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Aldehydes fixation was accidentally discovered in the early 20th century and soon became a widely adopted practice in the histological field, due to an excellent staining enhancement in tissues imaging. However, the fixation process itself entails cell proteins denaturation and crosslinking. The possible presence of artifacts, that depends on the specific system under observation, must therefore be considered to avoid data misinterpretation. This contribution takes advantage of scanning electron assisted-dielectric microscopy (SE-ADM) and Raman 2D imaging to reveal the possible presence and the nature of artifacts in unstained, and paraformldehyde, PFA, fixed MNT-1 cells. The high resolution of the innovative SE-ADM technique allowed the identification of globular protein clusters in the cell cytoplasm, formed after protein denaturation and crosslinking. Concurrently, SE-ADM images showed a preferential melanosome adsorption on the cluster's outer surface. The micron-sized aggregates were discernible in Raman 2D images, as the melanosomes signal, extracted through 2D principal component analysis, unequivocally mapped their location and distribution within the cells, appearing randomly distributed in the cytoplasm. Protein clusters were not observed in living MNT-1 cells. In this case, mature melanosomes accumulate preferentially at the cell periphery and are more closely packed than in fixed cells. Our results show that, although PFA does not affect the melanin structure, it disrupts melanosome distribution within the cells. Proteins secondary structure, conversely, is partially lost, as shown by the Raman signals related to α-helix, ß-sheets, and specific amino acids that significantly decrease after the PFA treatment.

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RESUMEN

Electron microscopes can observe samples with a spatial resolution of 10 nm or higher; however, they cannot observe samples in solutions due to the vacuum conditions inside the sample chamber. Recently, we developed a scanning electron-assisted dielectric microscope (SE-ADM), based on scanning electron microscope, which enables the observation of various specimens in solution. Until now, the SE-ADM system used a custom-made SE-ADM stage with a built-in amplifier and could not be linked to the scanning electron microscopy (SEM) operation system. Therefore, it was necessary to manually acquire images from the SE-ADM system after setting the EB focus, astigmatism, and observation field-of-view from the SEM operating console. In this study, we developed a general-purpose dielectric constant imaging unit attached to commercially available SEMs. The new SE-ADM unit can be directly attached to the standard stage of an SEM, and the dielectric signal detected from this unit can be input to the external input terminal of the SEM, enabling simultaneous observation yielding SEM and SE-ADM images. Furthermore, 4.5 nm spatial resolution was achieved using a 10 nm thick silicon nitride film in the sample holder in the observation of aggregated PM2.5. We carried out the observation of cultured cells, PM2.5, and clay samples in solution.

RESUMEN

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

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