Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging.     

Engineered microsphere contrast agents for optical coherence tomography

Contrast agents are utilized in virtually every imaging modality to enhance diagnostic capabilities. We introduce a novel class of optical contrast agent, namely, encapsulating microspheres, that are based not on fluorescence but on scattering nanoparticles within the shell or core. The agents are suitable for reflection- or scattering-based techniques such as optical coherence tomography, light microscopy, and reflectance confocal microscopy. We characterize the optical properties of gold-, melanin-, and carbon-shelled contrast agents and demonstrate enhancement of optical coherence tomography imaging after intravenous injection of such an agent into a mouse.     

Spatial and temporal coherence effects in interference microscopy and full‐field optical coherence tomography

Low‐coherence optical microscopy or optical coherence microscopy uses light with short coherence length. The well‐known case is: “white‐light interferometry”, which became recently more known as: “optical coherence tomography”. However, when lenses and microscope objectives are used to create interferometric images, in what is known classically as “interference microscopy” or today as “full‐field optical coherence tomography” the spatial coherence starts to play a critical role. In this article the coherence effects in low‐coherence optical microscopy are reviewed. As this technology is becoming increasingly publicized due to its importance in three‐dimensional imaging, particularly of scattering biological media and optical metrology, the understanding of the fundamental physics behind it is essential. The interplay between longitudinal spatial coherence and temporal coherence and the effects associated with them are discussed in detail particularly when high numerical apertures are used. An important conclusion of this study is that a high‐contrast, high‐resolution system for imaging of multilayered samples is the one that uses narrowband illumination and high‐NA objectives with an index‐matching fluid. Such a system, when combined with frequency‐domain operation, can reveal nearly real‐time three‐dimensional images, and is thus competitive with confocal microscopy.     

Multimodal nonlinear optical microscopy

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second‐harmonic generation, and coherent anti‐Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using a CARS microscope as a platform to develop more advanced NLO modalities such as electronic‐resonance‐enhanced four‐wave mixing, stimulated Raman scattering, and pump‐probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed.     

生物医学领域中超连续激光光谱技术的研究进展

超连续谱激光指的是当泵浦激光穿过特殊光波导时,一系列的非线性效应引起入射激光束的光谱展宽,从而输出宽光谱激光束。随着超快激光和光子晶体光纤技术的发展,利用超短脉冲在光子晶体光纤中的传播链产生相干的且亮度高的超连续谱激光成为了一种理想的白光源。自从超连续谱激光源投入应用以来,其应用领域越来越广。尤其在生物医学的细胞、血液等样品分析当中,荧光光谱学、流式细胞仪、共焦显微、光学相干层析等技术都是强有力的分析工具,在采用这些先进技术的科学仪器中,超连续谱激光源成为了一种主要光学部件。首先对超连续谱激光源的国际研究进展作了详细介绍,然后对超连续激光光谱技术在显微成像、流式细胞仪、荧光寿命成像显微、荧光共振能量转移、光学相干层析、共焦显微生物医学分析等生物医学领域中的发展及应用作了综合阐述。对超连续激光光谱技术在非接触式血液制品鉴别的需求、方案及研究进展进行了重点论述,包括覆盖400～2 000 nm光谱范围的光纤化轻型超连续谱激光光源研究;采用超连续谱激光光谱方法探索不同物种血液的种属特征;根据大数据的血液样品光谱特征元数据库分析建立数学模型,利用数学模型实现对血液样品种属光谱学判定;血液鉴别光谱分析便携式整机系统研发等。对超连续激光光谱技术在生物医学领域的应用前景作了展望。     

Improving signal levels in intravital multiphoton microscopy using an objective correction collar

Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.     

Near-infrared dyes as contrast-enhancing agents for spectroscopic optical coherence tomography

Optical coherence tomography (OCT) images of biological tissues often have low contrast. Spectroscopic optical coherence tomography (SOCT) methods have been developed to enhance contrast but remain limited because most tissues are not spectrally active in the frequency bands of laser sources commonly used in OCT. Near-infrared (NIR) dyes with absorption spectra features within the OCT source spectrum can be used for enhancing contrast in this situation. We introduce and demonstrate the use of NIR dyes as contrast agents for SOCT. Contrast-enhanced images are compared with fluorescence microscopy, demonstrating a link between SOCT and fluorescence imaging.     

Photoacoustic microscopy

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high‐resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering‐based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence‐based methods, such as wide‐field, confocal, and multi‐photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state‐of‐the‐art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies.     

Programmable Colored Illumination Microscopy (PCIM): A practical and flexible optical staining approach for microscopic contrast enhancement

Programmable colored illumination microscopy (PCIM) has been proposed as a flexible optical staining technique for microscopic contrast enhancement. In this method, we replace the condenser diaphragm of a conventional microscope with a programmable thin film transistor-liquid crystal display (TFT-LCD). By displaying different patterns on the LCD, numerous established imaging modalities can be realized, such as bright field, dark field, phase contrast, oblique illumination, and Rheinberg illuminations, which conventionally rely on intricate alterations in the respective microscope setups. Furthermore, the ease of modulating both the color and the intensity distribution at the aperture of the condenser opens the possibility to combine multiple microscopic techniques, or even realize completely new methods for optical color contrast staining, such as iridescent dark-field and iridescent phase-contrast imaging. The versatility and effectiveness of PCIM is demonstrated by imaging of several transparent colorless specimens, such as unstained lung cancer cells, diatom, textile fibers, and a cryosection of mouse kidney. Finally, the potentialities of PCIM for RGB-splitting imaging with stained samples are also explored by imaging stained red blood cells and a histological section.     

