The singlet-oxygen-sensitized delayed fluorescence in mammalian cells: a time-resolved microscopy approach

Abstract

The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 μM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 μs and a TPPS4 triplet lifetime of τT = 22 ± 5 μs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems.

Imaging of singlet oxygen feedback delayed fluorescence and lysosome permeabilization in tumor in vivo during photodynamic therapy with aluminum phthalocyanine

“…In addition, it is interesting to note that the DF lifetime is slightly shortened during the PDT, which is unexpected, because lengthening of lifetime due to oxygen depletion is rather anticipated. 21 Figure 5(b) shows that a relatively small radiant exposure (6 J∕cm 2 ), can lead, on the contrary, to a remarkable increase in DF signal (by 30% to 100% in different animals, n ¼ 3). In this specific case, this exposure did not induce an appreciable lysosome permeabilization in the tumor, as can be seen from the absence of a lifetime increase in the PF lifetime image.…”

“…14 Recently, we proposed a completely different approach to monitoring singlet oxygen. [20][21][22] It is based on the mechanism of singlet oxygen feedback delayed fluorescence (SOFDF), [23][24][25] which is intrinsic to a number of different photosensitizers (protoporphyrin IX, ionic porphyrins, porphycenes, eosin, Rose Bengal, and others), 20,[26][27][28] and hence no additional probe molecule is required. The triplet state of a photosensitizer (T 1 ) can interact with the previously generated 1 O 2 , which leads to a reverse intersystem crossing (RISC) of the photosensitizer molecule and repopulation of the S 1 state that can subsequently emit a photon.…”

“…In vivo typically τ T ≫ τ Δ , which allows us to express the SOFDF emission rate as 20,21 E Q -T A R G E T ; t e m p : i n t r a l i n k -; e 0 0 2 ; 1 1 6 ; 5 6 7…”

“…However, given that intracellular lifetimes of 1 O 2 are likely below 1 μs, 29 it can be challenging to resolve the rise of SOFDF kinetics, especially if background signals are present. 21 Triplet lifetimes and hence SOFDF decay times are often dictated by the concentrations of 3 O 2 that acts as an efficient quencher.…”

Photosensitized singlet oxygen generation and detection: Recent advances and future perspectives in cancer photodynamic therapy

“…Recently, time‐resolved microscopic detection of SOSDF from single living 3T3 mouse fibroblasts incubated with 100 µM TPPS4 and TMPyP has been successfully achieved, which suggests that SOSDF can be considered as a promising semi‐direct method for 1 O 2 detection in biological environments. Although this semi‐direct method generally exhibits much stronger signal than NIR 1 O 2 luminescence for detection, the specificity for 1 O 2 detection is still a great challenge . Therefore, only direct 1 O 2 luminescence measurement will be discussed in detail here.…”