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1.
    
This work describes application of coherent anti‐Stokes Raman scattering (CARS) microscopy technique for analytical characterization of microstructured materials based on chitosan. We demonstrate that nitrogen–hydrogen vibration band in the high wavenumber region of CARS spectrum prevails over response from oxygen–hydrogen vibrations and can be used as a spectral marker of chitosan. The chemically selective imaging is experimentally demonstrated by applying CARS microscopy to discriminate between chitosan and polystyrene microparticles. CARS microscopy was shown to be a valuable tool for characterization of polluted chitosan fibre from utilized engine filter material. A possibility to observe foreign material pieces on the surface of the polluted chitosan fibre is demonstrated and discussed.  相似文献   

2.
Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.  相似文献   

3.
We demonstrate the application of coherent anti-Stokes Raman scattering microscopy for the rapid, label-free chemical imaging of waterborne pathogens. Chemically selective images of cryptosporidium were acquired in just a few seconds using coherent anti-Stokes Raman scattering microscopy, demonstrating its capability for the rapid detection of cryptosporidium at the single oocyst level. We discuss the applicability of such a technique in a near-real time automated water testing system.  相似文献   

4.
    
We demonstrate for the first time the imaging of unstained breast tissue biopsies using third‐harmonic generation (THG) microscopy. As a label‐free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour‐associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright‐field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.  相似文献   

5.
    
Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ~480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ~26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.  相似文献   

6.
    
Coherent anti‐Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high‐power picosecond fiber‐based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber‐based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7–8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm?1. We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro‐crustaceans), on which we distinguish morphological details and lipid reserves. Microsc. Res. Tech. 77:422–430, 2014. © 2014 Wiley Periodicals, Inc.  相似文献   

7.
  总被引:1,自引:1,他引:0  
Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.  相似文献   

8.
Measuring medium‐range order is a challenging and important problem in the structural study of disordered materials. We have developed a new technique, fluctuation x‐ray microscopy, that offers quantitative insight into medium‐range correlations in disordered materials at nanometre and larger length scales.In this technique, which requires a spatially coherent x‐ray beam, a series of speckle patterns are measured at a large number of locations in a sample using various illumination sizes. Examination of the speckle variance as a function of the illumination spot size allows the structural correlation length to be measured. To demonstrate this technique we have studied polystyrene latex spheres, which serve as a model for a dense random‐packed glass, and for the first time have measured the correlation length in a disordered system by fluctuation X‐ray microscopy. We discuss data analysis and procedures to correct for shot noise and detector noise. This approach could be used to explore medium‐range order and subtle spatial structural changes in a wide range of disordered materials, from soft matter to nanowire arrays, semiconductor quantum dot arrays and magnetic materials.  相似文献   

9.
Sun CK  Chu SW  Tai SP  Keller S  Abare A  Mishra UK  DenBaars SP 《Scanning》2001,23(3):182-192
Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.  相似文献   

10.
    
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11.
    
In this study, second‐harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low‐grade intraepithelial neoplasia to high‐grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second‐harmonic imaging microscopy has the potential in label‐freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.  相似文献   

12.
    
Nonlinear optical microscopy (NLOM) was used as a noninvasive and label‐free tool to detect and quantify the extent of the cartilage recovery. Two cartilage injury models were established in the outer ears of rabbits that created a different extent of cartilage recovery based on the presence or absence of the perichondrium. High‐resolution NLOM images were used to measure cartilage repair, specifically through spectral analysis and image texture. In contrast to a wound lacking a perichondrium, wounds with intact perichondria demonstrated significantly larger TPEF signals from cells and matrix, coarser texture indicating the more deposition of type I collagen. Spectral analysis of cells and matrix can reveal the matrix properties and cell growth. In addition, texture analysis of NLOM images showed significant differences in the distribution of cells and matrix of repaired tissues with or without perichondrium. Specifically, the decay length of autocorrelation coefficient based on TPEF images is 11.2 ± 1.1 in Wound 2 (with perichondrium) and 7.5 ± 2.0 in Wound 1 (without perichondrium), indicating coarser image texture and faster growth of cells in repaired tissues with perichondrium (p < 0.05). Moreover, the decay length of autocorrelation coefficient based on collagen SHG images also showed significant difference between Wound 2 and 1 (16.2 ± 1.2 vs. 12.2 ± 2.1, p < 0.05), indicating coarser image texture and faster deposition of collagen in repaired tissues with perichondrium (Wound 2). These findings suggest that NLOM is an ideal tool for studying cartilage repair, with potential applications in clinical medicine. NLOM can capture macromolecular details and distinguish between different extents of cartilage repair without the need for labelling agents.  相似文献   

13.
    
