming the signal strength encountered in the experiment. The comparison of the theoretical with the experimental PSFs/OTFs showed that our setup operated close to theory in both imaging modes. Although I5M provided about 10 times brighter raw image data as compared to (2PE) 4Pi-microscopy, the I5M data could not be entirely cleared of artefacts. In conclusion, with the current aperture angles and fluorescence signal strengths, it is not advisable to trade in the suppression of the sidelobes for a larger image signal.

Fluorescence in situ hybridization coupled with far-field fluorescence microscopy is a commonly used technique to visualize chromosomal aberrations in diseased cells. To obtain the best possible results, chromatin integrity must be preserved to ensure optimal hybridization of fluorescence in situ hybridization probes. However, biological samples are known to degrade and storage conditions can be critical. This study concentrates its investigation on chromatin stability as a function of time following fluorescence in situ hybridization type denaturing protocols. This issue is extremely important because chromatin integrity affects the fluorescence response of the chromosome. To investigate this, metaphase chromosome spreads of human lymphocytes were stored at both -20 and -80 degrees C, and were then imaged using scanning near-field optical microscopy over a nine month period. Using the scanning near-field optical microscope's topography mode, chromosome morphology was analysed before and after the application of fluorescence in situ hybridization type protocols, and then as a function of storage time. The findings revealed that human chromosome samples can be stored at -20 degrees C for short periods of time (approximately several weeks), but storage over 3 months compromises chromatin stability. Topography measurements clearly show the collapse of the stored chromatin, with variations as large as 60 nm across a chromosome. However, storage at -80 degrees C considerably preserved the integrity with variations in topography significantly reduced. We report studies of the fluorescent response of stored chromosomes using scanning near-field optical microscopy and their importance for gaining further understanding of chromosomal aberrations.

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

We describe the theory and implementation of a frequency-domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications-type, radio-frequency electronics are used to provide inexpensive modulation references and to perform phase-sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.

Time-resolved microspectrofluorometry in live cells, based on time- and space-correlated single-photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal-to-noise ratio of 10(6), and a spectral resolution of 30 (Deltalambda/Lambda). As an example, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal-to-noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal-to-noise ratio or optimize a newly defined 'figure of merit', which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.

In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, phiFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360 degrees range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide-field frequency-domain FLIM system based on an acousto-optic modulator for modulation of the excitation and a microchannel-plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to +/- 29 degrees and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP-transfected HeLa cells were as high as 100%. With phiFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far-red and near-infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low-light or 'night vision' applications. The intensifier converts far-red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real-time viewing by eye of normally invisible far-red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright-field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.

A novel compact illumination device in variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell-substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four-layer model, as well as a Gaussian-shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell-substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time-resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).

Bent near-field optical probes for biological applications have been fabricated using a combination of a two-step chemical etching method and focused ion beam milling to create a well-defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (lambda) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/lambda)3 relationship. The double-etched probes are compared to pulled probes fabricated from highly GeO2-doped dispersion compensating fibre and a standard single-mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double-etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase-separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double-etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye-labelled lipid. The images obtained with the double-etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.

Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in fungal hyphae loaded with chloromethyl aminocoumarin (CMAC), and then anhydrously freeze-substituted, without any aldehyde fixation, are similar to those seen in living hyphae. Probe loss into the mounting medium (Spurr's resin) with CMAC and five other probes tested indicated that some unwanted solubilization of probe occurred during embedding, but nevertheless vacuoles could be imaged by their retention of probe.

We report on the construction details of a compact autocorrelator set-up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two-photon excitation fluorescence imaging. One of the novelties of the set-up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non-linear least-square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread-board (10 x 20 cm); it is PC-controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over-expression of enhanced green fluorescent protein-tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge-coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass-water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s-polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p-polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam-splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x-y plane with s-polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.

A non-enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context 'low temperature' means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 degrees C. For LTFISH, the DNA probe had to be single-stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near-field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH-procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single-stranded DNA probes to double-stranded DNA targets are discussed.

We demonstrate adaptive aberration correction for depth-induced spherical aberration in a multiphoton scanning microscope with a micromachined deformable mirror. Correction was made using a genetic learning algorithm with two-photon fluorescence intensity feedback to determine the desired shape for an adaptive mirror. For a 40x/0.6 NA long working distance objective, the axial scanning range was increased from 150 mm to 600 mm.

By physical rotation of the sample, axial tomography enables the acquisition of otherwise inaccessible spatial information from an object. In combination with confocal microscopy, the method can fundamentally improve the effective three-dimensional (3D) resolution. In this report we present a novel method for high resolution reconstruction of confocal axial tomographic data. The method automatically determines the relative angles of rotation, aligns the data from different rotational views and reconstructs a single high resolution 3D dataset. The reconstruction makes use of a known point spread function and is based on an unconstrained maximum likelihood deconvolution performed simultaneously from multiple (in our case three) angular views. It was applied to simulated as well as to experimental confocal datasets. The gain in resolution was quantified and the effect of choice of overrelaxation factors on the speed of convergence was investigated. A clearly improved 3D resolution was obtained by axial tomography together with reconstruction as compared with reconstruction of confocal data from only a single angular view.

A combination of atomic force microscopy (AFM) and near field scanning optical microscopy has been used to study domain formation in dipalmitoylphosphatidylcholine (DPPC)/cholesterol monolayers with cholesterol concentrations ranging from 0 to 50%. The results show a clear evolution from a mixture of liquid expanded and liquid condensed phases for cholesterol concentrations < 10% to a mixture of liquid expanded and two cholesterol-containing phases at intermediate concentrations, and finally to a single homogeneous liquid ordered phase for 33% cholesterol. Mixtures of the various phases are clearly identified by height differences in AFM and in some cases by fluorescence imaging for samples containing 0.5% BODIPY dye, which localizes preferentially in the more fluid phase. Note that fluorescence imaging, at least with the dye used here, is unable to distinguish between the cholesterol-rich and cholesterol-poor phases detected at intermediate cholesterol concentrations. The combination of fluorescence and AFM imaging provides a more complete picture of the phase evolution for cholesterol/DPPC monolayers than could be obtained by either technique alone, and presents substantial advantages over conventional fluorescence microscopy in that submicrometre-sized domains can be readily detected.

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 micrometer but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

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.

A real-time two-photon fluorescence microscope with a microlens-array scanner using a regenerative amplifier is presented. The regenerative amplifier generates pulsed laser lights with extremely high peak power and produces brighter two-photon-induced fluorescence compared with that produced without the amplifier. Experimental results obtained from the observation of some biological samples with the proposed microscope are given.

Photometric techniques are commonly used to monitor the output from fluorescent indicators during the study of cellular signalling. At the single-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present study reports an improved aperture controller which adjusts the area for fluorescence measurement, whilst allowing objects throughout the field of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filters which block > 99.9% of the fluorescence emission at 480-600 nm. A 780-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectable by a standard CCD camera. This allows detection of morphological events throughout the field of view and facilitates manipulation of extracellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable for use with a range of other fluorescent indicators, including those routinely used to detect Ca2+, Cl-, Na+ and pH. Data are presented which demonstrate the use of this controller to measure ADP-evoked [Ca2+]i increases in single human erythroleukaemia cells loaded with the Ca2+ indicator fura-2.