Recent advances in electron microscopy for the diagnosis and research of glomerular diseases

( Kazuho Honda ),( Takashi Takaki ),( Dedong Kang ) 대한신장학회 2023 Kidney Research and Clinical Practice Vol.42 No.2

Recent technical advances in the detection of backscattered electrons during scanning electron microscopy (SEM) have improved resolution and have provided several new technologies for research and clinical practice in kidney disease. The advances include three-dimensional (3D) electron microscopy (3D-EM), correlative light and electron microscopy (CLEM), low-vacuum SEM (LVSEM), and scanning transmission electron microscopy (STEM). 3D-EM analysis used to be laborious, but recently three different technologies, serial block-face SEM, focused ion beam SEM, and array tomography, have made 3D-EM easier by automating sectioning and the subsequent image acquisition in an SEM. CLEM is a method to correlate light microscopic images, especially immunofluorescent and electron microscopy images, providing detailed ultrastructure of the area of interest where the immunofluorescent marker is located. LVSEM enables the use of SEM on materials with poor electron conductivity. For example, LVSEM makes it possible for high resolution, 3D observation of paraffin sections. Finally, STEM is a method to observe ultrathin sections with improved resolution by using the focused electron beam scanning used in SEM and not the broad electron beam used in transmission electron microscopy. These technical advances in electron microscopy are promising to provide plenty of novel insights for understanding the pathogenesis and diagnosis of various glomerular diseases.

Toward High-Resolution Cryo-Electron Microscopy: Technical Review on Microcrystal-Electron Diffraction

Lee, Sangmin,Chung, Jeong Min,Jung, Hyun Suk Korean Society of Microscopy 2017 Applied microscopy Vol.47 No.4

Cryo-electron microscopy (cryo-EM) is arguably the most powerful tool used in structural biology. It is an important analytical technique that is used for gaining insight into the functional and molecular mechanisms of biomolecules involved in several physiological processes. Cryo-EM can be separated into the following three groups according to the analytical purposes and the features of the biological samples: cryo-electron tomography (cryo-ET), cryo-single-particle reconstruction, and cryo-electron crystallography. Cryo-tomography is a unique EM technique that is used to study intact biomolecular complexes within their original environments; it can provide mechanistic insights that are challenging for other EM-methods. However, the resolution of reconstructed three-dimensional (3D) models generated by cryo-ET is relatively low, while single-particle reconstruction can reproduce biomolecular structures having near-atomic resolution without the need for crystallization unless the samples are large (>200 kDa) and highly symmetrical. Cryo-electron crystallography is subdivided into the following two categories according to the types of samples: one category that deals with two-dimensional (2D) crystalline arrays and the other category that uses 3D crystals. These two categories of electron-crystallographic techniques use different diffraction data obtained from still diffraction and continuous-rotation diffraction. In this paper, we review crystal-based cryo-EM techniques and focus on the recently developed 3D electron-crystallographic technique called microcrystal-electron diffraction.

The Effects of Electron Beam Exposure Time on Transmission Electron Microscopy Imaging of Negatively Stained Biological Samples

Kim, Kyumin,Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Korean Society of Microscopy 2015 Applied microscopy Vol.45 No.3

Negative staining electron microscopy facilitates the visualization of small bio-materials such as proteins; thus, many electron microscopists have used this conventional method to visualize the morphologies and structures of biological materials. To achieve sufficient contrast of the materials, a number of imaging parameters must be considered. Here, we examined the effects of one of the fundamental imaging parameters, electron beam exposure time, on electron densities generated using transmission electron microscopy. A single site of a negatively stained biological sample was illuminated with the electron beam for different times (1, 2, or 4 seconds) and sets of micrographs were collected. Computational image processing demonstrated that longer exposure times provide better electron densities at the molecular level. This report describes technical procedures for testing parameters that allow enhanced evaluations of the densities of electron microscopy images.

