Cell‐Nanoparticle Interactions at (Sub)–Nanometer Resolution Analyzed by Electron Microscopy and Correlative Coherent Anti‐Stokes Raman Scattering

Abstract

A wide variety of nanoparticles are playing an increasingly important role in drug delivery. Label‐free imaging techniques are especially desirable to follow the cellular uptake and intracellular fate of nanoparticles. The combined correlative use of different techniques, each with unique advantages, facilitates more detailed investigation about such interactions. The synergistic use of correlative coherent anti‐Stokes Raman scattering and electron microscopy (C‐CARS‐EM) imaging offers label‐free, chemically‐specific, and (sub)‐nanometer spatial resolution for studying nanoparticle uptake into cells as demonstrated in the current study. Coherent anti‐Stokes Raman scattering (CARS) microscopy offers chemically‐specific (sub)micron spatial resolution imaging without fluorescent labels while transmission electron microscopy (TEM) offers (sub)‐nanometer scale spatial resolution and thus visualization of precise nanoparticle localization at the sub‐cellular level. This proof‐of‐concept imaging platform with unlabeled drug nanocrystals and macrophage cells revealed good colocalization between the CARS signal and electron dense nanocrystals in TEM images. The correlative TEM images revealed subcellular localization of nanocrystals inside membrane bound vesicles, showing multivesicular body (MVB)−like morphology typical for late endosomes (LEs), endolysosomes, and phagolysosomes. C‐CARS‐EM imaging has much potential to study the interactions between a wide range of nanoparticles and cells with high precision and confidence.

Nanoscopy for endosomal escape quantification

“…For a process such as endosomal escape -that is fast, rare and at the nanoscale -it may seem challenging to answer the various questions projected using individual methods. But as we have seen in this review, we are no longer restricted to a 'one method at a time' approach (70,74,142,(144)(145)(146). The benefits of correlative imaging -especially of SRM-EM -are of tremendous relevance in obtaining quantitative information on NP endosomal trafficking.…”

“…A more distinct approach was achieved by Saarinen et al 70 who used correlative coherent anti-Stokes Raman scattering and TEM (C-CARS-EM) to image glibenclamide-nanocrystals (GLI-NCs) in macrophages ( Fig. 2c , right).…”

“…With a near atomic resolution (57), EM is an irreplaceable tool in studying the physio-chemical properties of NP and quantifying their voyage through the endo-lysosomal pathway (30,37,(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74). EM can even detect a low number (few hundreds) of single nanoparticles escaping endosomal structures, and since it is a label-free method, it will localise and quantify NP generally untraceable by standard light microscopy methods.…”

In vitro Label Free Raman Microspectroscopic Analysis to Monitor the Uptake, Fate and Impacts of Nanoparticle Based Materials

“…Although currently only commercially available in limited frequency ranges, full spectral coherent Raman microspectroscopic imaging has already been demonstrated (Camp et al, 2014), promising label free high content spectroscopic analysis of sub-cellular processes in real-time, and hitherto unrivaled visualization of cellular processes and function. Indeed, CARS has been employed, in conjunction with electron microscopy to explore cellnanoparticle interactions at nanometer resolution (Saarinen et al, 2019), while SRS using polymer nanoparticles has been employed for multiplexed live-cell imaging (Hu et al, 2017).…”

SESIUM KLORIDA (CsCl) : TRANSPORT ION DALAM LARUTAN

“…akan tetapi saat ion bergerak melalui pelarut, maka ion akan mengalami gaya gesekan sehingga memperlambat Ie ' yang sebanding dengan kecepatannya. Jika diasumsikan bahwa hukum Stokes (187)(188)(189)(190)(191) untuk bola radius a dan s berlaku pada skala mikroskopis ,maka kita dapat menuliskan gaya perlambatan ini sebagai:…”