Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy

Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (170.4730) Optical pathology, (170.6935) Tissue characterization     

High-NA two-photon single cell imaging with remote focusing using a diffractive tunable lens

Fast, volumetric structural and functional imaging of cellular and sub-cellular dynamics inside the living brain is one of the most desired capabilities in the neurosciences, but still faces serious challenges. Specifically, while few solutions for rapid 3D scanning exist, it is generally much easier to facilitate fast in-plane scanning than it is to scan axially at high speeds. Remote focusing in which the imaging plane is shifted along the optical axis by a tunable lens while maintaining the position of the sample and objective is a promising approach to increase the axial scan speed, but existing techniques often introduce severe optical aberrations in high-NA imaging systems, eliminating the possibility of diffraction-limited single-cell imaging. Here, we demonstrate near diffraction-limited, volumetric two-photon fluorescence microscopy in which we resolve the deep sub-micron structures of single microglia cells with axial scanning performed using a novel high-NA remote focusing method. Image contrast is maintained to within 7% compared to mechanical sample stepping and the focal volume remains nearly diffraction-limited over an axial range greater than 86 µm.     

Optimal lens design and use in laser-scanning microscopy

In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of superior quality that fully make use of high-NA low-magnification objectives, offering diffraction-limited imaging over a large FOV and wavelength range. We constructed a two-photon laser-scanning microscope with optimized custom lenses which had a near diffraction limit point-spread-function (PSF) with less than 3.6% variation over a 400 µm FOV and less than 0.5 µm lateral color between 750 and 1050 nm.OCIS codes: (180.0180) Microscopy, (180.1790) Confocal microscopy, (180.4315) Nonlinear microscopy, (220.3620) Lens system design, (220.3630) Lenses     

Changes of collagen ultrastructure in breast cancer tissue determined by second-harmonic generation double Stokes-Mueller polarimetric microscopy

Second-harmonic generation (SHG) double Stokes-Mueller polarimetric microscopy is applied to study the alteration of collagen ultrastructure in a tissue microarray containing three pathological human breast cancer types with differently overexpressed estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Kleinman symmetry is experimentally validated in breast tissue for 1028 nm laser wavelength and it has been shown that measurements with only linearly polarized incoming and outgoing states can determine molecular nonlinear susceptibility tensor component ratio, average in-plane orientation of collagen fibers and degree of linear polarization of SHG. Increase in the susceptibility ratio for ER, PgR, HER2 positive cases, reveals ultrastructural changes in the collagen fibers while the susceptibility ratio increase and decrease in degree of linear polarization for ER and PgR positive cases indicate alteration of the ultrastructure and increased disorder of the collagen fibers within each focal volume. The study demonstrates a potential use of polarimetric SHG microscopy for collagen characterization and cancer diagnostics.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (190.4160) Multiharmonic generation, (110.5405) Polarimetric imaging     

Motion-artifact-robust,polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.OCIS codes: (170.1870) Dermatology, (170.3880) Medical and biological imaging, (190.4160) Multiharmonic generation, (180.4315) Nonlinear microscopy     

Precise,motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.OCIS codes: (170.6935) Tissue characterization, (190.2620) Harmonic generation and mixing, (180.4315) Nonlinear microscopy, (180.6900) Three-dimensional microscopy, (110.5405) Polarimetric imaging     

Effect of molecular organization on the image histograms of polarization SHG microscopy

Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.OCIS codes: (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing     

Second harmonic generation double stokes Mueller polarimetric microscopy of myofilaments

The experimental implementation of double Stokes Mueller polarimetric microscopy is presented. This technique enables a model-independent and complete polarimetric characterization of second harmonic generating samples using 36 Stokes parameter measurements at different combinations of incoming and outgoing polarizations. The degree of second harmonic polarization and the molecular nonlinear susceptibility ratio are extracted for individual focal volumes of a fruit fly larva wall muscle.OCIS codes: (180.4315) Nonlinear microscopy, (110.5405) Polarimetric imaging, (170.3880) Medical and biological imaging     

The structural origin of second harmonic generation in fascia

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.OCIS codes: (180.4315) Nonlinear microscopy, (190.4160) Multiharmonic generation     

Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy,applied to neurotransmitter imaging with GPCR-based sensors

Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.     

