Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.

Nearly all arthropod-associated Wolbachia contain intact and/or genomic remnants of phage WO, temperate bacteriophages that facilitate horizontal gene transfer, genomic rearrangement of the bacterial chromosome, and symbiotic interactions between Wolbachia and their arthropod hosts. Integrated prophage WO genomes produce active, lytic particles; but the lack of a cell-free culturing system for Wolbachia render them difficult to purify and study. This chapter describes polyethylene glycol (PEG) precipitation of phage particles from Wolbachia-infected arthropods, followed by confirmation of phage WO isolation and purification using electron microscopy and PCR.

Transmission electron microscopy (TEM) has revolutionized our understanding of protein structures by enabling atomic-resolution visualization without the need for crystallography, thanks to advancements in cryo-TEM and single particle analysis methods. However, conventional electron microscopy remains relevant for studying stained samples, as it allows the practical determination of optimal conditions through extensive experimentation. TEM also facilitates the examination of supramolecular complexes encompassing proteins, lipids, and nucleic acids. In this study, we investigated the applicability of lanthanoid reagents as electron-staining alternatives to uranyl acetate, which is globally regulated as a nuclear fuel material. We focus on a model biomembrane vesicle system, the chromatophores from the purple photosynthetic eubacterium Rhodospirillum rubrum, which integrate proteins and lipids. Through density distribution analysis of electron micrographs, we evaluated the efficacy of various lanthanoid acetates and found that triacetates of neodymium, samarium, and gadolinium exhibited similar staining effectiveness to uranyl acetate. Additionally, triacetates of praseodymium, erbium, and lutetium, followed by europium show promising results as secondary candidates. Our findings suggest that lanthanoid transition heavy metal acetates could serve as viable alternatives for electron staining in TEM, offering potential advantages over uranyl acetate.

Knowledge of the clinical liver anatomy has evolved with advanced imaging modalities and laparoscopic surgery. Therefore, precise anatomical resection knowledge has become the standard treatment for primary and secondary liver cancer. Segmentectomy, a parenchymal-preserving approach, is regarded as an option for anatomical resections in patients with impaired liver. Indocyanine green (ICG) staining is a promising method for understanding the anatomical borders of the liver segments. There are two methods of ICG staining (positive and negative), and the superiority of either approach has not been determined to date.
This is a prospective randomised controlled superiority clinical trial performed in a single centre tertiary hospital in Japan. A comparison between the accuracy of positive and negative ICG staining in guiding laparoscopic anatomical liver resection is planned in this study. Possible candidates are patients with liver malignant tumours in whom laparoscopic monosegmentectomy or subsegmentectomy is planned. Fifty patients will be prospectively allocated into the following two groups: group A, ICG-negative staining group, and group B, ICG-positive staining group. The optimal dose of ICG for positive staining will be determined during the preparation phase. To assess the ability of the ICG fluorescence guidance in anatomical resection, the primary endpoint is the success rate of ICG staining, which consists of a SOS based on three components: superficial demarcation in the liver surface, visualisation of the parenchymal borders and consistency with the preoperative three-dimensional simulation. The secondary endpoints are the evaluation of short-term surgical outcomes and recurrence-free survival.
The study was approved by Ageo Central General Hospital Clinical Research Ethical Committee (No: 1044) and it carried out following the Declaration of Helsinki (2013 revision). Informed consent will be taken from the patients before participating. The findings will be disseminated through peer-reviewed publications, scientific meetings and conferences.
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Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

Many members of the phylum Bacteroidota (formerly called Bacteroidetes) adhere to and move on solid surfaces. This type of bacterial motility is called gliding and does not involve the conventional bacterial motility machinery, such as flagella and pili. To understand the mechanism of gliding motility of some Bacteroidota bacteria such as a soil bacterium Flavobacterium johnsoniae and a marine bacterium Saprospira grandis, the gliding motility machines of these two bacteria have been analyzed by electron microscopy with negative staining. Here, we describe methods to directly observe the gliding motility machinery in Bacteroidota by transmission electron microscopy.

