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.     

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.     

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.     

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.     

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.     

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.     

Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice

A straightforward and transparent model, based on Gaussian beam optics, for the axial r ₀ resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope.     

The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.     

Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two-detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal-to-noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.     

Wavelength division scanning for two-photon excitation fluorescence imaging

We investigate wavelength division scanning for two‐photon excitation fluorescence imaging. Two‐photon imaging using lateral wavelength division scanning is demonstrated. In addition, we theoretically analyse the spatial and temporal properties of a femtosecond laser beam focused by a Fresnel lens and investigate the feasibility of axial scanning using wavelength division.     

MEMS-based fast scanning probe microscopes

Scanning probe microscopy is a frequently used nanometer-scale surface investigation technique. Unfortunately, its applicability is limited by the relatively low image acquisition speed, typically seconds to minutes per image. Higher imaging speeds are desirable for rapid inspection of samples and for the study of a range of dynamic surface processes, such as catalysis and crystal growth. We have designed a new high-speed scanning probe microscope (SPM) based on micro-electro mechanical systems (MEMS). MEMS are small, typically micrometer size devices that can be designed to perform the scanning motion required in an SPM system. These devices can be optimized to have high resonance frequencies (up to the MHz range) and have very low mass (10⁻¹¹ kg). Therefore, MEMS can perform fast scanning motion without exciting resonances in the mechanical loop of the SPM, and hence scan the surface without causing the image distortion from which conventional piezo scanners suffer. We have designed a MEMS z-scanner which we have integrated in commercial AFM (atomic force microscope) and STM (scanning tunneling microscope) setups. We show the first successful AFM experiments.