Multiplex Raman imaging of organelles in endothelial cells

Abstract

Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Twodimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.

Biomedical applications, perspectives and tag design concepts in the cell – silent Raman window

“…32 A multiplex approach using both EdU and BADY has been adopted by Matuszyk et al to explore endothelial dysfunction in in vitro models through detection of lipid-rich organelle in endothelial cells. 33 In other cases, diynes have been conjugated with sphingomyelin, a cell membrane lipid, 34 and employed as a Raman label in lipid raft measurements monitoring the characteristic 2263 cm À1 Raman band. 35 Jamieson et al employed alkyne tagged fatty acids to monitor their uptake and distribution in single cells using conventional Raman microscopy.…”

Analytical Techniques for Single-Cell Biochemical Assays of Lipids

“…An alkyne-tagged cholesterol labeled LDs in a liver cancer cell line, yielding the molecular identification of as many as seven different constituents of LDs, as well as the spectral isolation of three structurally different lipid species (110). Falcarinol, an anti-inflammatory polyacetylene that naturally scatters in the silent region, has been used to investigate cellular changes resulting from induced endothelial dysfunction (111). Deuterium is also commonly used as a Raman tag to visualize lipids (112,113).…”

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function

“…An alternative, potentially more robust approach to RM is to use so-called Raman probes (Rp) to increase the selectivity and sensitivity of Raman imaging. Labeled RM takes advantage of the silent Raman spectral region (1800–2800 cm –1 ), where no characteristic signal from biomolecules occurs. – For this purpose, mitochondria-targeted moiety lipophilic triphenylphosphonium cation (TTP + ) combined with bisarylbutadiyne (BADY) (MitoBADY) is most often used. The signal from MitoBADY colocalizes with cytochrome c when it is used in low concentration and short incubation time to avoid nonspecific accumulation.…”