Scanning near field optical/atomic force microscopy of bromodeoxyuridine-incorporated human chromosomes

The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCI with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.     

Doppler optical coherence imaging of converging flow

The experimental methods of Doppler optical coherence tomography are applied for two-dimensional flow mapping of highly scattering fluid in flow with complex geometry. Converging flow (die entry) is used to demonstrate non-invasive methods to map varying velocity profiles before and after the entry. Complex geometry flow is scanned with approximately 10 x 10 x 10 microm3 spatial resolution. Structural images of the phantom and specific velocity images are demonstrated. A variety of velocity profiles have been obtained before and after the entry. Concave, blunted, parabolic and triangular profiles are obtained at different distances after the entry. Application of the technique to the study of blood circulation is discussed.     

The cortical projection to the nucleus reticularis tegmenti pontis in the rhesus monkey

Summary The projection from the cerebral cortex to the nucleus reticularis tegmenti pontis (NRT) has been studied in the rhesus monkey with silver impregnation methods. The majority of cortical afferents to NRT arise in the precentral motor cortex (area 4), while more modest contributions come from the premotor region (area 6), the somatosensory cortex (areas 3, 1, and 2) and area 5. Very few, if any, degenerating fibres were observed in the NRT after lesions of other parts of the cortex. The cortical afferents terminate throughout NRT except in its dorsomedial part. There is a topographical arrangement so that fibres from area 6 terminate most medially in the NRT, fibres from area 5 most laterally (in the processus tegmentosus lateralis), while fibres from the motor cortex end in between and cover the largest terminal field. In addition, the projection from the motor cortex is somatotopically organized with the leg represented ventrally and the arm dorsally in the NRT. There is, however, ample overlap between the terminal regions of fibres from the various cortical areas. The results are discussed in relation to the termination of other afferent contingents to the NRT, and it is concluded that even if there is a high degree of convergence, various parts of the NRT appear not to receive identical sets of afferent inputs.     

Growth-related alterations in motor endplates of type-identified diaphragm muscle fibres

Summary Using a double-labelling technique, and dual-channel confocal microscopy, we examined the three-dimensional and two-dimensional morphologies of motor endplates on type I and II muscle fibres of 21-day-old and adult rat diaphragms. Motor endplates were visualized with fluorescein-conjugated -bungarotoxin, and muscle fibre type was immunocytochemically determined using an anti-fast (type II) myosin antibody with a Cy5-conjugated label. Surface (three-dimensional) and planar (two-dimensional) areas were obtained from three-dimensional reconstructions of confocal optical sections of labelled endplates. Muscle fibre diameters were also measured. Total branch lengths were measured from projection images of the three dimensional reconstructions. The surface and planar areas of endplates on type I fibres at day 21 were larger than those on type II fibres, and this difference increased with maturation. In adults, the surface area of endplates was positively correlated to muscle fibre size, but such a correlation was not found at day 21. When normalized for fibre diameter, the surface areas of endplates on type I fibres were still significantly larger than those on type II fibres in both age groups. The normalized endplate surface area for type II fibres remained constant with maturation, whereas for type I fibres, the increase in endplate surface area was disproportionate to fibre growth.     

Fibre type regionalisation in lower hindlimb muscles of rabbit, rat and mouse: a comparative study