Molecular Structure of Muscle Filaments Determined by Electron Microscopy

Craig, Roger Korean Society of Microscopy 2017 Applied microscopy Vol.47 No.4

Electron microscopy and X-ray diffraction have together played a key role in our understanding of the molecular structure and mechanism of contraction of muscle. This review highlights the role of electron microscopy, from early insights into thick and thin filament structure by negative staining, to studies of single myosin molecule structure, and finally to recent high-resolution structures by cryo-electron microscopy. Muscle filaments are designed for movement. Their labile structures thus present challenges to obtaining near-atomic detail, which are also discussed.

Recent Advances in Electron Crystallography

Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Korean Society of Microscopy 2017 Applied microscopy Vol.47 No.3

Electron crystallography has been used as the one of powerful tool for studying the structure of biological macromolecules at high resolution which is sufficient to provide details of intramolecular and intermolecular interactions at near-atomic level. Previously it commonly uses two-dimensional crystals that are periodic arrangement of biological molecules, however recent studies reported a novel technical approach to electron crystallography of three-dimensional crystals, called micro electron-diffraction (MicroED) which involves placing the irregular and small sized protein crystals in a transmission electron microscope to determine the atomic structure. In here, we review the advances in electron crystallography techniques with several recent studies. Furthermore, we discuss the future direction of this structural approach.

주사 전자 현미경에서 전자빔 프르브 생성을 위한 하드웨어 설계

임선종,이찬홍 한국공작기계학회 2007 한국공작기계학회 추계학술대회논문집 Vol.2007 No.-

Electron probe refers to the focused electron beam at the specimen. It generates the various image signals. Image of SEM is dependent upon the beam parameters: electron-probe size, electron-probe-current and electron probe convergence angle. In this paper, we presented hardware design for generation of electron probe.

Electron-Microscope Contributions to Autophagy Research and the Nobel Prize in Physiology or Medicine 2016

Rhyu, Im Joo Korean Society of Microscopy 2017 Applied microscopy Vol.47 No.1

Professor Yoshinori Ohsumi received the 2016 Nobel Prize in Physiology or Medicine for his contribution to autophagy research, which was first studied using electron microscopy. To celebrate and commemorate this historical moment, I describe the role of electron microscopy in autophagy research and suggest a role for next-generation electron microscopy in this research field.

Nano-Resolution Connectomics Using Large-Volume Electron Microscopy

Kim, Gyu Hyun,Gim, Ja Won,Lee, Kea Joo Korean Society of Microscopy 2016 Applied microscopy Vol.46 No.4

A distinctive neuronal network in the brain is believed to make us unique individuals. Electron microscopy is a valuable tool for examining ultrastructural characteristics of neurons, synapses, and subcellular organelles. A recent technological breakthrough in volume electron microscopy allows large-scale circuit reconstruction of the nervous system with unprecedented detail. Serial-section electron microscopy-previously the domain of specialists-became automated with the advent of innovative systems such as the focused ion beam and serial block-face scanning electron microscopes and the automated tape-collecting ultramicrotome. Further advances in microscopic design and instrumentation are also available, which allow the reconstruction of unprecedentedly large volumes of brain tissue at high speed. The recent introduction of correlative light and electron microscopy will help to identify specific neural circuits associated with behavioral characteristics and revolutionize our understanding of how the brain works.

Implant Surface Changes Observed with Confocal and Electron Microscopy After Probing with Metal and Plastic Periodontal Probes

진승호,박준범,손주완,고영경 대한구강악안면임플란트학회 2023 대한구강악안면임프란트학회지 Vol.27 No.4

Purpose: Probing around dental implants is recommended for the monitoring of peri-implant tissue health, but there are some risks associated with this procedure. There is a paucity of concrete evidence that contact with periodontal probes may leave remnants or indentations on the implant surface. The objective of this study was to provide visual evidence of the effects of probing around a rough-surface implant with metal and plastic periodontal probes, as assessed by confocal and scanning electron microscopy. Materials and Methods: Rough surface implants were placed in a pig mandible to simulate 8 mm of peri-implant bone loss. Probing around the entire circumference of the implants was performed using a metal and a plastic periodontal probe. Surface analysis was performed using confocal and scanning electron microscopy. Results: Confocal microscopy showed plastic remnants left on the thread peaks of the implant. Scanning electron microscopy showed smoothening of the sandblasted and acid-etched implant after probing. Conclusion: Contact with a periodontal probe leads to changes in the surface of rough-surface implants. The use of plastic probes may leave plastic remnants on the implant surface.

