Combined phase-sensitive acoustic microscopy and confocal laser scanning microscopy

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.     

Three-dimensional integral imaging of micro-objects

We propose a method for displaying micro-objects in space that is based on three-dimensional (3D) integral imaging, in which elemental images are calculated from a two-dimensional sampling of the optical field along different depths by use of confocal scanning microscopy. Experimental results are presented to demonstrate that a uniformly magnified 3D biological specimen can be displayed in space, and thus integral imaging can be used for 3D display of confocal microscopy. To the best of our knowledge, this is the first report of 3D integral imaging of (semitransparent) micro-objects.     

Sub-diffraction-limit imaging based on the topographic contrast of differential confocal microscopy

Using the nanometer depth sensitivity of differential confocal microscopy, we detect surface features of lateral dimensions smaller than the diffraction limit without fluorescence labeling. The lateral resolution of the topographic images is further enhanced by a maximum-likelihood estimation algorithm. Based on the comparison of signal and noise at high spatial frequency, we estimate the best lateral resolution of the enhanced images to be 0.15 lambda. In addition, on composite samples this technique can simultaneously display sub-diffraction-limit topographic features and reflectivity heterogeneity.     

A study of the image contrast in Fresnel zone plate imaging of extended sources

The image contrast is experimentally demonstrated to be a particular function of various parameters including the area of the source divided by the area of the central Fresnel zone. Over 150 images were analyzed and the best fit parameters are reported. It is also shown that the best contrast is obtained by a new technique in which the projection transparency is photographically reversed before image reconstruction.     

Quantitative studies of pyrocarbon‐coated materials using synchrotron radiation

Phase‐contrast imaging provides enhanced image contrast and is important for non‐destructive evaluation of structural materials. In this paper, experimental results on in‐line phase‐contrast imaging using a synchrotron source (ELETTRA, Italy) for objects required in material science applications are discussed. Experiments have been carried out on two types of samples, pyrocarbon‐coated zirconia and pyrocarbon‐coated alumina microspheres. These have applications in both reactor and industrial fields. The phase‐contrast imaging technique is found to be very useful in visualizing and determining the coating thickness of pyrocarbon on zirconia and alumina microspheres. The experiments were carried out at X‐ray energies of 16, 18 and 20 keV and different object‐to‐detector distances. The results describe the contrast values and signal‐to‐noise ratio for both samples. A comprehensive study was carried out to determine the thickness of the pyrocarbon coating on zirconia and alumina microspheres of diameter 500 µm. The advantages of phase‐contrast images are discussed in terms of contrast and resolution, and a comparison is made with absorption images. The results show considerable improvement in contrast with phase‐contrast imaging as compared with absorption radiography.     

Application of fuzzy c-means segmentation technique for tissue differentiation in MR images of a hemorrhagic glioblastoma multiforme

The application of a raw data-based, operator-independent MR segmentation technique to differentiate boundaries of tumor from edema or hemorrhage is demonstrated. A case of a glioblastoma multiforme with gross and histopathologic correlation is presented. The MR image data set was segmented into tissue classes based on three different MR weighted image parameters (T₁-, proton density-, and T₂-weighted) using unsupervised fuzzy c-means (FCM) clustering algorithm technique for pattern recognition. A radiological examination of the MR images and correlation with fuzzy clustering segmentations was performed. Results were confirmed by gross and histopathology which, to the best of our knowledge, reports the first application of this demanding approach. Based on the results of neuropathologic correlation, the application of FCM MR image segmentation to several MR images of a glioblastoma multiforme represents a viable technique for displaying diagnostically relevant tissue contrast information used in 3D volume reconstruction. With this technique, it is possible to generate segmentation images that display clinically important neuroanatomic and neuropathologic tissue contrast information from raw MR image data.     

Virtual non-contrast enhanced magnetic resonance imaging (VNC-MRI)

PurposeApplication of contrast agents (CA) is widely used in various clinical fields like oncology. Similar to approaches used in computed tomography, virtual non-contrast enhanced (VNC) images can be generated with the goal to supersede true non-contrast enhanced (TNC) images.MethodsIn MRI a T1-mapping sequence with variable flip angle (VFA) was used to acquire two images with different image contrast at the same time. To generate VNC images postprocessing based on this technique, an image-space based material decomposition algorithm was used. The inverse of a sensitivity matrix, consisting of intensity values for both VFA images and every material respectively, was used to determine the three material fractions and to calculate the final VNC images. The technique was tested on a 3 T scanner using a phantom and two in-vivo scans of patients with glioma and glioblastoma respectively. In all these cases the required six values were manually derived from the respective material or the background from both VFA images.ResultsPostprocessing results of the phantom show that the chosen materials can be separated and visualized individually and unwanted materials can be suppressed. In the VNC images of in-vivo scans the signal of the CA is removed successfully.ConclusionIt was shown that VNC images that match the visual impression of the TNC images can be generated, resulting in possibly reduced scan times and avoided mismatches due to movement of the patient.     

Improving contrast and sectioning power in confocal imaging by third harmonic generation in SiOx nanocrystallites

We present a new optical microscope in which the light transmitted by a sample-scanned transmission confocal microscope is frequency-tripled by SiOx nanocrystallites in lieu of being transmitted by a confocal pinhole. This imaging technique offers an increased contrast and a high scattered light rejection. It is demonstrated that the contrast close to the Sparrow resolution limit is enhanced and the sectioning power are increased with respect to the linear confocal detection mode. An experimental implementation is presented and compared with the conventional linear confocal mode.     

Gated magnetic resonance imaging of acute myocardial ischemia in dogs: application of multiecho techniques and contrast enhancement with GD DTPA

ECG gated magnetic resonance images were obtained in six canines prior to and immediately following occlusion of either the LAD or circumflex coronary artery using a surgically placed snare. Multiecho and single-echo acquisition techniques were utilized 0.25 mmol/kg Gd DTPA was injected as an IV bolus 1 hr following coronary artery ligation. In two animals, the region of ischemic myocardium was clearly visualized on multiecho technique without the use of intravenous contrast. The ischemic zone could be best identified on images with a long TE of 120 msec. Contrast enhancement with Gd DTPA enabled visualization of the ischemic myocardium in all six canines. Administration of Gd DTPA, a perfusion agent, improved both detectability and definition of the myocardial lesions.     

Fiber-optic confocal microscopy using a spatial light modulator

We present a novel fiber-optic confocal microscope in which the scanning operation is achieved by use of a spatial light modulator (SLM) to sequentially illuminate individual fibers or patterns of multiple fibers. Experimental images are presented, and the optical-sectioning capability of the device is demonstrated. The novel SLM-based system is more optically efficient, achieves higher contrast, and has improved optical-sectioning capabilities compared with those of other proposed instruments for confocal microendoscopy.     

Two‐Photon Imaging of a Cellular Line Using Organic Fluorescent Nanoparticles Synthesized by Laser Ablation

A comparative study of the optical properties of organic fluorescent nanoparticles fabricated by laser ablation (NPs‐LA), reprecipitation (NPs‐RP), and microemulsion (NPs‐ME) methods is presented. These nanoparticles contain a fluorene‐based p‐conjugated molecule (BT2). Distinctive electronic transitions are observed in samples due to the specific way in which the molecule BT2 is assembled in each type of nanoparticles; for instance, transitions involved in absorption and emission spectra of NPs‐LA result in blueshifting with respect to the molecular solution of BT2, whereas redshifting is observed in NPs‐RP and NPs‐ME. Further, the results show that under infrared excitation, the aqueous suspensions of NPs‐LA exhibit the highest fluorescence induced by two‐photon absorption (≈790 GM at 740 nm), as well as the best photostability, compared with aqueous suspensions of NPs‐RP and NPs‐ME. The nanoparticles synthetized by the three aforementioned methods are employed as exogenous agents for the visualization of human cervical cancer cell line (HeLa) using confocal and two‐photon microscopy. Under similar experimental conditions, it is found that microscopy images of the best quality are obtained with NPs‐LA. These results show that laser ablation is a suitable technique for the fabrication of organic fluorescent nanoparticles used as contrast agents for in vitro fluorescence microscopy.     

Ex vivo MRI in extracorporeal liver surgery

Extracorporeal resection of hepatic tumors that were considered inoperable in the past is now possible in selected cases. Such procedures require high-quality preoperative images for the exact delineation of the tumor extent and for an optimal planning of the line of parenchymal division. In-vivo CT and MRI can not always depict the tumor adequately. In such cases, ex-vivo MRI may be a useful additional technique. It combines a high spatial resolution with the best possible soft tissue contrast, as was learned from previous studies on donor livers destined for transplantation. Ex-vivo MRI favours both a sufficiently radical resection as well as sufficient hepatic functional reserve to be present for reimplantation. A case history is reported together with details on the technical procedure.     

High-contrast mapping of basal cell carcinomas

Because of low optical contrast in the visible spectral range, accurate detection of basal cell carcinomas (BCC) remains a challenging problem. In this letter, we experimentally demonstrate that reflectance confocal imaging in the vicinity of 1300 nm can be used for the detection of BCC without exogenous contrast agents. We present high-contrast reflectance confocal images of thick fresh skin tissues with clearly delineated cancer and discuss possible reasons for causing decreased scattering of BCC. Comparison with histopathology confirms that tumors scatter less and exhibit lower pixel values in the images, as compared to benign skin structures. The results demonstrate the feasibility of real-time noninvasive detection of BCC using intrinsic differences in scattering between tumors and normal skin.     

Some efficient methods to correct confocal images for easy interpretation

In this paper we have explained some efficient methods to correct artefacts in confocal laser beam scanning microscope (CLSM) images. The main aim is to enhance object features such that they become clearly visible for interactive evaluation and to reduce the overall noise so that the automatic segmentation and feature measurement can be done easily. A simple automatic-thresholding technique, and a straightforward method to restore the light intensity along the depth of the image stack are proposed. Another problem associated with the CLSM is the non-isotropic resolution. We have presented an interpolation technique based on XOR contouring and morphing to virtually insert the image slices in the image stack for improving the axial resolution. This interpolation technique has the merits of both contour- and intensity-based interpolations. Results of application of these methods on CLSM data are shown.     

一种实现共焦显微镜空间微分成像的新方法

提出了一种实现共焦显微镜空间微分成像的新方法。为了获得空间微分图像,首先利用时间分辨技术结合互补调制技术,获得两束相位相反的调制光,然后利用这两束相位相反的调制光结合共焦扫描技术,实现共焦显微镜的空间微分成像。实验表明:这种空间微分技术可以准确实现共焦显微镜的空间微分成像,从而获得成像物体的边缘轮廓和实现边缘增强。     

Spectrally encoded slit confocal microscopy

A simple and cost-effective method for real-time imaging in confocal microscopy is proposed. Spectrally encoded slit confocal microscopy (SESCoM) uses a spectral encoding technique together with a confocal slit aperture to achieve two-dimensional images. Simulation and experimental results of the SESCoM's axial and lateral performances are presented. The measured FWHM of the axial response is 1.15 mum when an objective with a NA of 0.95 is used. FWHMs of the lateral line spread functions are measured to be 236 and 244 nm along the x and y directions, respectively. Both the axial and the lateral experimental results agree well with the simulation results.     

双光子激光扫描显微镜中折射率失配引起的图象变形研究

双光子激光扫描显微镜(TPLSM)在观察生理溶液中的生物样品的结构时,水浸物镜是最好的选择.但是TPLSM在进行生物样品的三维结构观察时,需要样品和物镜在Z轴方向上做相对移动,这种移动很容易引起样品在水溶液中飘动.为了防止这种样品的漂移,往往要在水物镜和样品之间加入盖波片.由于盖波片和水及样品间的折射率失配将会导致TPLSM荧光图象的变形,给观察结果带来误差.本文主要利用TPLSM的外源探测器(ex-ternal detection)观察荧光小球的TPLSM的荧光图象,比较插入盖波片前后小球荧光图象的变化情况,研究水浸物镜在插入盖波片后折射率失配引起TPLSM图象的变形程度及共焦小孔对改进图象变形的作用.结果显示盖波片的折射率失配不仅导致TPLSM荧光图象的畸变,同时还导致TPLSM图象的荧光强度和信噪比的下降.进一步的研究表明共焦小孔可以部分改进由于折射率失配引起畸变的TPLSM荧光图象的质量.     

Comparison of FAIR technique with different inversion times and post contrast dynamic perfusion MRI in chronic occlusive cerebrovascular disease

The purpose of this study was to examine the signal change occurring with different inversion times (TIs) of the flow-sensitive alternating inversion recovery (FAIR) technique and to compare with the perfusion image obtained with Gd-DTPA injection. The subjects were 11 patients with unilateral occlusive cerebrovascular disease. Two FAIR images with different TIs (800 ms and 1600 ms) were measured for each patient and dynamic perfusion MRI was performed to produce four kinds of parameter maps: mean transit time (MTT), time to peak (TTP), relative cerebral blood flow (rCBF) and relative cerebral blood volume (rCBV) maps. Asymmetry ratios (ARs) between the affected and contra-lateral vascular sides were measured in both FAIR images and the four dynamic parameter maps. The AR of the MTT map of the four parameters showed the highest correlation with that of the FAIR images, especially with that of TI = 1600 ms (r = 0.829), and the AR of the rCBV map revealed the worst correlation with the FAIR images. The AR of the FAIR image with TI = 800 ms was less correlated with that of MTT than that with TI = 1600 ms. These results suggested that the signal intensity of the FAIR image was influenced by flow transition time and the change in TI could be used to select the flow with a different transition time. Our study suggested that a longer TI in the FAIR technique might be more useful than a shorter TI for evaluating chronic occlusive cerebrovascular disease in the clinical setting.     

衍射增强成像中的折射角与边界可见度

X射线衍射增强成像技术可以精确得到样品内部结构的折射角信息, 对提高轻元素物质的成像衬度有着重要的意义. 本文对楔型和圆形两种模型样品进行了DEI实验的研究, 对3种不同能量的X射线均精确得到了样品的折射角信息, 与理论计算值吻合的很好. 利用边界可见度(Edge Visibility)对楔形样品进行了定量的分析. 结果表明, 对于特定折射角的界面, 能精确计算其最佳可见度的位置, 并与实验结果吻合的较好. 最后对一块实际医学肿瘤样品边界处的可见度进行了定量的研究, 讨论了获得最佳成像衬度的条件.