SHG Microscope Research Articles

Dual-LC PSHG microscopy for imaging collagen type I and type II gels with pixel-resolution analysis.

Collagen of type I (Col I) and type II (Col II) are critical for cartilage and connective tissues in the human body, and several diseases may alter their properties. Assessing the identification and quantification of fibrillar collagen without biomarkers is a challenge. Advancements in non-invasive polarization-resolved second-harmonic generation (PSHG) microscopy have provided a method for the non-destructive investigation of collagen molecular level properties. Here we explored an alternative polarization modulated approach, dual-LC PSHG, that is based on two liquid crystal devices (Liquid crystal polarization rotators, LPRs) operating simultaneously with a laser scanning SHG microscope. We demonstrated that this more accessible technology allows the quick and accurate generation of any desired linear and circular polarization state without any mechanical parts. This study demonstrates that this method can aid in improving the ability to quantify the characteristics of both types of collagen, including pitch angle, anisotropy, and circular dichroism analysis. Using this approach, we estimated the effective pitch angle for Col I and Col II to be 49.7° and 51.6°, respectively. The effective peptide pitch angle for Col II gel was first estimated and is similar to the value obtained for Col I gel in the previous studies. Additionally, the difference of the anisotropy parameter of both collagen type gels was assessed to be 0.293, which reflects the different type molecular fibril assembly. Further, our work suggests a potential method for monitoring and differentiating different collagen types in biological tissues, especially cartilage or connective tissue.

PA36 SHG顕微鏡による液晶の極性凝集構造の観察(物理・物性)

PA36 SHG顕微鏡による液晶の極性凝集構造の観察(物理・物性)

PPM Detection Limits in PXRD by Integrating Nonlinear Optics and Synchrotron XRD

SHG microscopy allows rapid and selective identification of trace chiral crystals within amorphous media, enabling targeted XRD using a 5-10 micrometer diameter "minibeam". The sensitivity of PXRD is increased substantially by reducing the background scattering contributions of amorphous material otherwise encountered with a larger beam. In addition, performing diffraction only at the locations most likely to produce diffraction greatly reduced the overall beam-time required to perform the PXRD analyses. Integration of the SHG microscope directly into a synchrotron X-ray beamline at Argonne National Laboratory recovered high spatial registry between the regions of interest identified by SHG for positioning within the X-ray beam. Using this approach, diffraction was performed on individual griseofulvin nanocrystals suspended within an amorphous polymer, corresponding to a total of ~20 fg of total crystalline material. Additional measurements for ritonavir in hydroxypropylmethylcellulose (HPMC) were also performed, in which a bulk API concentration of 100 ppm produced diffraction peaks with a signal to noise ratio of >3000. Among other applications, sensitive detection of trace crystallinity can inform the design of amorphous formulations, in which the bioavailability of active pharmaceutical ingredients (APIs) is enhanced by maintaining them in an amorphous state. However, the long-term stability of a final dosage form can be negatively impacted by spontaneous transitioning to the typically more stable crystalline forms of the APIs, such that extensive quantitative characterization of the crystallization behaviors of amorphous formulations is routinely performed.

MO‐D‐141‐08: Multi‐Scale Analysis of Collagen Architecture for Classifying Tumor and Healthy Breast Tissue Images

Purpose: To quantify differences in collagen microarchitecture between healthy human breast tissue and breast cancer images using extended field of view second harmonic generation (EFOV SHG) microscopy coupled with analysis by the curvelet transform. Methods: Two frozen samples of normal associated human breast tissue (NAT) and invasive ductal carcinoma (IDC) were obtained through the University of Wisconsin BioBank. Samples were thawed, fixed, embedded in agar, cut into 300 micron thick sections using a vibratome and imaged using a custom inverted SHG microscope designed to allow for EFOV imaging. Individual SHG Images were captured at 0.79 micron resolution and a FOV of approximately 200 microns. The FOV was extended by stage scanning and image stitching to produce submicron resolution fields of view up to 2 cm2. NAT and IDC tissues were imaged, stitched using the Grid/Collection Stitching ImageJ plugin, and analyzed using features derived from the curvelet transform including variance in orientation and prevalence at various scales. These novel features were tested using support vector machine (SVM) cross validation. Results: IDC tissue contained linearly aligned, crisscrossing collagen while NAT tissue contained wavy, dense collagen. The mean curvelet alignment value for the normal images was approximately 2X higher than that for IDC. In addition, normal images had approximately 20% fewer closely correlated curvelets compared to IDC. Three‐fold cross validation of a linear SVM classifier revealed average sensitivity and specificity of 0.994 and 0.969 respectively. Conclusion: These results demonstrate the excellent potential for collagen architecture quantification based on features derived from the curvelet transform. We have also shown the value of EFOV SHG imaging to capture both large and small scale features necessary for quantifying collagen morphology. Future work will improve EFOV SHG imaging throughput and test classification capabilities of the curvelet based feature set on a large sample population. NIH R01 grants CA114462 and CA136590 to P.J.K. and K.W.E., T32 CA009206 to J.S.B and the Morgridge Institute for Research

