Label-Free, Real-Time Measurement of Metabolism of Adherent and Suspended Single Cells by In-Cell Fourier Transform Infrared Microspectroscopy.

Tommaso Vannocci,Giulia Milordini,Antonio De Riso,Annalisa Pastore,Magda Wolna,Gianfelice Cinque,Luca Quaroni

doi:10.3390/ijms221910742

Abstract

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

Highlights

We used IR microscopy to monitor the metabolic activity of suspended versus adhered living HEK-derived cells by relying on the intrinsic IR light absorption from multiple molecular species in the sample
Whereas adherent cells displayed the hallmarks of a metabolism relying on glycolysis followed by lactic fermentation, suspended cells did not display any metabolic activity within the detection limits of IR microscopy
We identified a spectral effect that appears to arise from the interplay of synchrotron infrared radiation and the cells and manifests itself as oscillations in the spectral domain that slowly develop over time

Summary

Introduction

Addressing the interplay of cellular metabolism and morphology calls for analytical methods with the capability to quantitatively monitor metabolite concentration in time and space across living cells at single-cell resolution. Mass spectrometry provides the highest sensitivity and specificity, but the application to live cell sampling, using ambient ionization methods, requires at last partial cytoplasmic extraction, preventing recording of time-resolved events [5]. An optimal method should be quantitative, non-invasive, require minimal or no sample pre-treatment, and be applicable to cell culture models and living organisms. It should afford molecular specificity and the capability to simultaneously detect multiple molecular species, ideally without a priori target selection

Methods

Results

Conclusion