고압전자현미경을 이용한 소뇌 조롱박세포 가지돌기가시 관찰

유임주,이계주,서영석,Rhyu, Im-Joo,Lee, Kea-Joo,Suh, Young-Suk 한국현미경학회 2001 Applied microscopy Vol.31 No.1

신경세포 가지돌기가시의 형태를 분석하는 것은 신경세포의 기능을 이해하는데 중요하다. 가지돌기가시는 광학현미경 해상도의 한계근처에 있는 구조물로 투과전자현미경 및 공초점헌미경 등을 이용한 연구들이 보고 되고 있다. 고압전자현미경은 높은 해상도와 투과능력 덕분에 두꺼운 절편의 관찰이 용이하여 신경세포의 가지돌기가시 등을 관찰하는데 유용한 것으로 알려져 있다. 고압전자현미경을 이용하여 신경세포의 가지돌기가시를 효과적으로 관찰하는방법을 확인하고 기본적인 형태학적 자료를 축적하고자 하였다. 생쥐 소뇌에 위치하는 조롱박세포의 가지돌기가시를 anti-calbindin 28kD항체 및 Golfi 염색으로 표지한 후 $4{\mu}m$두께의 절편을 제작하여 impregnation방법으로 각각 처리하여 표본을 제작한 후, 초고압전자현미경으로 관찰하여 효과적인 관찰방법을 찾고, 영상분석 기법을 이용하여 가지돌기가시의 밀도와 가시의 길이를 측정하였다. 초고압전자현미경 관찰 결과, 면역조직화학법과 Golgi법 모두 조롱박세포의 가지돌기가시를 관찰할 수 있었으나 Golgi법으로 준비된 표본이 가시를 정량적으로 분석하기에 더욱 적합하였다. 명상분석 결과로는 가지돌기 가시의 평균밀도가 $24.5{\pm}3.6$개/$10{\mu}m$였고, 가시의 평균길이는 $1.12{\pm}0.22{\mu}m$였다. 본 연구를 통해서 Gogli 법으로 염색된 조롱박세포를 고압전자현미경으로 관찰할 경우, 가지돌기가시를 정량적으로 관찰할만한 만족스러운 영상을 얻을 수 있었고, 추후 경사를 주어 촬영한 두 장의 사진을 이용하여 3차원적으로 분석하면 좀 더 정확한 결과를 얻을 수 있을 것으로 판단되며, 이는 소뇌의 신경가소성을 이해하는데 중요한 자료가 될 것이다. The morphological features of neuronal dendritic spines are changed their shapes, sizes and density in response to physiological or pathological conditions . Therefore, exact analysis of spines warrants understanding of neuronal function. The size of the spine is at the borderline of resolution with light microscopy. High voltage electron microscopy Provide excellent resolution of the spines with proper stain techniques thanks to its higher resolution and penetration power. We evaluated more effective staining method for observing dendritic spines after labeling Purkinje cells with anti-calbindin 28 kD immunohistochemistry or Golgi staining methods. 4 fm thickness sections were observed with high voltage electron microscopy and some morphometric analyses were performed. Both Golgi staining and immunohistochemistry revealed the detail structures of the Purkinje cell such as soma, dendrites, and dendritic spines. High voltage electron micrographs with Golgi staining provide more precise morphology and are easy to measure. Average density of spine is $24.5{\pm}3.6/10{\mu}m$ and its length is $1.12{\pm}0.22{\mu}m$. For quantitative analysis of the spines, high voltage electron, micrographs with Golgi staining are more effective. This preliminary result is expected to be useful for further study of spine plasticity in various conditions.