Laser‐scanning imaging techniques are frequently used to probe the molecule spatial orientation in a sample of interest by exploiting selection rules depending on the polarisation of the excitation light. For the successful implementation of these techniques the precise control of the polarisation at the sample level is of fundamental importance. Polarisation distortions induced by the optical elements are often the main limitation factor for the maximum size of the field‐of‐view in polarisation‐resolved (PR) laser‐scanning microscopy, since for large scanning angles the polarisation distortions may mask the real sample structure. Here we shall demonstrate the implementation of large‐field‐of‐view PR microscopy and show PR CARS imaging of mouse spinal cord thanks to a careful design of the laser‐beam optical path. We shall show that this design leads to strongly suppressed distortions and quantify their effects on the final images. Although the focus of this work is on CARS imaging, we stress that the approaches described here can be successfully applied to a wide range of PR laser‐scanning techniques.  相似文献   

14.
    
In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

15.
ZnO薄膜非线性光学特性的实验研究   总被引:1,自引:1,他引:0       下载免费PDF全文
利用金属有机化学气相沉积(MOCVD)技术在蓝宝石衬底上生长一层高质量的ZnO薄膜。为了考察沉积温度对样品的非线性特性的影响,在200~500℃生长了一系列ZnO薄膜。用X射线衍射谱(XRD)及扫描电镜(SEM)对样品结构进行了评价。以Nd:YAG激光器输出的1.06μm的激光为基频光,对ZnO薄膜样品的二阶及三阶非线性光学特性进行了实验研究。实验发现,对于250℃沉积温度的样品有较强的非线性效应,实验测得的二阶非线性极化张量χ(2)ZZZ=9.2pm/V,三阶有效非线性系数χ(3)=5.28×10-20m2/V2。  相似文献   

16.
    
Strained silicon (Si) technology enables improvements in complementary metal oxide semiconductor (CMOS) performance and functionality via replacement of the bulk, cubic-crystal Si substrate with an Si substrate that contains a tetragonally distorted, biaxially strained Si thin film at the surface. Here we use Raman spectroscopy to allow us to characterise growth processes of strained si, and to characterise the resulting level of strain/stress in the si and the effect it has on the underlying layer of graded SiGe.  相似文献   

17.
    
We describe a novel two‐photon fluorescence microscopy system capable of producing high‐quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes. Microsc. Res. Tech. 77:368–373, 2014. © 2014 Wiley Periodicals, Inc.  相似文献   

18.
    
Huang Z  Zhuo S  Chen J  Chen R  Jiang X 《Scanning》2008,30(6):452-456
The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.  相似文献   

19.
    
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20.
    
Papulonodular mucinosis (PM) is a cutaneous clue to the presence and activity of silent lupus erythematosus (LE), but the exact pathogenesis is still under secret. Moreover, the currently available treatments for PM are not satisfactory. To demonstrate the possibility of multiphoton microscopy (MPM) to trace the pathological state of PM and evaluate the treatment efficacy, epidermal and dermal alteration in skin lesion with PM before and after treatment was examined using MPM. Microstructure of epidermis as well as content and distribution of collagen and elastin in dermis were quantified to characterize the pathological states of PM. The results showed significant morphological difference between skin lesion before and after treatment, indicating the possibility of MPM to assess the therapeutic efficacy. With the advancement on MPM miniaturization and enhancement of contrast and depth of imaging, the MPM technique can be applied in in vivo tracking PM formation and progression, and leading the better understanding the PM pathogenesis and mechanism of response to any treatment, helping to establish novel effective therapies for PM. SCANNING 35:22‐27, 2013. © 2012 Wiley Periodicals, Inc.  相似文献   

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