Replacing critical point drying with a low-cost chemical drying provides comparable surface image quality of glandular trichomes from leaves of Millingtonia hortensis L. f. in scanning electron micrograph

Bhattacharya, Raktim; Saha, Sulagna; Kostina, O. V.; Muravnik, Lyudmila E.; Mitra, Adinpunya

doi:10.1186/s42649-020-00035-6

Cited by 31 publications

(13 citation statements)

References 15 publications

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“…Apart from a long solvent exchange process, water can also be chemically converted to methanol and acetone by a fast reaction with 2,2-dimethoxypropane (DMP) at pH below 6 [67,86]. Since the removal of these solvents by evaporation still does not preserve the morphology well, they should be further exchanged with some water-immiscible liquids such as tetramethylsilane (Me 4 C, TMS) or hexamethyldisilazane (Me 3 -Si-NH-Si-Me 3 , HMDS) [85,[87][88][89][90][91]]. An even better option is to exchange them with CO 2 at supercritical conditions (above 31 • C and 7,3 •10 6 Pa), for which the surface tension vanishes [67,92].…”

Section: Dehydrationmentioning

confidence: 99%

Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids

et al. 2021

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Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every cell and present in biofluids. Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions. However, a prerequisite for such usage of EVs is their detailed characterisation, including visualisation which is mainly achieved by atomic force microscopy (AFM) and electron microscopy (EM). Here we summarise the EV preparation protocols for AFM and EM bringing out the main challenges in the imaging of EVs, both in their natural environment as biofluid constituents and in a saline solution after EV isolation. In addition, we discuss approaches for EV imaging and identify the potential benefits and disadvantages when different AFM and EM methods are applied, including numerous factors that influence the morphological characterisation, standardisation, or formation of artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. Here presented comparison of approaches to EV imaging should help to estimate the current state in morphology research of EVs from human biofluids and to identify the most efficient pathways towards the standardisation of sample preparation and microscopy modes.

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Section: Dehydrationmentioning

confidence: 99%

Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids

et al. 2021

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“…To avoid the collapse and shrinkage of E. coli cells, samples were carefully dehydrated following the protocol of biological sample preparations for scanning electron microscopy. 22 From topography imaging in Figure 4a, we observed that the E. coli cell has a rod shape of approximately 0.9 μm in width and 1.7 μm in length. For PFIR imaging, we first acquired dual-color images of E. coli simultaneously at 1744 and 1248 cm −1 shown in Figure 4c,d, respectively.…”

Section: ■ Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Dual-Color Peak Force Infrared Microscopy

Xie

Wiemann

et al. 2021

Anal. Chem.

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Peak force infrared (PFIR) microscopy achieves nanoscale infrared imaging at sub-10 nm spatial resolution through photothermal mechanical detection of atomic force microscopy (AFM). However, it suffers from a major limitation that only one infrared frequency can be scanned for an AFM frame at a time. To overcome this limitation, we report here dual-color PFIR microscopy that enables simultaneous imaging at two infrared frequencies. This dual-color PFIR microscopy bypasses the limitations of frame drift and distortion of AFM when comparing two images of different infrared frequencies. We benchmark the performance and spatial resolution of this method using structured polymers exhibiting phase separation. We further demonstrate the application of this technique in imaging biological samples by mapping the cell wall of Escherichia coli (E. coli) bacteria. The presence of a bacterial outer membrane was detected without extrinsic labels. This dual-color PFIR microscopy enables simultaneous nondestructive chemical nanoimaging of multiple chemical components and will be useful for potential applications such as in situ dual-channel monitoring of chemical reactions.

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“…Following fixation, samples were rinsed with 1× PBS buffer for 10 min and subjected to a series of ethanol dehydration: 50%, 70%, 80%, and 100% ethanol, a 1:1 ethanol and hexamethyldisilazane (HMDS) solution, and HMDS absolute for 15–20 min for each step at room temperature. Samples then were laid on filter paper in mini glass petri dish with absolute HDMS overnight [ 26 , 27 ]. Samples were mounted on aluminum stubs with carbon adhesive tape and colloidal silver paint (Electron Microscopy Sciences, Hatboro, PA, USA) followed by sputter coating with gold-palladium using a Desk IV thin film coating system (Denton Vacuum, Moorestown, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

Localization of the Swainsonine-Producing Chaetothyriales Symbiont in the Seed and Shoot Apical Meristem in Its Host Ipomoea carnea

et al. 2022

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Several species of fungi from the orders Chaetothyriales and Pleosporales have been reported to produce swainsonine and be associated as symbionts with plants of the Convolvulaceae and Fabaceae, respectively. An endosymbiont belonging to the Chaetothyriales produces swainsonine and grows as an epibiont on the adaxial leaf surfaces of Ipomoea carnea, but how the symbiont passes through plant growth and development is unknown. Herein, different types of microscopy were used to localize the symbiont in seeds and in cross sections of plant parts. The symbiont was found in several tissues including the hilum, the sclereids, and the hypocotyl of seeds. In five-day old seedlings and mature plants, the symbiont was found in the shoot apical meristem (SAM) and the adaxial surface of immature folded leaves. The mycelia generally formed a close association with peltate glandular trichomes. This report provides further data explaining the relationship between the seed transmitted Chaetothyriales symbiont and Ipomoea carnea. These results provide a possible explanation for how this symbiont, and others like Periglandula may persist and are transmitted over time.

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Replacing critical point drying with a low-cost chemical drying provides comparable surface image quality of glandular trichomes from leaves of Millingtonia hortensis L. f. in scanning electron micrograph

Cited by 31 publications

References 15 publications

Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids

Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids

Dual-Color Peak Force Infrared Microscopy

Localization of the Swainsonine-Producing Chaetothyriales Symbiont in the Seed and Shoot Apical Meristem in Its Host Ipomoea carnea

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