Identifying and localizing intracellular nanoparticles using Raman spectroscopy

Abstract

Raman microscopy is employed to spectroscopically image biological cells previously exposed to fluorescently labelled polystyrene nanoparticles and, in combination with Kmeans clustering and Principal Component Analysis (PCA), is demonstrated to be capable of localising the nanoparticles and identifying the subcellular environment based on the molecular spectroscopic signatures. The neutral nanoparticles of 50 nm or 100 nm, as characterised by dynamic light scattering, are shown to be non-toxic to a human lung adenocarcinoma cell-line (A549), according to a range of cytotoxicity assays including Neutral Red, Alamar Blue, Coomassie Blue and (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal fluorescence microscopy identifies intracellular fluorescence due to the nanoparticle exposure, but the fluorescence distribution is spatially diffuse, potentially due to detachment of the dye from the nanoparticles, and the technique fails to unambiguously identify the distribution of the nanoparticles within the cells. Raman spectroscopic mapping of the cells in combination with K-means cluster analysis is used to clearly identify and localise the polystyrene nanoparticles in exposed cells, based on their characteristic spectroscopic signatures. PCA identifies the local environment as rich in lipidic signatures which are associated with localisation of the nanoparticles in the endoplasmic reticulum. The importance of optimised cell growth conditions and fixation processes is highlighted. The preliminary study demonstrates the potential of the technique to unambiguously identify and locate nonfluorescent nanoparticles in cells and to probe not only the local environment but also changes to the cell metabolism which may be associated with cytotoxic responses.

Raman spectroscopic mapping for the analysis of solar radiation induced skin damage

“…It is a routine technique for fingerprinting and identifying chemicals and acts as a standard method of analytical pharmacy and chemistry 8 . The potential of vibrational spectroscopy for diagnostic applications has been demonstrated, notably in dermal applications [9][10][11] , as well as for in vitro screening of toxicological effects of ionising radiation 12 , nanoparticles 13,14 and the action of chemotherapeutic agents 15 . Raman and infrared (IR) spectroscopy additionally provide detailed information of the molecular structure and composition of the tissue, ultimately promising an analysis of disease origin.…”

Cellular discrimination using in vitro Raman micro spectroscopy: the role of the nucleolus

“…14,15 In comparison to Infrared absorption spectroscopy; Raman micro spectroscopy offers the possibility to study biological matrices in an aqueous environment due to the weak contribution from water. 16,17 Thus, the potential applications extend beyond disease diagnostics to the label free in vitro screening of cytological processes, such as drug or nanoparticle uptake and mechanisms of interaction, and toxicology. 16,[18][19][20] There has been a wide range of studies to date demonstrating the potential of Raman micro spectroscopy to map live and fixed cells with subcellular resolution, [21][22][23][24][25] profile the distribution of anticancer agents [26][27][28][29][30] and nanoparticles in cells 16,31,32 and monitor subcellular processes 33 and toxicological responses.…”

Label‐free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines

“…Confocal Raman microscopy has been introduced recently as a promising tool for label free analysis of cells and 3D tissue cultures 37, and has been used to detect stem cell differentiation 38, tumor cells 39 and particle uptake 40. In the field of biotechnology and bioengineering, Raman spectroscopy has been used to monitor CHO cell characteristics for on‐line process monitoring 41, 42.…”