Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Abstract

Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution.

Keywords: Raman spectroscopy; SRS microscopy; alkyne tags; glucose uptake; imaging agents.

Figure 1

Structures of existing D-glucose analogues and synthesis of the new 3-O-propargyl-D-glucose. (a) [¹⁸F]2-Fluoro-2-deoxy-D-glucose ([¹⁸F]FDG). (b) [¹¹C]3-O-methyl-D-glucose ([¹¹C]3-OMG). (c) 2-NBD-2-deoxy-D-glucose (2-NBDG). (d) Synthetic route of 3-OPG 1. a) NaH, propargyl bromide, DMF, RT, 12 h, 90%; b) Dowex® 50WX8, H₂O, 80°C, 20 h, 96%.

Figure 2

Uptake of 3-OPG in live mammalian cells. (a) Spontaneous Raman spectra of 25 mM 3-OPG solution in PBS (black) and HeLa cells incubated with 25 mM 3-OPG for 4 hours (red). (b) SRS imaging of 3-OPG uptake (2129 cm⁻¹) in live HeLa cells incubated with 8 mM 3-OPG for 1 hour. The average intracellular concentration is estimated to be 3–5 mM. Images at 2000 cm⁻¹ (off-resonance) and 1655 cm⁻¹ (protein amide I) show the same area of cells. The 2129 cm⁻¹ color bars in all images correspond to a linear 3-OPG concentration range of 0–26 mM, unless otherwise noted. Scale bar: 20 µm.

Figure 3

Glucose transporter dependence of 3-OPG uptake in mammalian cells. Representative SRS images of live HeLa cells incubated with 3-OPG in the presence of (a) 0 mM D-glucose, (b) 10 mM D-glucose, (c) 50 mM D-glucose, and (d) 50 mM L-glucose. (e, f) HeLa cells were transfected with 10 nM negative control siRNA (e) or GLUT1 siRNA (f) before incubation with 3-OPG. (g, h) HeLa cells were incubated with 3-OPG in the presence of DMSO vehicle (g) or 10 µM cytochalasin B (h). Scale bar: 20 µm. (i) Relative SRS intensity of 3-OPG uptake in HeLa cells under the conditions above. Data are shown as Mean + standard deviation (SD) (n≥3 replicates for each group). *, p<0.05; ***, p<0.001 by Student’s t-test. p<0.05 is considered statistically significant.

Figure 4

SRS imaging of 3-OPG uptake in U-87 MG tumor xenograft tissues. (a) Strong uptake of 3-OPG in the proliferating region of tumor. (b) Little uptake is seen in the tumor region with necrotic cell morphology. The 2129 cm⁻¹ color bars in (a, b) correspond to a 3-OPG concentration range of 0–53 mM. (c) Sharp contrast of 3-OPG uptake at the interface of two tumor regions. Scale bar: 40 µm.

Figure 5

SRS imaging of 3-OPG uptake in neuronal systems. (a) SRS images of cultured mouse primary hippocampal neurons incubated with 3-OPG. Scale bar: 20 µm. (b) SRS images of mouse brain tissue slice cultured ex vivo with 3-OPG. Significant uptake of 3-OPG is seen at the granule cell layer of dentate gyrus. Scale bar: 40 µm.