Multidimensional custom-made non-linear microscope: from ex-vivo to in-vivo imaging

We have built a custom-made multidimensional non-linear microscope equipped with a combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging. The optical system was mounted on a vertical honeycomb breadboard in an upright configuration, using two galvo-mirrors relayed by two spherical mirrors as scanners. A double detection system working in non-descanning mode has allowed both photon counting and a proportional regime. This experimental setup offering high spatial (micrometric) and temporal (sub-nanosecond) resolution has been used to image both ex-vivo and in-vivo biological samples, including cells, tissues, and living animals. Multidimensional imaging was used to spectroscopically characterize human skin lesions, as malignant melanoma and naevi. Moreover, two-color detection of two photon excited fluorescence was applied to in-vivo imaging of living mice intact neocortex, as well as to induce neuronal microlesions by femtosecond laser burning. The presented applications demonstrate the capability of the instrument to be used in a wide range of biological and biomedical studies. PACS  87.64.mn; 78.47.Cd; 87.19.lw     

In‐line Bragg magnifier based on V‐shaped germanium crystals

In this work an X‐ray imaging system based on a recently developed in‐line two‐dimensional Bragg magnifier composed of two monolithic V‐shaped crystals made of dislocation‐free germanium is presented. The channel‐cut crystals were used in one‐dimensional and in two‐dimensional (crossed) configurations in imaging applications and allowed measurement of phase‐contrast radiograms both in the edge‐enhanced and in the holographic regimes. The measurement of the phase gradient in two orthogonal directions is demonstrated. The effective pixel size attained was 0.17 µm in the one‐dimensional configuration and 0.5 µm in the two‐dimensional setting, offering a twofold improvement in spatial resolution over devices based on silicon. These results show the potential for applying Bragg magnifiers to imaging soft matter at high resolution with reduced dose owing to the higher efficiency of Ge compared with Si.     

Experimental and theoretical studies on the one‐photon and two‐photon properties of a series of carbazole derivatives containing styrene

Symmetric‐type carbazole derivatives show great potential for application in two‐photon absorption (TPA) materials and organic light‐emitting diodes. The absorption spectra and fluorescence emission spectra of three different N‐alkyl symmetric‐type carbazole derivatives were investigated. The density functional theory (DFT) time‐dependent‐DFT//Becke, three‐parameter, Lee–Yang–Parr/6‐31 G* method has been used to theoretically study one‐photon absorption properties. The computational results are in good agreement with the available experimental values. The two‐photon excited fluorescence of the compounds was surveyed by 120 fs pulse at 790 nm Ti: sapphire laser operating at 1 kHz repetition rate. Two‐photon excited fluorescence was obtained in the range of 380–600 nm, and TPA cross‐sections were calculated. The TPA properties of the series of compounds were investigated by the ZINDO/single and double electronic excitation configuration interaction method. The influence of the chemical structure of the compounds on two‐photon optical properties was discussed. The results show how the different changes in one‐photon absorption and TPA properties on the basis of lengthening the conjugated bridge and the different carbazole N‐alkyl substituents are attributed to the transition dipole moment in the excited process. Copyright © 2011 John Wiley & Sons, Ltd.     

A multiplexed high‐resolution imaging spectrometer for resonant inelastic soft X‐ray scattering spectroscopy

The optical design of a two‐dimensional imaging soft X‐ray spectrometer is described. A monochromator will produce a dispersed spectrum in a narrow vertical illuminated stripe (～2 µm wide by ～2 mm tall) on a sample. The spectrometer will use inelastically scattered X‐rays to image the extended field on the sample in the incident photon energy direction (vertical), resolving the incident photon energy. At the same time it will image and disperse the scattered photons in the orthogonal (horizontal) direction, resolving the scattered photon energy. The principal challenge is to design a system that images from the flat‐field illumination of the sample to the flat field of the detector and to achieve sufficiently high spectral resolution. This spectrometer provides a completely parallel resonant inelastic X‐ray scattering measurement at high spectral resolution (～30000) over the energy bandwidth (～5 eV) of a soft X‐ray absorption resonance.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of a sCMOS‐based high‐resolution imaging system

The detection system is a key part of any imaging station. Here the performance of the novel sCMOS‐based detection system installed at the ID17 biomedical beamline of the European Synchrotron Radiation Facility and dedicated to high‐resolution computed‐tomography imaging is analysed. The system consists of an X‐ray–visible‐light converter, a visible‐light optics and a PCO.Edge5.5 sCMOS detector. Measurements of the optical characteristics, the linearity of the system, the detection lag, the modulation transfer function, the normalized power spectrum, the detective quantum efficiency and the photon transfer curve are presented and discussed. The study was carried out at two different X‐ray energies (35 and 50 keV) using both 2× and 1× optical magnification systems. The final pixel size resulted in 3.1 and 6.2 µm, respectively. The measured characteristic parameters of the PCO.Edge5.5 are in good agreement with the manufacturer specifications. Fast imaging can be achieved using this detection system, but at the price of unavoidable losses in terms of image quality. The way in which the X‐ray beam inhomogeneity limited some of the performances of the system is also discussed.     