Enhanced Numerical Method for the Design of 3-D-Printed Holographic Acoustic Lenses for Aberration Correction of Single-Element Transcranial Focused Ultrasound

The correction of transcranial focused ultrasound aberrations is a relevant issue for enhancing various non-invasive medical treatments. The emission through multi-element phased arrays has been the most widely accepted method to improve focusing in recent years; however, the number and size of transducers represent a bottleneck that limits the focusing accuracy of the technique. To overcome this limitation, a new disruptive technology, based on 3-D-printed acoustic lenses, has recently been proposed. As the submillimeter precision of the latest generation of 3-D printers has been proven to overcome the spatial limitations of phased arrays, a new challenge is to improve the accuracy of the numerical simulations required to design this type of ultrasound lens. In the study described here, we evaluated two improvements in the numerical model applied in previous works for the design of 3-D-printed lenses: (i) allowing the propagation of shear waves in the skull by means of its simulation as an isotropic solid and (ii) introduction of absorption into the set of equations that describes the dynamics of the wave in both fluid and solid media. The results obtained in the numerical simulations are evidence that the inclusion of both s-waves and absorption significantly improves focusing.     

Differential microscopy for fluorescence-detected nonlinear absorption linear anisotropy based on a staggered two-beam femtosecond Yb:KGW oscillator

We present a new laser system and nonlinear microscope, designed for differential nonlinear microscopy. The microscope features time-correlated single photon counting of multiphoton fluorescence generated by an alternating pulse-train of orthogonally polarized pulses. The generated nonlinear signal is separated using home-built electronics. Results are presented on fluorescence-detected nonlinear absorption linear anisotropy (FDNALA) of chloroplasts in Asparagus Sprengerii Regel and of Congo Red-stained cellulose.     

Nonlinear optical microscopy of early stage (ICRS Grade-I) osteoarthritic human cartilage

In a synovial joint, the articular cartilage is directly affected during the progression of Osteoarthritis (OA). The characterization of early stage modification in extra-cellular matrix of cartilage is essential for detection as well as understanding the progression of disease. The objective of this study is to demonstrate the potential and capability of nonlinear optical microscopy for the morphological investigation of early stage osteoarthritic cartilage. ICRS Grade-I cartilage sections were obtained from the femoral condyle of the human knee. The surface of articular cartilage was imaged by second harmonic generation and two-photon excited fluorescence microscopy. Novel morphological features like microsplits and wrinkles were observed, which would otherwise not be visible in other clinical imaging modalities (e.g., CT, MRI, ultrasound and arthroscope. The presence of superficial layer with distinct collagen fibrils parallel to the articular surface in 4 specimens out of 14 specimens, indicates that different phases of OA within ICRS Grade-I can be detected by SHG microscopy. All together, the observed novel morphologies in early stage osteoarthritic cartilage indicates that SHG microscopy might be a significant tool for the assessment of cartilage disorder.OCIS codes: (190.1900) Diagnostic applications of nonlinear optics, (170.4580) Optical diagnostics for medicine, (180.4315) Nonlinear microscopy     

Stokes vector based polarization resolved second harmonic microscopy of starch granules

We report on the measurement and analysis of the polarization state of second harmonic signals generated by starch granules, using a four-channel photon counting based Stokes-polarimeter. Various polarization parameters, such as the degree of polarization (DOP), the degree of linear polarization (DOLP), the degree of circular polarization (DOCP), and anisotropy are extracted from the 2D second harmonic Stokes images of starch granules. The concentric shell structure of a starch granule forms a natural photonic crystal structure. By integration over all the solid angle, it will allow very similar SHG quantum efficiency regardless of the angle or the states of incident polarization. Given type I phase matching and the concentric shell structure of a starch granule, one can easily infer the polarization states of the input beam from the resulting SH micrograph.OCIS codes: (180.4315) Nonlinear microscopy, (120.5410) Polarimetry, (320.0320) Ultrafast optics, (120.0120) Instrumentation, measurement, and metrology, (160.1435) Biomaterials     

