Three-dimensional super-resolution correlation-differential confocal microscopy with nanometer axial focusing accuracy

We present a correlation-differential confocal microscopy (CDCM), a novel method that can simultaneously improve the three-dimensional spatial resolution and axial focusing accuracy of confocal microscopy (CM). CDCM divides the CM imaging light path into two paths, where the detectors are before and after the focus with an equal axial offset in opposite directions. Then, the light intensity signals received from the two paths are processed by the correlation product and differential subtraction to improve the CM spatial resolution and axial focusing accuracy, respectively. Theoretical analyses and preliminary experiments indicate that, for the excitation wavelength of λ = 405 nm, numerical aperture of NA = 0.95, and the normalized axial offset of uM = 5.21, the CDCM resolution is improved by more than 20% and more than 30% in the lateral and axial directions, respectively, compared with that of the CM. Also, the axial focusing resolution important for the imaging of sample surface profiles is improved to 1 nm

Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration

A new super-resolution image restoration confocal Raman microscopy method (SRIR-RAMAN) is proposed for improving the spatial resolution of confocal Raman microscopy. This method can recover the lost high spatial frequency of the confocal Raman microscopy by using Poisson-MAP super-resolution imaging restoration, thereby improving the spatial resolution of confocal Raman microscopy and realizing its super-resolution imaging. Simulation analyses and experimental results indicate that the spatial resolution of SRIR-RAMAN can be improved by 65% to achieve 200 nm with the same confocal Raman microscopy system. This method can provide a new tool for high spatial resolution micro-probe structure detection in physical chemistry, materials science, biomedical science and other areas

Confocal Raman image method with maximum likelihood method

With the increasing interest in nano microscopic area, such as DNA sequencing, micro structure detection of molecular nano devices, a higher requirement for the spatial resolution of Raman spectroscopy is demanded. However, because of the weak Raman signal, the pinhole size of confocal Raman microscopy is usually a few hundreds microns to ensure a relatively higher spectrum throughput, but the large pinhole size limits the improvements of spatial resolution of confoal Raman spectroscopy. As a result, the convential confocal Raman spectroscopy has been unable to meet the needs of science development. Therefore, a confocal Raman image method with Maximum Likelihood image restoration algorithm based on the convential confocal Raman microscope is propose. This method combines super-resolution image restoration technology and confocal Raman microscopy to realize super-resolution imaging, by using Maximum Likelihood image restoration algorithm based on Poisson-Markov model to conduct image restoration processing on the Raman image, and the high frequency information of the image is recovered, and then the spatial resolution of Raman image is improved and the super-resolution image is realized. Simulation analyses and experimental results indicate that the proposed confocal Raman image method with Maximum Likelihood image restoration algorithm can improve the spatial resolution to 200 nm without losing any Raman spectral signal under the same condition with convential confocal Raman microscopy, moreover it has strong noise suppression capability. In conclusion, the method can provide a new approach for material science, life sciences, biomedicine and other frontiers areas. This method is an effective confocal Raman image method with high spatial resolution

Synchronous nanoscale topographic and chemical mapping by differential-confocal controlled Raman microscopy

Confocal Raman microscopy is currently used for label-free optical sensing and imaging within the biological, engineering, and physical sciences as well as in industry. However, currently these methods have limitations, including their low spatial resolution and poor focus stability, that restrict the breadth of new applications. This paper now introduces differential-confocal controlled Raman microscopy as a technique that fuses differential confocal microscopy and Raman spectroscopy, enabling the point-to-point collection of three-dimensional nanoscale topographic information with the simultaneous reconstruction of corresponding chemical information. The microscope collects the scattered Raman light together with the Rayleigh light, both as Rayleigh scattered and reflected light (these are normally filtered out in conventional confocal Raman systems). Inherent in the design of the instrument is a significant improvement in the axial focusing resolution of topographical features in the image (to ∼1 nm ), which, when coupled with super-resolution image restoration, gives a lateral resolution of 220 nm. By using differential confocal imaging for controlling the Raman imaging, the system presents a significant enhancement of the focusing and measurement accuracy, precision, and stability (with an antidrift capability), mitigating against both thermal and vibrational artefacts. We also demonstrate an improved scan speed, arising as a consequence of the nonaxial scanning mode

