Shengde Liu

Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis.

Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

Quantitative refractive index distribution of single cell by combining phase-shifting interferometry and AFM imaging

Cell refractive index, an intrinsic optical parameter, is closely correlated with the intracellular mass and concentration. By combining optical phase-shifting interferometry (PSI) and atomic force microscope (AFM) imaging, we constructed a label free, non-invasive and quantitative refractive index of single cell measurement system, in which the accurate phase map of single cell was retrieved with PSI technique and the cell morphology with nanoscale resolution was achieved with AFM imaging. Based on the proposed AFM/PSI system, we achieved quantitative refractive index distributions of single red blood cell and Jurkat cell, respectively. Further, the quantitative change of refractive index distribution during Daunorubicin (DNR)-induced Jurkat cell apoptosis was presented, and then the content changes of intracellular biochemical components were achieved. Importantly, these results were consistent with Raman spectral analysis, indicating that the proposed PSI/AFM based refractive index system is likely to become a useful tool for intracellular biochemical components analysis measurement, and this will facilitate its application for revealing cell structure and pathological state from a new perspective.

Improved phase retrieval method of dual-wavelength interferometry based on a shorter synthetic-wavelength

In dual-wavelength interferometry (DWI), by combing the advantage of the shorter synthetic-wavelength and the immune algorithm of phase ambiguity, we propose an improved phase retrieval method with both high accuracy and large measurement range, which is a pair of contradiction in the reported DWI method. First, we calculate the height of measured object at longer synthetic-wavelength through using the wrapped phases of two single-wavelengths. Second, by combining the immune algorithm of phase ambiguity and the height of measured object at longer synthetic-wavelength, we can perform the phase unwrapping of the larger one of the two single-wavelengths, then achieve accurate height at single-wavelength named as the transition height. Finally, we perform phase unwrapping of shorter synthetic-wavelength through using the immune algorithm of phase ambiguity and the transition height, and then the height at shorter synthetic-wavelength can be achieved. Compared with the reported method, in addition to maintaining the advantage of high accuracy, the proposed method does not need the additional wavelength, so the corresponding measurement procedures is greatly simplified. Simulation and experimental results demonstrate the performance of proposed method.

Nanoscale Relationship Between CD4 and CD25 of T Cells Visualized with NSOM/QD-Based Dual-Color Imaging System

In this study, by using of near-field scanning optical microscopy (NSOM)/immune-labeling quantum dot (QD)-based dual-color imaging system, we achieved the direct visualization of nanoscale profiles for distribution and organization of CD4 and CD25 molecules in T cells. A novel and interesting finding was that though CD25 clustering as nanodomains were observed on the surface of CD4+CD25high regulatory T cells, these CD25 nanodomains were not co-localized with CD4 nanodomains. This result presented that the formation of these CD25 nanodomains on the surface of CD4+CD25high T cells were not associated with the response of T cell receptor (TCR)/CD3-dependent signal transduction. In contrast, on the surface of CD4+CD25low T cells, CD25 molecules distributed randomly without forming nanodomains while CD4 clustering as nanodomains can be observed; on the surface of CD8+CD25+ T cells, CD25 clustering as nanodomains and co-localization with CD8 nanodomains were observed. Collectively, above these results exhibited that TCR/CD3-based microdomains were indeed required for TCR/CD3-mediated T cells activation and enhanced the immune activity of CD4+CD25low T cells or CD8+CD25+ T cells. In particular, it was found that the formation of CD25 nanodomains and their segregation from TCR/CD3 microdomains were the intrinsic capability of CD4+CD25high T cells, suggesting this specific imaging feature of CD25 should be greatly associated with the regulatory activity of CD4+CD25high T cells. Importantly, this novel NSOM/QD-based dual-color imaging system will provide a useful tool for the research of distribution-function relationship of cell-surface molecules.

NSOM/QD-based visualization of GM1 serving as platforms for TCR/CD3 mediated T-cell activation.