Morphological characteristics of liposomes, such as size and lamellarity directly impact their quality and biological performance of encapsulated drug. Gaining insights into these parameters may also help ensure identification and utilization of most efficient process parameters for liposomes manufacturing. Direct imaging of such self-assembling colloidal structures, although challenging, is feasible through transmission electron microscopy (TEM) which uses nanometer scale wavelength of electrons for illumination, enabling an accurate assessment of the morphological characteristics of liposomes. This chapter will provide background information on the working principle and general sample preparation procedure for the two most commonly used TEM techniques for imaging liposomes, viz. negative staining transmission electron microscopy and cryogenic transmission electron microscopy.

The Golgi apparatus is a highly dynamic organelle that controls lipid and protein sorting in the endocytic and exocytic cellular pathways. Perturbation of the lipid homeostasis or of the molecular machineries that regulate membrane remodeling/trafficking events on the Golgi membranes can dramatically change the morphology and functions of the Golgi apparatus. So far, several approaches have been described to characterize and define the Golgi morphology in intact cells and in vitro. Here, we describe the application of negative staining (NS) electron microscopy (EM) on purified Golgi membranes from HeLa cells. This approach allows to quantify and functionally characterize membrane remodeling events upon specific treatments that alter the Golgi morphology.

To minimize the loss of functional liver volume in cases of severe cirrhosis and repeat hepatectomy for recurrence of hepatocellular carcinoma (HCC), anatomical hepatectomy is gradually extended from major to minor hepatectomy (Miyama et al. in Cancers (Basel):13, 2021; Ishizawa et al. in Ann Surg 256:959-964, 2012). For local located HCC, (sub)segmentectomy can yet be regarded as a choice instead of hemihepatectomy. Indocyanine green (ICG) has been used for tumor location, navigation of resected margin and liver segment, and identification of bile leakage. Negative stain that ICG dye was administered intravenously after occluding the target portal pedicle is more applicable to sectionectomy or hemihepatectomy, especially in cases where multiple target pedicles exist or portal vein puncture is difficult to carry out to achieve anatomic resection. Herein, we present a video of laparoscopic segmentectomy III and IV with ICG fluorescence negative stain using Glisson Pedicle approach.
A 49-year-old woman with hepatitis B related cirrhosis for 2 years was referred for treatment of a single nodule in segment IV invading the umbilical portion of left portal vein. The preoperative alpha-fetoprotein (AFP) was 442 ng/ml and protein induced by vitamin K absence or antagonist-II (PIVKA-II) was 122 mAu/ml. Liver function was Child-Pugh A and indocyanine green retention test at 15 min (ICG-R15) was 9.2%. The surgical procedure involved the following steps: (1) Extrahepatic Glisson pedicle dissection based on Laennec\'s s capsule (Sugioka et al. in J Hepatobiliary Pancreat Sci 24:17-23, 2017) was performed for isolation of the pedicles towards segments III and IV in the umbilical fossa. (2) Demarcation line was revealed and ICG (1 ml, 5 mg/l) was administered intravenously for the negative stain after dividing the target pedicles. (3) Parenchyma transection was performed along the border of the negative staining area in the cranial and caudal direction.
Operative time was 220 min and blood loss was 150 ml with no transfusion. HCC sized 2.5 cm*1.7 cm*1.2 cm was confirmed in histopathology with a free margin and no microvascular invasion. The fibrosis of the liver parenchyma was S4 based on Ishak system. The patient was discharged on the postoperative day 6 without any complications. No recurrence in residual liver was noted on the CT scan at 9 months during follow-up.
Laparoscopic segmentectomy III and IV is an effective procedure for HCC especially in cases with demands of hepatic parenchymal preservation. ICG navigation and Glisson Pedicle approach may be particularly helpful.