The topographical distribution of different fibre types in muscles of the lower hindlimb in rabbits and mice was quantitatively determined. The results were compared to those previously obtained, using the same new quantification methods, in homologous muscles of the rat. Type I fibres ('slow') were identified using myofibrillar ATPase histochemistry and mapped out at the mid proximo-distal level for 11 'fast' muscles in the rabbit and 7 'fast' muscles in the mouse. For the slow soleus muscle the procedure was undertaken for the type II fibres. Furthermore, for 5 of the 'fast' muscles from each animal species (extensor digitorum longus; flexor digitorum and hallucis longus; gastrocnemius medialis; peroneus longus; tibialis anterior), several more proximal and distal cross-sectional levels were also analysed. All the investigated 'fast' muscles showed a significant degree of topographical eccentricity in the midlevel distribution of type I fibres. For most muscles, the direction of this 'vector regionalisation' of type I fibres was similar between the three animal species. For homologous muscles, the degree of vector regionalisation was significantly different: mouse > rat > rabbit. The relative area of the region containing the type I fibres, inversely related to the degree of 'area regionalisation', was also significantly different: mouse < rat < rabbit. Also within each animal species, muscles with a marked degree of vector regionalisation tended to show a marked area regionalisation. Proximo-distal differences in type I fibre density were observed in all the three species of animals; also these patterns showed marked inter-species differences. The findings demonstrate the general occurrence of, and systematic relationships between, different aspects of type I fibre regionalisation. The observed interspecies differences suggest that the expression of this phenomenon is adapted to differing functional needs.     

Imaging of non-parabolic velocity profiles in converging flow with optical coherence tomography

The optical coherence tomography method was explored for two-dimensional flow mapping of a highly scattering fluid in flow with complex geometry. Converging flow (capillary entry) with 4:1 constriction was used for demonstration of non-invasive and remote methods of mapping varying velocity profiles. Downstream of the geometry was scanned with approximately 10 x 10 x 10 microm3 spatial resolution and structural imaging of the lumen and images of one particular velocity were acquired. Stable concave, blunted and parabolic profiles are obtained at different distances of the inlet length. Application of the technique for the blood circulation is also discussed.     

Do shorter wavelengths improve contrast in optical mammography?

The detection of tumours with time-resolved transmittance imaging relies essentially on blood absorption. Previous theoretical and phantom studies have shown that both contrast and spatial resolution of optical images are affected by the optical properties of the background medium, and high absorption and scattering are generally beneficial. Based on these observations, wavelengths shorter than presently used (680-780 nm) could be profitable for optical mammography. A study was thus performed analysing time-resolved transmittance images at 637, 656, 683 and 785 nm obtained from 26 patients bearing 16 tumours and 15 cysts. The optical contrast proved to increase upon decreasing wavelengths for the detection of cancers in late-gated intensity images, with higher gain in contrast for lesions of smaller size (<1.5 cm diameter). For cysts either a progressive increase or decrease in contrast with wavelength was observed in scattering images.     

Late emergence of Thy-1 on climbing fibres demonstrates a gradient of maturation from the fissures to the folial convexities in developing rat cerebellum

Summary In the third week of postnatal life, Thy-1 staining of Purkinje cells starts to decrease, first in the depths of the fissures and then progressively, over the next 10 weeks, up the walls of the fissures to the convexities of the folia. This is accompanied by a far more striking appearance of high levels of Thy-1 on a network of fibres whose distribution strongly suggests they are climbing fibres. They acquire the antigen in the same topographical gradient of maturation, and at the same time, as Purkinje cells lose it. That these are climbing fibres was confirmed by destroying the inferior olive with 3-acetylpyridine, which also eliminated the intense Thy-1 staining in the cerebellum. At a stage (18 days) when only some climbing fibres in the molecular layer are seen to be Thy-1-positive, only a proportion of inferior olivary neurons are Thy-1-posirive with intense antigen labelling over the Nissl substance. The possibility that Thy-1 also appears on mossy fibres, and at relatively low levels on parallel fibres, is discussed.     

Diameter measurement from images of fluorescent cylinders embedded in tissue

Absorption and scattering of light by tissue as well as limitations in the resolution of the optical system influence the appearance of tissue embedded objects in fluorescence images and may reduce the accuracy of measurements from these images. Although the principles of light scattering in tissue and optical resolution are well known, the interplay between the two in fluorescence imaging in an imaging cryomicrotome is not well understood. In this paper we present and investigate an image formation model in a reflection geometry, like an imaging cryomicrotome, that takes both light scattering by tissue as well as the point spread function of the lens into account. The validity of the model was investigated by comparison of diameter estimates of fluorescent cylinders as obtained from images acquired in a reflection geometry with those estimated from simulated images. The results reveal that diameter values estimated from the simulated images are in excellent agreement with the experimental estimates. Our approach in modeling the image formation process of embedded fluorescent structures allows for the prediction of accuracy of quantitative estimates from fluorescence images. The relationship between imaging parameters and bias can be applied to arrive at accurate diameter estimates of near cylindrical structures like blood vessels.     

