Abstract
O2 PLIM microscopy was employed in various studies, however current platforms have limitations in sensitivity, image acquisition speed, accuracy and general usability. We describe a new PLIM imager based on the Timepix3 camera (Tpx3cam) and its application for imaging of O2 concentration in various tissue samples stained with a nanoparticle based probe, NanO2-IR. Upon passive staining of mouse brain, lung or intestinal tissue surface with minute quantities of NanO2-IR or by microinjecting the probe into the lumen of small or large intestine fragments, robust phosphorescence intensity and lifetime signals were produced, which allow mapping of O2 in the tissue within 20 s. Inhibition of tissue respiration or limitation of O2 diffusion to tissue produced the anticipated increases or decreases in O2 levels, respectively. The difference in O2 concentration between the colonic lumen and air-exposed serosal surface was around 140 µM. Furthermore, subcutaneous injection of 5 µg of the probe in intact organs (a paw or tail of sacrificed mice) enabled efficient O2 imaging at tissue depths of up to 0.5 mm. Overall, the PLIM imager holds promise for metabolic imaging studies with various ex vivo models of animal tissue, and also for use in live animals.
Highlights
Quantitative monitoring of molecular oxygen ( O2) concentration and its spatial distribution is important for many physiological studies with cells, tissue samples and whole organisms including humans1–5. O2 is the key substrate and environmental parameter for living systems, and a useful functional readout for respiring samples and their metabolic responses related to oxygen consumption[6,7]
Imaging of macroscopic samples such as organs and whole animals in Phosphorescence Lifetime IMaging (PLIM) mode is still rare[35,36,37]; in a standard microscope setup, image area is too small for the analysis of O2 dynamics on the organ level
We have shown that the resolving power of our PLIM macro-imager, which utilizes a 256 × 256 pixels imaging chip, is 12.7 lp/mm[38]
Summary
Quantitative monitoring of molecular oxygen ( O2) concentration and its spatial distribution is important for many physiological studies with cells, tissue samples and whole organisms including humans1–5. O2 is the key substrate and environmental parameter for living systems, and a useful functional readout for respiring samples and their metabolic responses related to oxygen consumption[6,7]. Various phosphorescent probes including cell-permeable or impermeable, small molecule, supramolecular dendrimers, nano- or micro-particulate structures, as well as planar solid-state O2 sensors are available[6,9,10,11,12,13,14,15]. They allow sensing and imaging of O 2 in various types of samples in intensity, ratiometric and lifetime-based detection mode, including the time-gated and time-correlated single photon counting (TCSPC) imaging on wide-field, confocal or multi-photon microscopes[6,16]. Imaging of macroscopic samples such as organs and whole animals in PLIM mode is still rare[35,36,37]; in a standard microscope setup, image area is too small for the analysis of O2 dynamics on the organ level
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