Journal of microscopy最新文献

Metallic nanoparticle dimers have been used to enhance the excitation rate of single-quantum emitters. The interparticle distance (d) of the dimers has a crucial influence on the signal enhancement. Therefore, precise control of d is desired for optimal performance. However, statistical analysis of d has been often restricted to a small number of dimers due to the lack of reliable automatic software tools. For this reason, we developed a novel analysis tool for automatic dimer analysis. Our approach combines particle detection by circle Hough transformation (CHT) with custom classification routines optimised for distinct types of particles. We applied our tool to scanning electron microscopy (SEM) images and achieved great agreement in dimer detection, reaching an agreement of around 97% between automatic analysis and manual inspection for more than 3000 metallic nanoparticle dimers on DNA origami controlled by a combination of multiple DNA strands. Our study revealed the effects of the strand length (L) on the distribution of d. Based on geometric consideration, we expected a strong correlation between L and the standard deviation (σ) of d. We could verify this correlation by characterising four dimer designs with different L while analysing more than 1000 dimers per specimen.

Sidestream dark field (SDF) imaging is a tool for assessing microcirculation, commonly used for early diagnosis and monitoring of sepsis. In this study, we used SDF imaging to observe and assess the microcirculation of the intestinal mucosa during bowel surgery. We also compared different performance between normal mucosa and diseased mucosa using SDF imaging. SDF imaging was conducted in 13 patients to evaluate microcirculation parameters. All patients were assessed at distances of 0, 1, 2, 3 and 4 centimeters (cm) from the edge of the mesentery, respectively. Microcirculatory parameters such as microvascular flow index (MFI), proportion of perfused vessels (PPV), vascular density (VD), total vessel density (TVD), perfused vessel density (PVD) and heterogeneity index (HI) were measured in these patients. Compared to normal intestinal mucosa, the diseased intestinal mucosa exhibited higher values for VD (p = 0.044), TVD (p = 0.006) and PVD (p = 0.007). No significant differences in PPV, MFI and HI were observed between the two groups. The microcirculation parameters (MFI, PPV and PVD) of the intestine at the distal distance of 3 cm were significantly lower than those at a distance of 2 cm (MFI 1.5 (0.75) vs. 3 (0.5), PPV 57.6 (9.1) vs. 97.1 (8.6)% and PVD 11.395 (3.082) vs. 20.726 (4.115) mm/mm²). In conclusion, SDF imaging is an advanced technique that provide real-time visualization of intestinal mucosal microcirculation. It has the potential to assess the blood perfusion of the intestine during surgery.

Ex vivo x-ray angiography provides high-resolution, three-dimensional information on vascular phenotypes down to the level of capillaries. Sample preparation for ex vivo angiography starts with the removal of blood from the vascular system, followed by perfusion with an x-ray dense contrast agent mixed with a carrier such as gelatine or a polymer. Subsequently, the vascular micro-architecture of harvested organs is imaged in the intact fixed organ. In the present study, we present novel microscopic dual-energy CT (microDECT) imaging protocols that allow to visualise and analyse microvasculature in situ with reference to the morphology of hard and soft tissue. We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x-ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate-perfused specimens. A novel Sorensen-buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. The presented microDECT workflows are suited as tools to screen and quantify microvasculature and can be implemented in various correlative imaging pipelines to target regions of interest for downstream light microscopic investigation.

Auer, J. M. T., Murphy, L. C., Xiao, D., Li, D. U. & Wheeler, A. P., J. Microsc. 2023; 291, 1, p. 43–56.

In Acknowledgements, we did not acknowledge the support from Medical Research Scotland for Dong Xiao's time in this work.

We want to add ‘We also want to thank Medical Research Scotland for supporting Dong Xiao's research.’

In the author list, ‘David U. Li' should be corrected as' David D.-U. Li’.

We apologize for these two errors.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

Changes in the surrounding environment, if transmitted to the electron microscope, are frequently perceived as noise that diminishes the quality of the images. However, in fact, ‘noises’ contain rich information about the environment. This work reports a very rare event where aberration-corrected HAADF-STEM images were acquired during the impact of seismic waves, resulted from a mild earthquake. By analysing these images, we found that the drift and vibration of the sample are detectable and quantifiable. Despite many potential challenges, this work demonstrates the utilisation of electron microscopes in detecting and monitoring seismic waves with high spatial resolution, which may lead to unique applications in the low-frequency regime.