Organization of the projections from the subiculum to the ventral striatum in the rat. A study using anterograde transport of Phaseolus vulgaris leucoagglutinin

The projections of the subiculum, as the main output structure of the hippocampal formation, to the striatum were studied in the rat using the anterograde tracer Phaseolus vulgaris leucoagglutinin. It appears that not only the entire nucleus accumbens, part of the so-called ventral striatum, receives fibres from the subiculum, but that the hippocampal projection area in the striatum includes also the most medial, ventral, rostral and caudal parts of the caudate-putamen complex. Moreover, a relatively small number of fibres and terminals are present in the striatal elements of the medial part of the olfactory tubercle. The projections to the ventral and caudal parts of the caudate-putamen are predominantly derived from the ventral subiculum, whereas the projections to the rostral part of the caudate-putamen are derived from the dorsal subiculum. Furthermore, with respect to the subiculum-accumbens pathway a topographical organization could be established. Thus, the ventral or temporal part of the subiculum projects predominantly to the caudomedial part of the nucleus accumbens, and to a lesser degree to its rostromedial portion, whereas progressively more dorsal or septal parts of the subiculum send fibres to successively more lateral and rostral portions of the nucleus accumbens. Very sparse projections are found to the contralateral nucleus accumbens, arranged in a topographical manner similar to the ipsilateral projections. An important observation with respect to the structure of the nucleus accumbens is that the subicular terminations are inhomogeneously distributed, although a relation with earlier described mosaic patterns in the connectivity and neurochemical composition of the nucleus is not yet clear. Subicular fibres have their densest terminations in relatively cell-poor regions of the nucleus accumbens, and in particular tend to avoid small cell clusters.     

Ultrahigh-resolution imaging of human donor cornea using full-field optical coherence tomography

A feasibility study of ultrahigh-resolution full-field optical coherence tomography (FF-OCT) for a subcellular-level imaging of human donor corneas is presented. The FF-OCT system employed in this experiment is based on a white light interference microscope, where the sample is illuminated by a thermal light source and a horizontal cross-sectional (en face) image is detected using a charge coupled device (CCD) camera. A conventional four-frame phase-shift detection technique is employed to extract the interferometric image from the CCD output. A 95-nm-broadband full-field illumination yields an axial resolution of 2.0 microm, and the system covers an area of 850 microm x 850 microm with a transverse resolution of 2.4 microm using a 0.3-NA microscope objective and a CCD camera with 512 x 512 pixels. Starting a measurement from the epithelial to the endothelial side, a series of en face images was obtained. From detected en face images, the epithelial cells, Bowman's layer, stromal keratocyte, nerve fiber, Descemet's membrane, and endothelial cell were clearly observed. Keratocyte cytoplasm, its nuclei, and its processes were also separately detected. Two-dimensional interconnectivity of the keratocytes is visualized, and the keratocytes existing between collagen lamellaes are separately extracted by exploiting a high axial resolution ability of FF-OCT.     

Imaging using parallel integrals in optical projection tomography

We develop and demonstrate improved image-forming optics for optical projection tomography (OPT), with which the parallel integral throughout an object can be obtained. This method results in an improved resolution for OPT images, especially for the cross sections far from the optical axis of the image-forming optics. We find the optimal configuration used in our OPT system by use of a point spread function and simulation technique. The new method is validated by both numerical simulations and experimental results. The spatial resolution of the OPT system presented is approximately 40 microm.     

