Real‐time Raman and SRS imaging of living human macrophages reveals cell‐to‐cell heterogeneity and dynamics of lipid uptake

Abstract

Monitoring living cells in real‐time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real‐time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages.

Applications of vibrational tags in biological imaging by Raman microscopy

“…Matthäus et al used d 31 -palmitic acid and d 33 -oleic acid to quantify the fatty acid scavenging kinetics in human macrophages by Raman spectroscopic imaging 35 . Recently the same group applied SRS imaging with faster speed and revealed heterogeneous uptake dynamics across the cell population 45 . Not all fatty acids are metabolized in the same way.…”

Invited Article: Comparison of hyperspectral coherent Raman scattering microscopies for biomedical applications

“…Coherent Raman imaging experiments have been performed using a compact fiber laser pumped optical parametric oscillator (OPO, picoEmerald S, APE, Berlin, Germany). 13 The OPO is pumped by the second harmonic of an Yb-fiber laser emitting pulses at 80 MHz pulse repetition rate of 2 ps duration and 1032 nm central wavelength. This laser is also used as the Stokes beam in the imaging experiments.…”

“…For CRS imaging, the laser was coupled into a home-built laser scanning microscope equipped with a 20× microscope objective (20× NIR Apo, NA = 0.4, Mitutoyo, Japan) imaging the sample using 47 mW laser power at the sample. 13,14 Both CARS and SRS signals are detected in the forward direction. A dichroic longpass beam splitter at 735 nm (Semrock, USA) separates SRS and CARS signals.…”

Uptake of Retinoic Acid‐Modified PMMA Nanoparticles in LX‐2 and Liver Tissue by Raman Imaging and Intravital Microscopy

“…Consequently, the chemical properties remain unaltered, however, the spectroscopic features change significantly by shifting the observed scattering intensities of the CH/CD bonds by about 1000 wavenumbers. The concept of stable isotopic labeling has for instance been applied to study uptake dynamics of individual amino acids, deuterated lipids, and liposomal nanoparticles …”