7D43 小角X線散乱およびSHG顕微鏡観察下における電気化学法生成コラーゲンの引張試験(GS10 生体計測(光計測))

7D43 小角X線散乱およびSHG顕微鏡観察下における電気化学法生成コラーゲンの引張試験(GS10 生体計測(光計測))

408 SHG顕微鏡を用いたコラーゲンゲル再構築の動態イメージング(OS4-2:細胞と機械工学:計測とモデリングの最前線,オーガナイズドセッション4:細胞と機械工学:計測とモデリングの最前線)

408 SHG顕微鏡を用いたコラーゲンゲル再構築の動態イメージング(OS4-2:細胞と機械工学:計測とモデリングの最前線,オーガナイズドセッション4:細胞と機械工学:計測とモデリングの最前線)

Switching Processes and Formation of the Stable Artificial Domain Structure in Ferroelectric LaBGeO5

The processes of polarization switching in LaBGeO 5 (LBGO) crystals are studied in details for the triangle and pulse electric field in the wide temperature range from room temperature (RT) to Tc = 530°C. It is revealed that the temperature dependence of a coercive field (f = 0.1 Hz) has an exponential form and this field reaches a value 150 kV/cm at the RT. The temperature evolution of the switching time and the form of the switching current are obtained in the mode of the successive solitary pulses and in the steady conditions. The features of “non-classical” behavior are noted (the dependence of switching parameters on the prehistory of samples, frequency dependence of the switching time). It is shown that the stable artificial domain structure at RT with the periodicity of 200 μm can be obtained by applying about 300–350 V/mm near 300°C. The stable domain structure was contrasted by means of SHG microscope probing.

SHG interference microscope as a tool of nondestructive observation of ferroelectric 180° domain structures

Using the interference SHG microscope, ferroelectric 180° domain structure can be nondestructively observed. The principle of the microscope is to use the fact that up and down domains produce optical second harmonic waves with same amplitude but opposite phase. Then they are superposed with homogeneous SH wave from a standard SHG plate. The phase is controlled by a glass plate to get maximum contrast. This microscope was successfully applied to domain structure observations in BaTiO3 and domain reversal process in MgO:LiNbO3 under the pulsed-electric field. The possibility of the domain structure observation along the axial direction is also discussed.

Non-Destructive observations of the 180° ferroelectric domain using the nonlinear optical (SHG) microscope

Two-dimensional images of the 180° ferroelectric domain structure can be successfully obtained by using our recently developed SHG microscope/1/,/2/. The image is obtained as a result of the interference between second harmonic waves from a domain and those from homogeneously poled part. This is a quite general method for optical observation of 180° ferroelectric domains of any kind of crystal. As examples, second harmonic images are shown for BaTiO3, LiTaO3, LiNbO3 and V-LiNbO3. In particular, the successful observation of a periodically inverted antiparallel domain in LiTaO3 verified that the SHG microscopy provides a unique nondestructive method for characterizing the periodicity of inverted domains in quasi-phase matching devices. The two-dimensional Maker fringe pattern can be easily obtained by the SHG microscope with a wedge-shaped specimen. This method is also useful to evaluate the poling state of ferroelectric crystals.

Total reflection-type SHG microscope. -Principle and observation of MNA crystals grown on a TiO2 PRISM-

Abstract A new total reflection-type SHG microscope has been constructed where two-dimensional images are built from the second harmonic waves produced by the evanescent wave in the vicinity of a substrate surface. By using the microscope, the SHG image of an organic nonlinear optical crystal grown on a TiO2 prism was successfully obtained.

Optical second harmonic intensity image of multi-layered metal film patterns

The images of multi-layered metal film patterns have been observed using a scanning SHG microscope. The contrast between the SH intensities of one-layered and two layered domains was correlated with the contact potential difference between the top layer and the underlayer metals.

New nonlinear optical microscope and its application to the observation of ferroelectric domain structure

Abstract We have constructed a new nonlinear optical device called the SHG microscope. In the microscope, the fundamental wave with λ = 1.06 μm from a Nd:YAG laser enters a specimen and the second harmonic waves emitted from the specimen are collected by the IR objective lens and the two-dimen-sional image is detected by the CCD camera. By using the SHG microscope, we observed several kinds of specimens with inhomogeneous distribution of the nonlinear optical constant. In particular, we succeeded in observing 90° domains in BaTiO3 and a periodically inverted antiparallel ferroelectric domain in LiTaO3 in a nondestructive way. The general method for observing antiparallel ferroelectric domain structure is discussed.