Three-dimensional resolution-enhancement divided aperture correlation-differential confocal microscopy with nanometer axial focusing capability

Divided aperture confocal microscopy (DACM) provides an improved imaging depth, imaging contrast, and working distance at the expense of spatial resolution. Here, we present a new method-divided aperture correlation-differential confocal microscopy (DACDCM) to improve the DACM resolution and the focusing capability, without changing the DACM configuration. DACDCM divides the DACM image spot into two round regions symmetrical about the optical axis. Then the light intensity signals received simultaneously from two round regions by a charge-coupled device (CCD) are processed by correlation manipulation and differential subtraction to improve the DACM spatial resolution and axial focusing capability, respectively. Theoretical analysis and preliminary experiments indicate that, for the excitation wavelength of λ = 632.8 nm, numerical aperture NA = 0.8, and normalized offset vM = 3.2 of the two regions, the DACDCM resolution is improved by 32.5% and 43.1% in the x and z directions, simultaneously, compared with that of the DACM. The axial focusing resolution used for the sample surface profile imaging was also significantly improved to 2 nm

Confocal Raman spectroscopy method based on quadratic curve fitting

Raman spectroscopy plays an improtant role in analytical science because of its unique characteristics, such as non-contact and non-destructive detecting, fewer sample consumption, high sensitivity and other characteristics, and it provides a powerful analytical tool for the modern basic research fields. Because of the combination of confocal microscopy technology and Raman spectroscopy technology, confocal Raman microscopy has the advantage of both high resolution spectroscopy and chromatography detection, which inherits from confocal microscopy and raman spectroscopy. As a result, it is widely used in many fields, such as physical chemistry, materials science, biomedical, archaeological, cultural identification, and forensic science. But with the environmental changing, the system drifting or other issues, during the long detection process, the system turns to defocusing. As a result, during the hole scanning process, the system can not be focused on every detection point, and then it would lead to a mistake. Eventurally, conventional confocal Raman system could obtain the presence of measurement error even erroneous results in the long process. In this paper, on the basis of conventional confocal Raman system, a confocal Raman spectroscopy method based on quadratic curve fitting is proposed to solve this problem. Based on the principle that the maxium of the concal curve corresponding the system foucs, the steps to find system foucus as follows: fist, usesing quadratic curve to fit confocal curve; second, finding the maxium of the confocal curve; and last obtaining the system foucs. With this method, during the scanning process, every point should be focused, therefore, the effect of defocusing is eliminated efficiently, and accurate measurements of confocal Raman spectroscopy system is achieved.Through simulation and experimental results show that: the proposed method that confocal Raman spectroscopy method based on quadratic curve fitting can effectively eliminate the influence of system defocus on experimental result, and effectively improve the axial system of fixed focus accuracy, which could provide a guarantee for further application of confocal Raman spectroscopy. This anti-drift method is effective and accurate in focusing with great potential to be applied in broader areas

Medial reward and lateral non-reward orbitofrontal cortex circuits change in opposite directions in depression

