Wide-field optically sectioning fluorescence microscopy with laser illumination

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.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

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.     

An optical sectioning programmable array microscope implemented with a digital micromirror device

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x - z sections and less intensity drop-off with scanning depth.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays

We discuss how the strength of the optical sectioning property of the direct-view reflection microscope varies with the size and distribution of the pin-holes in the source and detector arrays. Finite-sized circular pin-holes are considered in both bright-field and fluorescence imaging and the results are compared with those of the corresponding scanning optical microscope. We discuss the effect of using both incoherent and coherent illumination.     

Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens

An imaging technique called orthogonal-plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three-dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high-resolution images of optical sections of an intact, excised guineapig cochlea. The lateral and axial resolutions of the images were 10 and 26 μm, respectively, within a 1·5-mm field of view.     

A light efficient optically sectioning microscope

We describe a simple method by which optically sectioned images may be obtained. The system geometry is similar to that of a tandem scanning microscope but a one-dimensional grid pattern is used rather than an array of pinholes. This produces a composite image consisting of an optically sectioned image superimposed on a conventional image. A blank sector on the disc is used to provide a wide-field image. Image subtraction yields the optically sectioned image in real time.     

Scanning optical fluorescence microscopy

A scanning optical fluorescence microscope is described which possesses several advantages over a conventional fluorescence microscope. These include improved resolution, a reduction in background- and auto-fluorescence, an increase in the available fluorescence spectrum and simple modification for automated fluorescence studies. Experimental results are included.     

Continuous serial thin sectioning for electron microscopy

The process of serial sectioning for electron microscopy has been refined such that loss of thin sections is kept below 0.1% and the series is continued at will. The method relies on microscopic control of all manipulative steps, Formvar casting on plate glass for coated slot grids, coating of the block with contact cement for reliable ribboning, pickup by a one-step method with grid support in the diamond knife trough, staining in LKB grid holders, gentle treatment of grids in the electron microscope, and a slight modification to the microscope for safe grid withdrawal. The results are particularly applicable to the reconstruction of neuronal microcircuits and larger volumes of neuropil.     

Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy

Layer‐by‐layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z‐response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z‐response of confocal, two‐photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z‐response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z‐response, off‐axis aberration, spherical aberration, etc.) of a three‐dimensional scanning system. The sectioned imaging property charts method needs a through‐focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long‐term monitoring of the z‐response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real‐time evaluation of the optical sectioning capabilities of different three‐dimensional scanning systems especially when coupled to sectioned imaging property charts.     

Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays

Direct-view microscopes use multiple-aperture arrays in the source and detector planes. We develop a theory for brightfleld and fluorescence direct-view microscopy which allows us to determine the optical sectioning strength for finite-sized, multiple-pinhole arrays with an arbitrary distribution of apertures. We specialize to the cases of square, hexagonal and interleaving Archimedean spiral arrays and consider the implications of the array configuration on both the optical sectioning strength and the light budget.     

Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration

We present a comparison between theoretical and experimental results for the axial response to a plane mirror specimen of the direct-view microscope employing multiple-pinhole arrays. The effects of pinhole size, pinhole spacing and array geometry are investigated in detail with a view to (i) achieving good optical sectioning characteristics and (ii) maximizing the amount of light available for imaging. The implications of our results for practical systems as regards pinhole-array design and fabrication are also discussed.     

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.     

Application of a laser scalpel to sectioning fragile tissues prior to optical sectioning microscopy and 3D reconstruction

A method is described for the cutting of fragile material with a laser scalpel which minimizes damage to friable materials, making the interior of structures accessible for optical sectioning microscopy or for high resolution X-ray microtomography followed by 3D reconstruction.     

Quantitative fluorescence in confocal microscopy

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen-stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations. In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re-absorption of the excitation light and emission light respectively. In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens. Under confocal observation, as measured with the BIO-RAD MRC-500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

Future trends in microscopy

Recent progress and current trends in microscopy are examined with particular reference to possible future developments. The scope for systematic improvement of the traditional optical or electron designs and procedures seems far from exhausted. However the introduction of novel, lens-free microscopes which, like the scanning tunnelling microscope, produce magnification using an electronic lever arm, could expand the boundaries of the subject most dramatically. The future microscopist may find himself displaced from the more routine tasks of focusing, alignment, image recording and even simple interpretation by the electronic robot. On the other hand, he will gain access to a wider and more sophisticated range of physical and chemical phenomena governing the interaction between the specimen and the radiations he uses to probe its microscopic structure and properties.     

A model based reconstruction technique for depth sectioning with scanning transmission electron microscopy

Depth sectioning in high angular annular dark field scanning transmission electron microscopy is considered a candidate for three-dimensional characterization on the atomic scale. However at present the depth resolution is still far from the atomic level, due to strong limitations in the opening angle of the beam. In this paper we introduce a new, parameter based tomographic reconstruction algorithm that allows to make maximal use of the prior knowledge about the constituent atom types and the microscope settings, so as to retrieve the atomic positions and push the resolution to the atomic level in all three dimensions.     

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

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