Miniaturization and defocus correction for objective-coupled planar illumination microscopy

Recently, a light sheet-based technique called objective-coupled planar illumination (OCPI) microscopy [Holekamp et al., Neuron 57, 661 (2008)] was shown to permit low-phototoxicity, high-speed, three-dimensional fluorescence imaging of extended tissue samples. Here, we introduce two major improvements in OCPI microscopy. First, we miniaturize the objective coupler by using a uniaxial gradient-index lens to produce the light sheet. Second, we demonstrate theoretically and experimentally that refractive index mismatch at the fluid/tissue interface causes a significant defocus aberration. By introducing the ability to tune the angle of the light sheet, we show that defocus correction in a miniaturized OCPI microscope leads to a significant improvement in image sharpness deeper into tissue.     

Current status of the TwinMic beamline at Elettra: a soft X‐ray transmission and emission microscopy station

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X‐ray microscopy station, is reported. The X‐ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy‐tailored using a collimated plane‐grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full‐field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X‐ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across‐absorption‐edge imaging, X‐ray absorption near‐edge structure or low‐energy X‐ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.     

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.     

Acoustic radiation force optical coherence elastography for elasticity assessment of soft tissues

Biomechanical properties of soft tissues are important indicators of tissue functions which can be used for clinical diagnosis and disease monitoring. Elastography, incorporating the principles of elasticity measurements into imaging modalities, provides quantitative assessment of elastic properties of biological tissues. Benefiting from high-resolution, noninvasive, and three-dimensional optical coherence tomography, optical coherence elastography (OCE) is an emerging optical imaging modality to characterize and map biomechanical properties of soft tissues. Recently, acoustic radiation force (ARF)–OCE has been developed for elasticity measurements of ocular tissues, detection of vascular lesions, and monitoring of blood coagulation based on remote and noninvasive ARF excitation to both internal and superficial tissues. Here, we describe the advantages of the ARF–OCE technique, the measurement methods in ARF–OCE, the applications in biomedical detection, current challenges, and advances. ARF–OCE technology has the potential to become a powerful tool for in vivo elasticity assessment of biological samples in a noncontact, noninvasive, and high-resolution nature.     

Transillumination spatially modulated illumination microscopy

The diagnostic utility of a conventional transillumination microscope, the most common imaging modality in clinical use today, is limited by the microscope's resolution. It is, however, possible to achieve lateral resolution well beyond the classical limit by using laterally structured illumination in a wide-field, nonconfocal microscope. In this method, the spatially modulated illumination (SMI) makes high-resolution information that is normally inaccessible visible in the observed image. Previously presented SMI microscopy systems operated in epifluorescence mode. We describe the design, construction, and testing of a novel transillumination SMI microscope. As transillumination is necessary for most medical applications, such as histopathologic evaluation of biopsy tissue and chromosomal analysis, such a system should have a significant diagnostic effect.     

Dark-field optical coherence microscopy

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.     

Sidelobe suppression in light-sheet fluorescence microscopy with Bessel beam plane illumination using subtractive imaging

The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy(LSFM) with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.     

Ultra-High Resolution Optical Coherence Tomography (OCT) Using a Halogen Lamp as the Light Source

The axial resolution of optical coherence tomography (OCT) is determined by the coherence length of the light source. We demonstrate for the first time high-resolution OCT of biological tissue using a halogen lamp as the light source for a low coherence interferometer. High-resolution OCT imaging with 3.5 μm resolution was performed successfully for onion and porcine skin, although the coherence light power for illumination of a sample is as small as 100 nW.     

Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.     

Combined scanning optical coherence and two-photon-excited fluorescence microscopy

We demonstrate simultaneous imaging by optical coherence microscopy (OCM) and two-photon-excited (TPE) fluorescence microscopy. A mode-locked Ti:sapphire laser is focused and scanned in three dimensions through a fixed sample, generating both backscattered light and fluorescence light, which are independently detected. Both imaging modes provide rapid en-face imaging with submicrometer resolution. High-power delivery into the sample yields an OCM sensitivity in excess of 130 dB at 100-kHz pixel rates. Simultaneous imaging of cell nuclei with OCM and TPE is demonstrated in live drosophila embryos.     

Total-internal-reflection fluorescence microscopy with W-shaped axicon mirrors

A scheme based on a W-shaped axicon mirror device for total-internal-reflection fluorescence microscopy (TIRFM) is presented. This approach combines the advantages of higher efficiency compared with traditional TIRFM, adjustable illumination area, and simple switching between wide-field and TIRF imaging modes. TIRF images obtained with this approach are free of shadow artifacts and of interference fringes. Example micrographs of fluorescently labeled polystyrene beads, of Convallaria majalis tissue, and of Propidium-iodide-labeled Chinese hamster ovary cells are shown, and the capabilities of the scheme are discussed.