生物物理学

Tracking macrophages by non-invasive molecular imaging can provide useful insights into the immunobiology of inflammatory disorders in preclinical disease models. Perfluorocarbon nanoemulsions (PFC-NEs) have been well documented in their ability to be taken up by macrophages through phagocytosis and serve as ¹⁹F magnetic resonance imaging (MRI) tracers of inflammation in vivo and ex vivo. Incorporation of near-infrared fluorescent (NIRF) dyes in PFC-NEs can help monitor the spatiotemporal distribution of macrophages in vivo during inflammatory processes, using NIRF imaging as a complementary methodology to MRI. Here, we discuss in depth how both colloidal and fluorescence stabilities of the PFC-NEs are essential for successful and reliable macrophage tracking in vivo and for their detection in excised tissues ex vivo by NIRF imaging. Furthermore, PFC-NE quality assures NIRF imaging reproducibility and reliability across preclinical studies, providing insights into inflammation progression and therapeutic response. Previous studies focused on assessments of colloidal property changes in response to stress and during storage as a means of quality control. We recently focused on the joint evaluation of both colloidal and fluorescence properties and their relationship to NIRF imaging outcomes. In this protocol, we summarize the key assessments of the fluorescent dye–labeled nanoemulsions, which include long-term particle size distribution monitoring as the measure of colloidal stability and monitoring of the fluorescence signal. Due to its simplicity and reproducibility, our protocols are easy to adopt for researchers to assess the quality of PFC-NEs for in vivo NIRF imaging applications.

Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in E. coli, the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in H₂O at ambient conditions, is label free, without any limitations in protein size and does not require any purification step.

Graphic abstract:

In-cell infrared difference spectroscopy on photoreceptors in living E. coli using attenuated total reflection.