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.

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.

Unilateral-shift-subtracting confocal microscopy with nanoscale axial focusing precision.

A novel unilateral-shift-subtracting confocal microscopy (USSCM) method with nanoscale axial focusing precision is proposed based on the optical arrangement of conventional confocal microscopy (CM). As the two segments of data on both sides of the confocal axial response curve are very sensitive to variations of the axial position, USSCM introduces an axial shift of S for one segment, to intersect it with the other segment. It then separately interpolates the two segments of intersecting data, subtracts the corresponding interpolated data, and selects the data that exhibit a good linearity from all of the subtracted data to fit a straight line. It calculates the zero position of the fitting line and offsets it by S/2, to precisely reveal the focus position of the confocal system, thereby achieving high-precision imaging of the three-dimensional sample's structure. Theoretical analyses and preliminary experiments indicate that, for excitation wavelength of λ=405 nm, numerical aperture of NA=0.95, and normalized axial shift of S=5.21, USSCM achieves an axial resolution of 3 nm and a repetitive focusing precision of 1.5 nm, while it does not change the lateral resolution of CM. Furthermore, compared with conventional CM, under the same noise condition, USSCM is less affected by system aberration, which leads to higher focusing precision. These findings demonstrate that USSCM is a very efficient method for imaging.

Laser differential fitting confocal microscopy with high imaging efficiency.

Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability.

Super-resolution radially polarized-light pupil-filtering confocal sensing technology.

Radially polarized beams can be focused to a tighter spot in the focal plane with a high numerical-aperture objective when combined with an optimally designed pupil filter. Based on the unique characters, a novel super-resolution radially polarized-light pupil-filtering confocal sensing technology (SRPCST) is proposed, and a sensor based on SRPCST is developed. By using a radially polarized beam and pupil-filtering technology, SRPCST can effectively improve its lateral resolution. In SRPCST a strong longitudinal field component can be generated in the focal plane by focusing a radially polarized light with a high numerical-aperture objective. Pupil-filtering technology will modify the pupil function of the optical system by optimally designing the parameters of pupil filter to higher resolution. Theoretical analyses and packaged SRPCST sensor experiments indicate that the lateral resolution of SRPCST can be improved by 15.23% and 32.12% through super-resolution image restoration compared with confocal microscopy under the same conditions.

Clinical Analysis of Myocardial Injury in Highlanders with Pulmonary Hypertension.

Background: Pulmonary hypertension (PH) is a prevalent adverse cardiovascular event at high-altitude environments. Prolonged exposure to high altitudes may result in myocardial injury, which is associated with poor clinical outcomes. This study aims to investigate the clinical characteristics of myocardial injury in patients with PH at high altitude. Methods: Consecutive patients admitted to a general tertiary hospital at the altitude of 3,650 m were selected into this retrospective study. Clinical and biochemical data were collected, as well as based on cardiac troponin I (cTnI) and echocardiography, patients were divided into myocardial injury group and non-myocardial injury group. Results: A total of 231 patients were enrolled, among whom 29 (12.6%) had myocardial injury. We found that body mass index, left ventricular end-diastolic dimension, and serum level of creatine kinase-MB (CK-MB) in myocardial injury group were significantly higher than non-myocardial injury group. Spearman correlation analysis revealed that cTnI has a significant positive correlation with CK-MB and lactic dehydrogenase instead of aspartate aminotransferase. A receiver operating characteristic curve was drawn to demonstrate that CK-MB could significantly predict the occurrence of myocardial injury with an area under the curve of 0.749, and a level of 3.035 (sensitivity = 59.3%, specificity = 90.5%) was optimal cutoff value. Conclusion: The incidence of myocardial injury in highlanders with PH is significant. CK-MB, as a convenient and efficient marker, has been found to be closely associated with cTnI and plays a predictive role in the occurrence of myocardial injury with PH in individuals exposed to high altitude.