Direct molecular imaging of nanoscale relationship between T-cell receptor complexes (TCR/CD3) and gangliosidosis GM1 before and after T-cell activation has not been reported. In this study, we made use of our expertise of near-field scanning optical microscopy(NSOM)/immune-labeling quantum dots- (QD-)based dual-color imaging system to visualize nanoscale profiles for distribution and organization of TCR/CD3, GM1, as well as their nanospatial relationship and their correlation with PKCθ signaling cascade during T-cell activation. Interestingly, after anti-CD3/anti-CD28 Ab co-stimulation, both TCR/CD3 and GM1 were clustered to form nanodomains; moreover, all of TCR/CD3 nanodomains were colocalized with GM1 nanodomains, indicating that the formation of GM1 nanodomains was greatly correlated with TCR/CD3 mediated signaling. Specially, while T-cells were pretreated with PKCθ signaling inhibitor rottlerin to suppress IL-2 cytokine production, no visible TCR/CD3 nanodomains appeared while a lot of GM1 nanodomains were still observed. However, while T-cells are pretreated with PKCα β signaling inhibitor GÖ6976 to suppress calcium-dependent manner, all of TCR/CD3 nanodomains were still colocalized with GM1 nanodomains. These findings possibly support the notion that the formation of GM1 nanodomains indeed serves as platforms for the recruitment of TCR/CD3 nanodomains, and TCR/CD3 nanodomains are required for PKCθ signaling cascades and T-cell activation

An advanced phase retrieval algorithm in N-step phase-shifting interferometry with unknown phase shifts

In phase-shifting interferometry with unknown phase shifts, a normalization and orthogonalization phase-shifting algorithm (NOPSA) is proposed to achieve phase retrieval. The background of interferogram is eliminated through using the orthogonality of complex sinusoidal function; and the influence of phase shifts deviation on accuracy of phase retrieval is avoided through both normalization and orthogonalization processing. Compared with the current algorithms with unknown phase shifts, the proposed algorithm reveals significantly faster computation speed, higher accuracy, better stability and non-sensitivity of phase shifts deviation.

General spatial phase-shifting interferometry by optimizing the signal retrieving function

A general spatial phase-shifting (GSPS) interferometry method is proposed to achieve phase retrieval from one-frame spatial carrier frequency interferogram. By optimizing the internal signal retrieving function of the spatial phase-shifting (SPS) method, the accuracy, anti-noise ability and speed of phase retrieval can be significantly improved, meanwhile the corresponding local calculation property is reserved. Especially, in the case that the ratio of the spatial carrier to the phase variation rate are small, the proposed method reveals obvious advantage in the accuracy improvement relative to the conventional SPS methods, so the more details of measured sample can be effectively reserved through introducing smaller spatial carrier frequency, and this will facilitate its application in interference microscopy. The principle analysis, numerical simulation and experimental result are employed to verify the performance of the proposed GSPS method.

A Rapid Spatial Carrier-Frequency Phase-Shifting Method Based on the Orthogonality of Diamond Diagonal Vectors

From a one-frame off-axis interferogram with unknown carrier-frequency, a rapid spatial carrier-frequency phase-shifting (SCFPS) method is proposed to achieve the phase measurement of the dynamic process. First, based on the SCFPS technique, four-frame phase-shifting subinterferograms are constructed from one-frame off-axis interferogram. Second, by using the orthogonality of diamond diagonal vectors, respectively, achieved through the subtraction and addition operations between two equal length vectors, which are generated from above four-frame phase-shifting subinterferograms, the accurate phase can be retrieved rapidly. Compared with current SCFPS methods, in addition to maintaining high accuracy of phase-shifting method, as well as no requirement for carrier-frequency calibration, the proposed method reveals an obvious advantage in processing speed of phase retrieval, and this will greatly facilitate its application prospect for dynamic phase measurement.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

Abstract. Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665  cm−1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

Theoretical Localized Electric Field Enhancement in Tip-Enhanced Spectroscopy Using Multi-Order Radially Polarized Modes

Multi-order radially polarized modes (RPMs), including Bessel-Gaussian, Gaussian, Super Gaussian, and multi-order hollow Gaussian are respectively utilized as the illumination laser to achieve tip-enhanced spectroscopy (TES). Based on the vector diffraction theory and finite difference time domain (FDTD) analysis, we achieve the optimization of RPM illuminated TES system, including the focal spot size, focal depth, and electric field enhancement factor, in which the focal spot size of 5th order hollow Gaussian RPM is smallest (0.54λ) and the focusing depth of super Gaussian RPM is longest (4.71λ). Specially, it is found that the multi-order hollow Gaussian RPM illuminated TES system with the tip cone angle of 45° reveals better focusing ability and 40~60-fold electric field enhancement factor compared to the linearly polarized mode (LPM) illuminated TES system. These results will supply a useful reference for spectral signal enhancement of TES system.