From anti‐Stokes photoluminescence to resonant Raman scattering in GaN single crystals and GaN‐based heterostructures

The progress on anti‐Stokes photoluminescence and Stokes and anti‐Stokes Raman scattering in GaN single crystals and GaN/AlN heterostructures is reviewed. Anti‐Stokes photoluminescence investigated in the past was primarily attributed to two‐photon absorption, three‐photon absorption, and phonon‐assisted absorption. On the other hand, anti‐Stokes Raman scattering was used to determine electron‐phonon scattering time and decay time constant for longitudinal‐optical phonons. In a typical high electron mobility transistor based on GaN/AlN heterostructures, strong resonances were reached for first‐order and second‐order Raman scattering processes. Therefore, both Stokes and anti‐Stokes Raman intensities were dramatically enhanced. The feasibility of laser cooling of a nitride structure has been demonstrated. Anti‐Stokes photoluminescence and Raman scattering have potential applications in upconversion lasers and laser cooling of nitride ultrafast electronic and optoelectronic devices.     

Full‐field X‐ray reflection microscopy of epitaxial thin‐films

Novel X‐ray imaging of structural domains in a ferroelectric epitaxial thin film using diffraction contrast is presented. The full‐field hard X‐ray microscope uses the surface scattering signal, in a reflectivity or diffraction experiment, to spatially resolve the local structure with 70 nm lateral spatial resolution and sub‐nanometer height sensitivity. Sub‐second X‐ray exposures can be used to acquire a 14 µm × 14 µm image with an effective pixel size of 20 nm on the sample. The optical configuration and various engineering considerations that are necessary to achieve optimal imaging resolution and contrast in this type of microscopy are discussed.     

Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

A plug‐and‐measure diagnostic tool for flux,resolution and beam size of a soft X‐ray beamline

A compact multi‐functional diagnostic tool has been installed for commissioning beamlines at the Taiwan Light Source. For a photon beam, the instrument can measure the photon flux, energy resolution and beam size, consecutively with a photodiode or gold mesh, a windowless gas cell and a movable fluorescence screen viewport. Two gratings with ruling densities of 350 and 1000 lines mm^?1 and dispersing photons of energies from 80 to 1200 eV were calibrated with a photon flux of 10¹¹ photon s^?1 at slit openings of 50 µm × 50 µm; a maximum resolving power of greater than 10000 was certified with an absorption spectra of gaseous samples. Pressure differences over four orders of magnitude were achieved between the ion chamber and the flux measurement chamber with a single capillary differential pumping stage. A sequence of beam profiles was measured by moving continuously in the vicinity of the nominal focal positions. This tool is useful in commissioning or trouble‐shooting at most beamlines in a synchrotron facility.     

Fiber‐based light sources for biomedical applications of coherent anti‐Stokes Raman scattering microscopy

During the past decade coherent anti‐Stokes Raman scattering (CARS) microscopy has evolved to one of the most powerful imaging techniques in the biomedical sciences, enabling the label‐free visualization of the chemical composition of tissue in vivo in real time. While the acquisition of high‐contrast images of single cells up to large tissue sections enables a wide range of medical applications from routine diagnostics to surgical guidance, to date CARS imaging is employed in fundamental research only, essentially because the synchronized multiple wavelength pulsed laser sources required for CARS microscopy are large, expensive and require regular maintenance. Laser sources based on optical fibers can overcome these limitations combining highest efficiency and peak powers with an excellent spatial beam profile and thermal stability. In this review we summarize the different fiber‐based approaches for laser sources dedicated to coherent Raman imaging, in particular active fiber technology and passive fiber‐based frequency conversion processes, i.e. supercontinuum generation, soliton self‐frequency shift and four‐wave mixing. We re‐evaluate the ideal laser parameters for CARS imaging and discuss the suitability of different laser concepts for turn‐key operation required for routine application in clinics.

