Intracellular oxygen: Similar results from two methods of measurement using phosphorescent nanoparticles

Abstract

The ability to resolve the spatio-temporal complexity of intracellular O 2 distribution is the \Holy Grail" of cellular physiology. In an e®ort to obtain a minimally invasive approach to the mapping of intracellular O 2 tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 m focused spot. Both methods utilized Ru coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium-sapphire Tsunami lasers (710-1050 nm).

Reactive oxygen species and redox compartmentalization

“…Nevertheless, it is not possible to exclude that alterations in the cell physiology occur due to introduction of a ~30 kDa protein as a fluorescent sensor or that the presence of a redox sensitive probe may interfere with the physiological redox signaling. In addition, tissue and cellular oxygen distribution may also play a role in ROS detection, since it is not homogeneous (Williams et al, 2012 ; Lloyd et al, 2014 ). These concerns need to be taken into account, and, when possible, appropriate controls should be performed.…”

Purely Organic Microparticles Showing Ultralong Room Temperature Phosphorescence

“…In the last years, small organic phosphorescent particles have been successfully used in a variety of devices for sensing and imaging . They can be incorporated in chemical sensors − or light-emitting diodes, − used in O 2 sensing, , for in vivo imaging, or in phosphorescent immunoassays. , Most of the organic particles discussed in the literature contain metal complexes such as Pt­(II), − Ir­(III), − Eu­(III), or others. , Since the heavy metal effect induces an efficient spin–orbit coupling, they have long been used to promote organic phosphorescence. , The aim for environmentally friendly alternatives, low cost, and easy processability led to a huge effort in the preparation of small purely organic particles showing room temperature phosphorescence (RTP). Several strategies can be pursued, including H-Aggregates, − encapsulation, , polymerization, , and host-guest doping. , The big advantage of RTP concerning in vivo imaging is the enhanced signal-to-noise ratio compared to fluorescence since in the afterglow emission, no luminescent background signal is present .…”

“…12,13 Most of the organic particles discussed in the literature contain metal complexes such as Pt(II), 14−16 Ir(III), 17−19 Eu(III), 20 or others. 21,22 Since the heavy metal effect induces an efficient spin−orbit coupling, they have long been used to promote organic phosphorescence. 23,24 The aim for environmentally friendly alternatives, low cost, and easy processability led to a huge effort in the preparation of small purely organic particles showing room temperature phosphorescence (RTP).…”

Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging

“…Moreover, many of these methods are dependent on injection of an organic compound or insertion of a needle or catheter into the desired location that may result in cytotoxicity, inflammation, injury to the tissue, and artifacts in the measurements. 12 The primary means of detecting [O 2 ] in the microscope have been either photoacoustic (point measurements with a few microns resolution) or phosphorescent. The latter relies on the Stern-Volmer quenching of a luminophore by collision, and the 10 s to 100 s of microseconds between such collisions are in reach of the phosphors lifetimes (τ p ).…”

“…Optical assessment of [O 2 ] using phosphorescence intensity alone can also be misleading because the concentration of phosphorescent dye and/or intensity of the excitation light are different in different regions inside a cell or tissue. 9,12,14,15 We sought to instead develop a fluorescence lifetime microscopy (FLIM) probe appropriate to intracellular targeting. Inspired by Thompson's use of protein Zn 2þ sensors coupled to fluorophores, 16 we sought to couple a reporter fluor to the natural [O 2 ] binding protein myoglobin.…”