Radioluminescence of some optical fibres

Measurement of radiation fields with small scintillators as detectors for radiation dose is an attractive method provided that the scintillator luminescence can be discriminated against other unwanted signals. In diagnostic radiology radioluminescence of the optical fibres required for a transmission of the luminescence to an optical detector is the only source for such an impairment of the signal. To find fibres with low radioluminosity a survey of ten commercial fibres including polystyrene (PS), polystyrene wavelength shifting fibres, poly-methylmetacrylate (PMMA) and silica as fibre core material was made. Measurements were made with two photomultipliers of different spectral response and a clinical x-ray unit. Lowest radioluminosity was obtained with PMMA fibres. Depending on spectral sensitivity of the multiplier clear PS fibres give five to ten times the luminescence of PMMA. From the silica fibres only one high-OH fibre gave comparable results for wavelength up to 520 nm. Wavelength shifting fibres and silica fibres with low OH-content show strong fluorescence and are not suitable fibre materials.     

Performance evaluation of an improved optical computed tomography polymer gel dosimeter system for 3D dose verification of static and dynamic phantom deliveries

The performance of a next-generation optical computed tomography scanner (OCTOPUS-5X) is characterized in the context of three-dimensional gel dosimetry. Large-volume (2.2 L), muscle-equivalent, radiation-sensitive polymer gel dosimeters (BANG-3) were used. Improvements in scanner design leading to shorter acquisition times are discussed. The spatial resolution, detectable absorbance range, and reproducibility are assessed. An efficient method for calibrating gel dosimeters using the depth-dose relationship is applied, with photon- and electron-based deliveries yielding equivalent results. A procedure involving a preirradiation scan was used to reduce the edge artifacts in reconstructed images, thereby increasing the useful cross-sectional area of the dosimeter by nearly a factor of 2. Dose distributions derived from optical density measurements using the calibration coefficient show good agreement with the treatment planning system simulations and radiographic film measurements. The feasibility of use for motion (four-dimensional) dosimetry is demonstrated on an example comparing dose distributions from static and dynamic delivery of a single-field photon plan. The capability to visualize three-dimensional dose distributions is also illustrated.     

Comprehensive data visualization for high resolution endovascular carotid arterial wall imaging

Carotid angioplasty and stenting is a minimally invasive endovascular procedure that may benefit from in vivo high resolution imaging for monitoring the physical placement of the stent and potential complications. The purpose of this pilot study was to evaluate the ability of optical coherence tomography to construct high resolution 2D and 3D images of stenting in porcine carotid artery. Four Yorkshire pigs were anaesthetized and catheterized. A state-of-the-art optical coherence tomography (OCT) system and an automated injector were used to obtain both healthy and stented porcine carotid artery images. Data obtained were then processed for visualization. The state-of-the-art OCT system was able to capture high resolution images of both healthy and stented carotid arteries. High quality 3D images of healthy and stented carotid arteries were constructed, clearly depicting vessel wall morphological features, stent apposition and thrombus formation over the inserted stent. The results demonstrate that OCT can be used to generate high quality 3D images of carotid arterial stents for accurate diagnosis of stent apposition and complications under appropriate imaging conditions.     

Longitudinal extent of dorsal root fibres in the spinal cord and brain stem of the frog

The longitudinal arrangement of dorsal root fibres was investigated with a modified cobalt labelling technique in the spinal cord and brain stem of frogs. The topographical order of dorsal root fibres in the dorsal white column closely resembles the well-known scheme of the mammalian spinal cord. A significant difference between frogs and mammals is the extension of fibres up to the cerebellar plate. The ascending fibres of different origin are organized in concentric rings in the medulla. An oval-shaped area and a triangular area in the dorsal horn, and the motor horn, receive fibre collaterals in the spinal cord. Thoracic dorsal root fibres terminate exclusively in the oval-shaped area. Fibre terminations clearly outline the dorsal column nuclei which begin in the obex region and end at the level of the glossopharyngeal nucleus. The spinal nucleus of the trigeminus is richly supplied by both thin and thick calibre dorsal root fibres in its entire rostrocaudal extension. Two parts of the reticular formation receive dorsal root fibres; the first is in the dorsal gray matter ventral and lateral to the solitary fascicle in the medulla, the second is the lateral reticular zone. In the vestibular region, the medial, lateral and superior vestibular nuclei are innervated by dorsal root fibres. The granular layer of the cerebellum receives a significant contingent of dorsal root fibres. Fibres terminating in the vestibular region and in the cerebellum arise from limb-innervating spinal ganglia.The results indicate a close similarity in the longitudinal arrangement of dorsal root fibres in frogs and in higher vertebrates. The several collaterals that terminate in the hindbrain may modulate the function of the receiving structures. On the basis of present and previous findings the aggregation of primary sensory fibres and the convergence of their terminations are surveyed in the hindbrain.     