The first brain-wide voxel-level resting state functional-connectivity neuroimaging analysis of depression is reported, with 421 patients with major depressive disorder and 488 controls. Resting state functional connectivity between different voxels reflects correlations of activity between those voxels and is a fundamental tool in helping to understand the brain regions with altered connectivity and function in depression. One major circuit with altered functional connectivity involved the medial orbitofrontal cortex BA 13, which is implicated in reward, and which had reduced functional connectivity in depression with memory systems in the parahippocampal gyrus and medial temporal lobe, especially involving the perirhinal cortex BA 36 and entorhinal cortex BA 28. The Hamilton Depression Rating Scale scores were correlated with weakened functional connectivity of the medial orbitofrontal cortex BA 13. Thus in depression there is decreased reward-related and memory system functional connectivity, and this is related to the depressed symptoms. The lateral orbitofrontal cortex BA 47/12, involved in non-reward and punishing events, did not have this reduced functional connectivity with memory systems. Second, the lateral orbitofrontal cortex BA 47/12 had increased functional connectivity with the precuneus, the angular gyrus, and the temporal visual cortex BA 21. This enhanced functional connectivity of the non-reward/punishment system (BA 47/12) with the precuneus (involved in the sense of self and agency), and the angular gyrus (involved in language) is thus related to the explicit affectively negative sense of the self, and of self-esteem, in depression. A comparison of the functional connectivity in 185 depressed patients not receiving medication and 182 patients receiving medication showed that the functional connectivity of the lateral orbitofrontal cortex BA 47/12 with these three brain areas was lower in the medicated than the unmedicated patients. This is consistent with the hypothesis that the increased functional connectivity of the lateral orbitofrontal cortex BA 47/12 is related to depression. Relating the changes in cortical connectivity to our understanding of the functions of different parts of the orbitofrontal cortex in emotion helps to provide new insight into the brain changes related to depression, which are considered in the Discussion

The immunosuppressive effects and mechanisms of loureirin B on collagen-induced arthritis in rats

IntroductionRheumatoid arthritis (RA) is a common disease mainly affecting joints of the hands and wrists. The discovery of autoantibodies in the serum of patients revealed that RA belonged to the autoimmune diseases and laid a theoretical basis for its immunosuppressive therapy. The pathogenesis of autoimmune diseases mainly involves abnormal activation and proliferation of effector memory T cells, which is closely related to the elevated expression of Kv1.3, a voltage-gated potassium (Kv) channel on the effector memory T cell membrane. Drugs blocking the Kv1.3 channel showed a strong protective effect in RA model animals, suggesting that Kv1.3 is a target for the discovery of specific RA immunosuppressive drugs.MethodsIn the present study, we synthesized LrB and studied the effects of LrB on collagen- induced arthritis (CIA) in rats. The clinical score, paw volume and joint morphology of CIA model rats were compared. The percentage of CD3+, CD4+ and CD8+ T cells in rat peripheral blood mononuclear and spleen were analyzed with flow cytometry. The concentrations of inflammatory cytokines interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-10 and IL-17 in the serum of CIA rats were analyzed with enzyme-linked immunosorbent assay. The IL-1b and IL-6 expression in joints and the Kv1.3 expression in peripheral blood mononuclear cells (PBMCs) were quantified by qPCR. To further study the mechanisms of immunosuppressive effects of LrB, western blot and immunofluorescence were utilized to study the expression of Kv1.3 and Nuclear Factor of Activated T Cells 1 (NFAT1) in two cell models - Jurkat T cell line and extracted PBMCs.ResultsLrB effectively reduced the clinical score and relieved joint swelling. LrB could also decrease the percentage of CD4+ T cells, while increase the percentage of CD8+ T cells in peripheral blood mononuclear and spleen of rats with CIA. The concentrations of inflammatory cytokines interleukin (IL)-1b, IL-2, IL-6, IL-10 and IL-17 in the serum of CIA rats were significantly reduced by LrB. The results of qPCR showed that Kv1.3 mRNA in the PBMCs of CIA rats was significantly higher than that of the control and significantly decreased in the LrB treatment groups. In addition, we confirmed in cell models that LrB significantly decreased Kv1.3 protein on the cell membrane and inhibited the activation of Nuclear Factor of Activated T Cells 1 (NFAT1) with immune stimulus.ConclusionIn summary, this study revealed that LrB could block NFAT1 activation and reduce Kv1.3 expression in activated T cells, thus inhibiting the proliferation of lymphocytes and the release of inflammatory cytokines, thereby effectively weakening the autoimmune responses in CIA rats. The effects of immunosuppression due to LrB revealed its potential medicinal value in the treatment of RA