Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy

Abstract

Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type-specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

Facilitated Transport of EGFR Inhibitors Plays an Important Role in Their Cellular Uptake

“…For example, SRS can directly probe the metabolic dynamics of single cells . It could also potentially image cell phenotypic changes . Together, SRS imaging could potentially provide a comprehensive characterization of cell drug exposure and drug response in tumor tissue.…”

Simultaneous Dual-Band Hyperspectral Stimulated Raman Scattering Microscopy with Femtosecond Optical Parametric Oscillators

“…These examples show the system’s capability of acquiring quantitative chemical information in the fingerprint region or cell silent region with Raman tags, such as deuterium labeling and alkyne tags, while simultaneously obtaining high-fidelity morphological features from the C–H stretching region. In addition, it is possible to tune the OPOs to simultaneously excite the O–H and C–H stretching modes for cell density and tissue lipid and protein concentration measurements. , With the three-dimensional imaging capability of SRS, we can also extend this technique to 3D tissue and in vivo applications. Furthermore, we showed the potential of the system in broadband SRS imaging with a dual-modulation configuration, which allows rapid collection of rich spectral information on major biochemical constituents, including proteins, lipids, and DNA.…”

“…In addition, it is possible to tune the OPOs to simultaneously excite the O−H and C−H stretching modes for cell density and tissue lipid and protein concentration measurements. 29,56 With the three-dimensional imaging capability of SRS, we can also extend this technique to 3D tissue and in vivo applications. Furthermore, we showed the potential of the system in broadband SRS imaging with a dualmodulation configuration, which allows rapid collection of rich spectral information on major biochemical constituents, including proteins, lipids, and DNA.…”

“…However, it only allows for the simultaneous detection of two Raman transitions within 100 cm –1 limited by the bandwidth of the pump beam . Other approaches, such as two-color SRS using a polarization encoding scheme, also have a limited separation between the two targeted Raman transitions of around 100–300 cm –1 . − …”

Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples

“…Magnetic levitation methods determine the density of single cells by balancing the cell's gravity and the buoyancy exerted by a paramagnetic medium 9,10 . Methods detecting dry-mass density (dry mass over total volume), such as quantitative phase microscopy (QPM) or Raman imaging coupled with cell volume measurements, provide alternative density measurements 11,12,13,14,15,16 . Although these methods provide subcellular resolution and single-cell tracking, experiments published thus far with mammalian cells contain tens to hundreds of single cells when measuring cell density.…”