Three-dimensional optical topography of brain activity in infants watching videos of human movement

We present 3D optical topography images reconstructed from data obtained previously while infants observed videos of adults making natural movements of their eyes and hands. The optical topography probe was placed over the temporal cortex, which in adults is responsible for cognitive processing of similar stimuli. Increases in oxyhaemoglobin were measured and reconstructed using a multispectral imaging algorithm with spatially variant regularization to optimize depth discrimination. The 3D optical topography images suggest that similar brain regions are activated in infants and adults. Images were presented showing the distribution of activation in a plane parallel to the surface, as well as changes in activation with depth. The time-course of activation was followed in the pixel which demonstrated the largest change, showing that changes could be measured with high temporal resolution. These results suggest that infants a few months old have regions which are specialized for reacting to human activity, and that these subtle changes can be effectively analysed using 3D optical topography.     

Monitoring the process of pulmonary melanoma metastasis using large area and label-free nonlinear optical microscopy

We performed large area nonlinear optical microscopy (NOM) for label-free monitoring of the process of pulmonary melanoma metastasis ex vivo with subcellular resolution in C57BL/6 mice. Multiphoton autofluorescence (MAF) and second harmonic generation (SHG) images of lung tissue are obtained in a volume of ≈ 2.2 mm × 2.2 mm × 30 μm. Qualitative differences in morphologic features and quantitative measurement of pathological lung tissues at different time points are characterized. We find that combined with morphological features, the quantitative parameters, such as the intensity ratio of MAF and SHG between pathological tissue and normal tissue and the MAF to SHG index versus depth clearly shows the tissue physiological changes during the process of pulmonary melanoma metastasis. Our results demonstrate that large area NOM succeeds in monitoring the process of pulmonary melanoma metastasis, which can provide a powerful tool for the research in tumor pathophysiology and therapy evaluation.     

Photoacoustic imaging with deconvolution algorithm

The impulse response of the ultrasonic transducer used for detection is crucial for photoacoustic imaging with high resolution. We demonstrate a reconstruction method that allows the optical absorption distribution of a sample to be reconstructed without knowing the impulse response of the ultrasonic transducer. A convolution relationship between photoacoustic signals measured by an ultrasound transducer and optical absorption distribution is developed. Based on this theory, the projection of the optical absorption distribution of a sample can be obtained directly by deconvolving the recorded PA signal originating from a point source out of that from the sample. And a modified filtered back projection algorithm is used to reconstruct the optical absorption distribution. We constructed a photoacoustic imaging system to validate the reconstruction method and the experimental results demonstrated that the reconstructed images agreed well with the original phantom samples. The spatial resolution of the system reaches 0.3 mm.     

Time-resolved diffuse optical tomography and its application to in vitro and in vivo imaging

This work reviews our research during the past several years on time-resolved (TR) near-infrared diffuse optical tomography (DOT). Following an introduction of the measuring modes, two proposed schemes of image reconstruction in TR-DOT are described: one utilizes the full TR data, and the other, referred to as the modified generalized pulse spectrum technique (GPST), uses the featured data extracted from the TR measurement. The performances of the two algorithms in quantitativeness and spatial resolution are comparatively investigated with 2-D simulated data. TR-DOT images are then presented for phantom experiments, which are obtained by using a 16-channel time-correlated single photon counting system, and the factors affecting the quantification of the reconstruction are discussed. Finally, in vitro and in vivo imaging examples are illustrated for validating the capibility of TR-DOT to provide not only the anatomical but also the physiological information of the objects.