Abstract
Understanding and quantifying the temporal acquisition of host cell molecules by intracellular pathogens is fundamentally important in biology. In this study, a recently developed holographic optical trapping (HOT)‐based Raman microspectroscopy (RMS) instrument is applied to detect, characterize and monitor in real time the molecular trafficking of a specific molecular species (isotope‐labeled phenylalanine (L‐Phe(D8)) at the single cell level. This approach enables simultaneous measurement of the chemical composition of human cerebrovascular endothelial cells and the protozoan parasite Toxoplasma gondii in isolation at the very start of the infection process. Using a model to decouple measurement contributions from host and pathogen sampling in the excitation volume, the data indicate that manipulating parasites with HOT coupled with RMS chemical readout was an effective method for measurement of L‐Phe(D8) transfer from host cells to parasites in real‐time, from the moment the parasite enters the host cell.
Highlights
IntroductionAll biological processes involve highly orchestrated interactions between cells at a molecular level
The other Raman bands have been extensively reported in the literature, and can be assigned to molecular vibrational modes of proteins, lipids, nucleic acids and carbohydrates
Time-course spectra of optically trapped T. gondii (Figure 1C) were acquired over a duration of 4 minutes to investigate potential spectral changes caused by adverse effects caused by the laser irradiation
Summary
All biological processes involve highly orchestrated interactions between cells at a molecular level. Understanding these complex processes requires the ability to manipulate cells and control their interactions without compromising. Tools are needed to measure the underlying molecular properties of cells to detect changes in their biochemical properties (eg, transfer of molecules involved in cell-cell signaling). The research problem addressed is how molecular trafficking events can be measured from the moment the parasite first encounters its surrogate host cell
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