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.     

Optical sectioning in fluorescence microscopy

We review the origins of optical sectioning in fluorescence microscopy in terms of the structure of the illumination used to generate the fluorescence within the specimen. We note that the conventional microscope using essentially uniform illumination does not exhibit optical sectioning whereas the confocal microscope using point (many spatial frequencies) illumination does. We show that the optical sectioning strength of a confocal microscope is not optimal and discuss the advantages of using a single spatial frequency for the structure of the illumination and the detection. In this case the optical sectioning strength is shown to be up to 25% narrower than in the ideal confocal case.     

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.     

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.     

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.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

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.     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

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.     

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.     

A sandwich-embedding method for oriented sectioning

High‐quality sections are indispensable for many scientific studies. Most published methods are often time‐consuming or require special devices. We present an easy, quick and low‐cost method for oriented embedding of thin structures using glycol methacrylate resin and self‐constructed, reusable embedding tools made of overhead transparencies. This technique allows for more flexibility in orientation than other methods, enabling precise transverse, longitudinal and even oblique sectioning.     

Frequency-domain fluorescence microscopy with the LED as a light source

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 µm 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)₂phe-C₁₂, 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.     

Time-lapse FRET microscopy using fluorescence anisotropy

We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein–protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.     

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.     

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.     

Time-resolved fluorescence microscopy of gunshot residue: an application to forensic science

Time‐resolved fluorescence microscopy has rapidly emerged as the technique of choice for many researchers aiming to gain specific insights into the dynamics of intricate biological systems. Although the unique advantages the technique provides over other methods have proven to be particularly useful in the biosciences, to date they have been largely unexploited by other research disciplines. In this paper, we demonstrate the capacity of time‐resolved fluorescence microscopy as a practical analytical tool in the forensic sciences via the imaging of gunshot residues that are expelled when a firearm is discharged. This information may prove to be useful for determination of the true sequence of events that took place in a firearm related crime.     

QD as a bifunctional cell-surface marker for both fluorescence and atomic force microscopy

Fluorescent quantum dots (QDs) are a new class of fluorescent label and have been extensively used in cell imaging. Streptavidin-conjugated QDs have a diameter of ca. 10–15 nm; therefore when used as probes to label cell-surface biomolecules, they can provide contrast enhancement under atomic force microscopy (AFM) and allow specific proteins to be distinguished from the background. In addition, the size and fluorescent properties potentially make them as probes in correlative fluorescence microscopy (FM) and AFM. In this study, we tested the feasibility of using QD-streptavidin conjugates as probes to label wheat germ agglutinin (WGA) receptors on the membrane of human red blood cells (RBCs) and simultaneously obtain fluorescence and AFM images. The results show that the distribution of QDs labeled on human RBCs was non-uniform and that the number of labeled QDs on different erythrocytes varied significantly, which perhaps indicates different ages of the erythrocytes. Thus, QDs may be employed as bifunctional cell-surface markers for both FM and AFM to quantitatively investigate the distribution and expression of membrane proteins or receptors on cell surface.     

Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain

Anatomical information at the cellular level is important in many fields, including organ systems development, computational biology and informatics. Creating data sets at resolutions that provide enough detail to reconstruct cellular structures across tissue volumes from 1 to 100 mm³ has proven to be difficult and time-consuming. In this paper, we describe a new method for staining and imaging large volumes of tissue at sub-micron resolutions. Serial sections are cut using an automated ultra-microtome, whereas concurrently each section is imaged through a light microscope with a high-speed line-scan camera. This technique, knife-edge scanning microscopy, allows us to view and record large volumes of tissue in a relatively small amount of time (approximately 7 mm² s⁻¹).
  The resolution and scanning speed of knife-edge scanning microscopy provides a new method for imaging tissue at sufficient resolution to reconstruct maps of cellular distribution and morphology. We show that these techniques preserve the alignment of serial sections accurately enough to allow for reconstruction of neuronal processes and microvasculature. Expanding these techniques to other tissues opens up the possibility of creating fully reconstructed cellular maps of entire organs.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.