Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.
Histological investigations of biological tissue benefited tremendously from staining different cellular structures
with various organic dyes. With the introduction of new imaging modalities such as second harmonic generation
(SHG) and third harmonic generation (THG) microscopy, the demand for novel dyes that enhance the harmonic
signals has arisen. The new labels with high molecular hyperpolarizability have recently been termed
harmonophores. In this study, we demonstrate that hematoxylin, the standard histological stain used in H&E
(hematoxylin and eosin) staining, enhances the microscopic THG signal. Hematoxylin has an affinity for
basophilic structures such as the cell nucleus, ribosomes and mitochondria, while eosin stains structures such as
the cytoplasm, collagen and red blood cells. The histological sections of H&E stained cancerous prostate tissue
found in transgenic adenocarcinoma of the mouse prostate (TRAMP) have been investigated with the multimodal
SHG, THG and multiphoton excitation fluorescence (MPF) microscope. Strong THG signal revealed intracellular
structures originating where the hematoxylin stain resides, while SHG imaging of the tissue showed the presence
of collagen fibrils in the extracellular matrix. The MPF was mostly present in the extracellular matrix. The
spectrally and temporally resolved MPF revealed that most of the fluorescence originates from the eosin. The
THG image did not correlate with MPF confirming that the harmonic signal originates from hematoxylin.
Multimodal nonlinear microscopy adds invaluable information about cellular structures to the widely used bright
field investigations of H&E stained histological sections, and can be efficiently used for morphological studies as
well as cancer diagnostics.
In recent years, semiconductor nanodots have been actively used for biolabeling. We propose using alternate composite nanostructures consisting of a semiconductor size-quantized core covered by a nanometer-thick Au shell, having two principal advantages over purely semiconducting nanodots: (i) reduction of toxicity due to a complete Au coverage of the cores containing potentially poisonous Cd, Se, or Pb; (ii) amplification of exciting and/or emitted light by plasmon effects in a metallic shell which will increase the imaging efficiency. Theoretical calculations show that the optical absorption and emission spectra have several peaks corresponding to interband transitions in the core, and the two plasmon modes in the Au shell. When the energy of interband transitions coincides with one of the plasmon peaks, the resonant electromagnetic field in the core is enhanced which should result in amplification of the luminescence
intensity. Especially effective amplification can be reached if the frequencies of the exciting and emitting light both match two plasmon peaks. Experimental measurements were performed with composite nanostructures containing CdSe-ZnS cores fabricated by the organo-metallic method, followed by deposition of the gold shell using thermal decomposition of a Au (I) precursor. These revealed a multimodal structure of the absorption and luminescence spectra, good tunability, high intensity, and narrow emission linewidth. The dependence of spectra on the thickness of Au shell was investigated. The measurements were performed in different biological media and demonstrated stability and environment-insensitivity - a prerequisite for biolabeling.
Chronic total occlusions (CTOs) are defined as complete occlusions of an artery older than one month. Minimally invasive catheter-based interventions commonly employed for partial occlusions (e.g., balloon angioplasty followed by stenting) are problematic in CTOs because of the phycisian's inability to pass the device through the occlusion without a significant risk of arterial wall perforations. Furthermore, successfully treated CTOs exhibit a high re-occlusion rate. As a result, these cases are mostly sent to bypass surgery. With the advent of drug-eluting stents that reduce the incidence of re-occlusion, and thus, eliminating the second problem, new devices have begun to emerge that aim to recanalize CTOs without the cost and trauma of bypass surgery. These devices, however, need effective image guidance methods to ensure successful crossing of the CTOs.
Optical coherence tomography (OCT) is being evaluated as an intravascular imaging modality for guiding catheter-based interventions of CTOs. Occluded ex vivo human arterial samples were used to produce longitudinal cross-sections using an OCT system. These OCT images were compared with histology to assess OCT's ability to identify different components of the occluded artery, evaluate the imaging depth, and determine the ability to detect the underlying vessel wall.
Given the inherent difficulties of creating a mechanically scanning OCT probe in the distal tip of a catheter for use in a stenotic artery, we directed our initial efforts towards developing a "motionless" fiber based OCT system using a single mode fiber array. We discuss design considerations for implementing a forward viewing intravascular OCT probe.
The work is devoted to photoluminescent investigation of arterial walls in order to create a new navigation method for minimally invasive treatment of cardiovascular decease in the presence of chronic total occlusions. The method uses the distinct photoluminescent properties of arterial wall and chronic total occlusion plaque to alert the interventionalist when a fiber-optic equipped catheter is in contact with the vessel wall. We conducted a study to compare the photoluminescence properties of healthy and stenosed vessel walls, and a typical chronic total occlusion plaque in the spectral range 300-700 nm. All samples were obtained from human tibial arteries. These groups of arterial samples showed easily differentiable luminescence amplitude and spectral characteristics. The photoluminescent properties of intact and intentionally damaged vessel walls were also investigated to permit detection of artery perforation that could take place during the revascularization. Using optical excitation of different wavelength gives additional opportunities of detecting arterial plaques requiring laser treatment. The results presented are complemented with micro-computed tomography images and histology of the segments analyzed.
Our aim is to use nanocrystals (NC) to study endothelial cell biology, in particular, the cell surface receptor Tie2. Tie2 is highly expressed in endothelial cells and is critical for angiogenesis and vascular maintenance. Conjugating NCs to the Tie2 ligands, the angiopoietins, and tracking their characteristic luminescence lines will allow us to study the regulation of Tie2 in vitro and in vivo. To study NC behavior in a relevant biological system, endothelial cells were grown and cultured in vitro. Two different types of NC were made and tested: two-color core-shell CdSe/ZnS and more complicated nanostructures containing also Au clusters. Measurements were performed in specially prepared media with different pH values, as well as in the cell growth medium. The positions of NC-related luminescence lines were not influenced by the kind of media which makes them suitable for biolabeling the cell surface receptor Tie2. At the same time, the relative magnitude of the NC peaks depends on the pH of the medium and can therefore be used for characterization of the latter. Confocal microscopic images show that NCs with different ligands demonstrate different distributions inside living cells.
In rotational tomotherapy a high energy (6 MeV) photon beam irradiates the patient. A CT detector placed behind the patient is used to establish the position of the patient and the dose delivered. A possible detector design uses amorphous selenium (a-Se) as the x-ray to charge conversion medium requiring a detailed investigation of the change in x-ray sensitivity due to exposure to radiation in a-Se. Our novel experimental method called x-ray time of flight provides instantaneous measurements of x-ray sensitivity and charge transport parameters in a-Se films. The method analyzes the current from a-Se samples in response to single short pulses of LINAC radiation. X-ray sensitivity was observed to decrease substantially with large exposures (e.g. more than 50% after 4 Gy accumulated over ~5min) and to completely recover over <48h. The mechanisms responsible were studied from the kinetics of the measured current waveforms. On the basis of experimental data, a model for dose dependent sensitivity was formulated taking into account carrier trapping, re-distribution of electric field in the a-Se film due to space charge and evolving recombination processes. In principle quantitative comparison of experimental and theoretical characteristics will permit the determination of the main material parameters (carrier mobility, deep trapping lifetime), as well as the generation rate of carriers by x-rays. Thus a basis for the development of a practical a-Se based megavoltage CT detector has been investigated.
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