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.OCIS codes: (180.4315) Nonlinear microscopy, (190.4180) Multiphoton processes     

Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution

Second harmonic generation (SHG) microscopy is a powerful tool for label free ex vivo or in vivo imaging, widely used to investigate structure and organization of endogenous SHG emitting proteins such as myosin or collagen. Polarization resolved SHG microscopy renders supplementary information and is used to probe different molecular states. This development towards functional SHG microscopy is calling for new methods for high speed functional imaging of dynamic processes. In this work we present two approaches with linear polarized light and demonstrate high speed line scan measurements of the molecular dynamics of the motor protein myosin with a time resolution of 1 ms in mammalian muscle cells. Such a high speed functional SHG microscopy has high potential to deliver new insights into structural and temporal molecular dynamics under ex vivo or in vivo conditions.OCIS codes: (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing, (170.2655) Functional monitoring and imaging     

High-performance imaging of cell-substrate contacts using refractive index quantification microscopy

Non-invasive imaging of living cells is an advanced technique that is widely used in the life sciences and medical research. We demonstrate a refractive index quantification microscopy (RIQM) that enables label-free studies of glioma cell-substrate contacts involving cell adhesion molecules and the extracellular matrix. This microscopy takes advantage of the smallest available spot created when an azimuthally polarized perfect optical vortex beam (POV) is tightly focused with a first-order spiral phase, which results in a relatively high imaging resolution among biosensors. A high refractive index (RI) resolution enables the RI distribution within neuronal cells to be monitored. The microscopy shows excellent capability for recognizing cellular structures and activities, demonstrating great potential in biological sensing and live-cell kinetic imaging.     

Vibrationally resonant sum-frequency generation microscopy with a solid immersion lens

We use a hemispheric sapphire lens in combination with an off-axis parabolic mirror to demonstrate high-resolution vibrationally resonant sum-frequency generation (VR-SFG) microscopy in the mid-infrared range. With the sapphire lens as an immersed solid medium, the numerical aperture (NA) of the parabolic mirror objective is enhanced by a factor of 1.72, from 0.42 to 0.72, close to the theoretical value of 1.76 ( = n_sapphire). The measured lateral resolution is as high as 0.64 μm. We show the practical utility of the sapphire immersion lens by imaging collagen-rich tissues with and without the solid immersion lens.OCIS codes: (170.0180) Microscopy, (180.4315) Nonlinear microscopy, (190.4223) Nonlinear wave mixing, (110.3080) Infrared imaging     

Chiral imaging of collagen by second-harmonic generation circular dichroism

We provide evidence that the chirality of collagen can give rise to strong second-harmonic generation circular dichroism (SHG-CD) responses in nonlinear microscopy. Although chirality is an intrinsic structural property of collagen, most of the previous studies ignore that property. We demonstrate chiral imaging of individual collagen fibers by using a laser scanning microscope and type-I collagen from pig ligaments. 100% contrast level of SHG-CD is achieved with sub-micrometer spatial resolution. As a new contrast mechanism for imaging chiral structures in bio-tissues, this technique provides information about collagen morphology and three-dimensional orientation of collagen molecules.OCIS codes: (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing     

Measurement of orientation and susceptibility ratios using a polarization-resolved second-harmonic generation holographic microscope

Three-dimensional second-harmonic fields, sample orientation, and susceptibility ratios of biological samples are measured using polarization-resolved second-harmonic generation (SHG) microscopy. The three-dimensional (3D) polarization is gathered by measurement of a series of holograms for which excitation and analyzer polarizations are systematically varied, and the 3D SHG field is recovered through numerical back propagation. Harmonophore orientation is resolved in 3D from a sub-set of polarization-resolved SHG holograms. We further expand on previous approaches for the determination of susceptibility ratios, adding the calculation of multiple ratio values to allow intrinsic verification.OCIS codes: (090.1995) Digital holography, (180.4315) Nonlinear microscopy, (110.3010) Image reconstruction techniques