Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging.

Abstract

Quantitative-phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative-phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a digital holographic microscope (DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how quantitative-phase digital holographic microscopy (QP-DHM) can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-DHM can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM.