Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two-dimensional (2-D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow-type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non-confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time-resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2-D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real-time or can use a cooled CCD at (very) low light level for image integration over an extended period.     

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.     

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy

We describe a technique for imaging enzyme activity through steady‐state fluorescence anisotropy measurements on a per‐pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy‐FL‐labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five‐fold. Fluorescence lifetime and time‐resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy‐FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy‐containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.     

New imaging modes for lenslet-array tandem scanning microscopes

The tandem scanning microscope permits confocal images to be obtained in real time and viewed directly by eye. The light budget of these instruments may be increased from a few percent to a few tens of percent by incorporating an array of microlenses so as to increase the amount of illumination light that reaches the specimen. These instruments are configured for fluorescence imaging together with laser illumination. We describe how the versatility of the instrument may be enhanced to permit the use of incoherent light sources as well as extending the imaging modes to include bright‐field reflection.     

Fibre-optic two-photon scanning fluorescence microscopy

Two geometries of a novel two‐photon fluorescence microscope incorporating single‐mode fibre optics for the delivery of ultrashort‐pulsed illumination to a remote sample are characterized. First, a 785 nm single‐mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non‐linear response of the fibre to intense ultrashort‐pulsed light. Second, a 785 nm single‐mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort‐pulsed laser beam caused by the non‐linear response of the fibre to ultrashort‐pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort‐pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low‐cost, compact instrument. Sets of three‐dimensional images of fluorescent polymer microspheres and biological material confirm these features.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Image scanning fluorescence emission difference microscopy based on a detector array

We propose a novel imaging method that enables the enhancement of three‐dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.     

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.     

Imaging in scanning microscopes with slit-shaped detectors

We consider the imaging in scanning microscopes employing point and slit-shaped detectors in both the bright-field and fluorescent mode. In particular we consider the three-dimensional aspects of the imaging and show inter alia that acceptable, albeit asymmetrical, images result from a slit detector system at low levels of defocus. The situation becomes worse as the defocus is increased although acceptable extended-focus and isometric images are still possible in some cases.     

Navigating transdermal diffusion with multiphoton fluorescence lifetime imaging

We demonstrate the potential of fluorescence lifetime imaging by time-correlated single-photon counting as a method for monitoring the transdermal diffusion pathway and diffusion rate of pharmaceuticals in human skin. The current application relies on observing subtle changes in the fluorescence lifetime of the intrinsic fluorophores present in the intracellular region between corneocytes of the stratum corneum. We have comprehensively characterized the measured fluorescence lifetimes from intracorneocyte junctions in three skin section types (dermatomed skin, epidermal membranes and stratum corneum) revealing statistically significant differences of the short lifetime component between each of the types, which we attribute to the sample preparation and imaging method. We show using epidermal membrane sections that application of a drug/solvent formulation consisting of ethinyl estradiol and spectroscopic grade ethanol to the surface gives rise to a slight but statistically significant shortening of the fluorescence lifetime of the long-lived emitting species present in the sample, from approximately 2.8 ns to 2.5 ns. The method may be useful for future studies where the kinetics and pathways of a variety of applied formulations could be investigated.     

Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy

Imaging properties of scanning confocal electron microscopy (SCEM) were studied by calculating simple model systems using the multislice method. A simple geometrical explanation was given, particularly for the difference between bright field (BF) and annular dark field (ADF) SCEM. It is demonstrated that the BF-SCEM image contrast consists of two features. One gradually changes over a wide defocus range and depends on the lateral size of the object. Another appears only near the focus and is independent of sample size. On the contrary, ADF-SCEM image contrast does not depend on the lateral size of the object. Therefore, the ADF-SCEM will provide more readily interpretable image contrast.     

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.     

Highly efficient upconverters for multiphoton fluorescence microscopy

A new generation of efficient two-photon absorbing fluorescent molecules has been developed and used effectively for two-photon laser scanning microscopy. Several examples of the use of these new fluorophores have been presented. In addition, issues relating to the two-photon absorption cross-section, excitation power, sample properties and resolution in two-photon laser scanning microscopy are discussed.     

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.     

Imaging 'intact' myofibrils with a near-field scanning optical microscope

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro , naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.     

Biological imaging using fast laser scanning heterodyne differential phase confocal microscopes

Differential phase microscopy has proved invaluable in the study of live, unstained, thin biological samples because of its ability to image changes in refractive index and topography. Similarly, because of its optical sectioning capability, confocal microscopy is now a well-established technique in the study of relatively thick live biological samples. This paper describes the development and application of two differential phase heterodyne confocal microscopes, and compares their performance. The use of these systems for imaging in-vitro cell and tissue cultures is considered and compared with confocal reflection microscopy. It is demonstrated that the differential phase capability reveals subcellular structural information not readily seen in the confocal reflection images. This technique opens up the possibility of imaging thick unstained live tissues, avoiding cell damage and artefacts associated with staining procedures. Furthermore, the differential phase images can be used to provide a visual frame within which stained features can be located.     

Intercomparison of scanning probe microscopes

Comparison measurements on reference standards are reported in which 13 partners with different instruments took part. A set of prototype standards which had been produced and calibrated within a European project were used for the measurements. Here, results of measurements on a 240 nm step height standard and a two-dimensional lateral standard with a nominal pitch of 1 μm are reported.     

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.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.