     

X‐ray spectra by means of Monte Carlo simulations for imaging applications

X‐ray tubes have a broad range of applications worldwide, including several techniques for atomic physics, like X‐ray fluorescence, as well as for medical imaging, like computed tomography. The performances of X‐ray imaging detectors have shown to be significantly sensitive to the incident beam spectrum. Therefore, an accurate knowledge of the X‐ray beam becomes necessary for the emission source characterization and the whole imaging process comprehension. Direct measurements and suitable Monte Carlo simulations may be used to establish the X‐ray spectra. Dedicated Monte Carlo simulation routines, based on the PENELOPE code, have been developed to determine the Bremsstrahlung X‐ray spectra generated by conventional X‐ray tubes. The simulated spectra have been validated by comparison with the corresponding experimental data showing an overall good agreement. The incorporation of a suitably designed virtual grid allowed to assess the angular distribution of Bremsstrahlung yield, showing a remarkable anisotropy. In addition, a dedicated program has been developed for virtual imaging, which enables to perform suitable X‐ray absorption contrast images. Also, the developed program includes a user‐friendly graphic interface to allow the upload of required input parameters, which include setup arrangement, beam characteristics, sample properties and image simulation parameters (spatial resolution, tracks per run, etc.). The software includes dedicated subroutines which handle the physical process from X‐ray generation up to detector signal acquisition. The aim of the developed program is to perform virtual imaging by means of absorption contrast and using conventional X‐ray sources, which may be a useful tool for the study the X‐ray imaging techniques in several research fields as well as for educational purposes. The performed comparisons with experimental data have shown good agreement. The obtained results for X‐ray imaging may constitute useful information for the comprehension and improvement of X‐ray image quality, like absorption contrast optimization, detail visualization, definition and detectability. Copyright © 2010 John Wiley & Sons, Ltd.     

Giant Three‐Photon Absorption in Monolayer MoS2 and Its Application in Near‐Infrared Photodetection

For decades, there has been extensive research on exploring fundamental physical mechanisms for strong and fast optical nonlinearities. One of the important nonlinear‐optical mechanisms is multiphoton absorption which has a wide range of photonic applications. Herein, a theoretical model is proposed for three‐photon absorption (3PA) in monolayer MoS₂. The model shows that the 3PA coefficients are on the order of 0.1 cm³/GW². As compared to bulk semiconductors, these coefficients are enhanced by several orders of magnitude due to excitonic effects. Such exciton‐enhanced 3PA is validated by light‐intensity‐dependent photocurrent measurements on a monolayer MoS₂ photodetector with femtosecond laser pulses. These results lay both theoretical and experimental foundation for developing sensitive near‐infrared MoS₂‐based three‐photon detectors.     

Frequency‐sensitive switching control effect induced by two‐photon resonance in an EIT‐based layered medium

The optical response of an atomic vapor can be coherently manipulated by tunable quantum interference occurring in atomic transition processes. A periodic layered medium whose unit cells consist of a dielectric and an EIT (electromagnetically induced transparency) atomic vapor is designed for light propagation manipulation. Such an EIT‐based periodic layered medium exhibits a flexible frequency‐sensitive optical response, where a very small change in probe frequency can lead to a drastic variation of reflectance and transmittance. As the destructive quantum interference relevant to two‐photon resonance arises in EIT atoms interacting with both control and probe fields, the controllable optical processes that depend sensitively on the external control field will take place in this EIT‐based periodic layered medium. Such a frequency‐sensitive and field‐controlled optical behavior of reflection and transmission in the EIT photonic crystal can be applicable to designs of new devices such as photonic switches, photonic logic gates and photonic transistors, where one laser field can be controlled by the other one, and would have potential applications in the areas of integrated optical circuits and other related techniques (e.g., all‐optical instrumentations).     

High‐efficiency coherence‐preserving harmonic rejection with crystal optics

This work reports a harmonic‐rejection scheme based on the combination of Si(111) monochromator and Si(220) harmonic‐rejection crystal optics. This approach is of importance to a wide range of X‐ray applications in all three major branches of modern X‐ray science (scattering, spectroscopy, imaging) based at major facilities, and especially relevant to the capabilities offered by the new diffraction‐limited storage rings. It was demonstrated both theoretically and experimentally that, when used with a synchrotron undulator source over a broad range of X‐ray energies of interest, the harmonic‐rejection crystals transmit the incident harmonic X‐rays on the order of 10^?6. Considering the flux ratio of fundamental and harmonic X‐rays in the incident beam, this scheme achieves a total flux ratio of harmonic radiation to fundamental radiation on the order of 10^?10. The spatial coherence of the undulator beam is preserved in the transmitted fundamental radiation while the harmonic radiation is suppressed, making this scheme suitable not only for current third‐generation synchrotron sources but also for the new diffraction‐limited storage rings where coherence preservation is an even higher priority. Compared with conventional harmonic‐rejection mirrors, where coherence is poorly preserved and harmonic rejection is less effective, this scheme has the added advantage of lower cost and footprint. This approach has been successfully utilized at the ultra‐small‐angle X‐ray scattering instrument at the Advanced Photon Source for scattering, imaging and coherent X‐ray photon correlation spectroscopy experiments. With minor modification, the harmonic rejection can be improved by a further five orders of magnitude, enabling even more performance capabilities.     

Sub‐microsecond‐resolved multi‐speckle X‐ray photon correlation spectroscopy with a pixel array detector

Small‐angle X‐ray photon correlation spectroscopy (XPCS) measurements spanning delay times from 826 ns to 52.8 s were performed using a photon‐counting pixel array detector with a dynamic range of 0–3 (2 bits). Fine resolution and a wide dynamic range of time scales was achieved by combining two modes of operation of the detector: (i) continuous mode, where data acquisition and data readout are performed in parallel with a frame acquisition time of 19.36 µs, and (ii) burst mode, where 12 frames are acquired with frame integration times of either 2.56 µs frame^?1 or 826 ns frame^?1 followed by 3.49 ms or 1.16 ms, respectively, for readout. The applicability of the detector for performing multi‐speckle XPCS was demonstrated by measuring the Brownian dynamics of 10 nm‐radius gold and 57 nm‐radius silica colloids in water at room temperature. In addition, the capability of the detector to faithfully record one‐ and two‐photon counts was examined by comparing the statistical distribution of photon counts with expected probabilities from the negative binomial distribution. It was found that in burst mode the ratio of 2 s to 1 s is markedly smaller than predicted and that this is attributable to pixel‐response dead‐time.     

第二讲 光电子技术在保健医疗和生物学应用中的进展

文章论述了激光与光电子技术在人类保健、医疗以及生命科学应用中的作用和意义.综述了光活检技术、光美容医疗和生物光子技术等光电子技术在保健、医疗和生物学领域中的具体应用.重点介绍了：（1）用于组织病理诊断的光活检技术,其中包括光活检的发展历史、技术优点、研究现状,以及荧光光谱和成像技术等实用光活检技术的临床应用;（2）非消融性光疗的基本作用机制和研究进展;（3）扫描共焦显微术、多光子荧光显微术、近场光学扫描显微术和光镊等显微生物成像技术的工作原理和应用.最后,展望了激光与光电子技术在生命科学中的应用前景.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Controlling fluorescence emission with split‐ring‐resonator‐based plasmonic metasurfaces

Metasurfaces, which consist of resonant metamaterial elements in the form of two‐dimensional thin planar structures, retain great capabilities in manipulating electromagnetic wave and potential applications in modifying interaction with fluorescent molecules. The metasurfaces with magnetic responses are favorable to weakening fluorescence quenching while less investigated in controlling fluorescence. In this paper, we demonstrate control over fluorescence emission by engineering the magnetic and electric modes in plasmonic metasurfaces consisting of 45‐nm‐thick gold split‐ring‐resonators (SRRs). The fluorescence emission exhibits an enhancement factor of ∼18 and is predominantly x‐polarized with assistance of the magnetic mode excited by oblique incidence with an x‐polarized electric field. The magnetic and electric modes excited by oblique incidence with a y‐polarized electric field contribute to the rotation of emission polarization with respect to the incident polarization. The results demonstrate manipulating the interaction of fluorescent emitters with different resonant modes of the SRR‐based metasurface at the nanoscale by the polarization of incident light, providing potential applications of metasurfaces in a wide variety of areas, including optical nanosources, fluorescence spectroscopy and compact biosensors.

     

Near‐room‐temperature photon‐noise‐limited quantum well infrared photodetector

With the modern development of infrared laser sources such as broadly tunable quantum cascade lasers and frequency combs, applications of infrared laser spectroscopy are expected to become widespread. Consequently, convenient infrared detectors are needed, having properties such as fast response, high efficiency, and room‐temperature operation. This work investigated conditions to achieve near‐room‐temperature photon‐noise‐limited performance of quantum well infrared photodetectors (QWIPs), in particular the laser power requirement. Both model simulation and experimental verification were carried out. At 300 K, it is shown that the ideal performance can be reached for typical QWIP designs up to a detection wavelength of 10 µm. At 250 K, which is easily reachable with a thermoelectric Peltier cooler, the ideal performance can be reached up to 12 µm. QWIPs are therefore suitable for detection and sensing applications with devices operating up to or near room temperature.