Fig 1.
Photophysical model of Cy5 with transitions 1 to 16.
General photophysical processes: 1 – photoexcitation (EXC), 2 – fluorescence (FLU), 3 – internal conversion (IC) + vibrational relaxation (VR), 4 – intersystem crossing (ISC) + VR, 5 – ISC + VR, 6 – isomerization (ISO), 7 – back-isomerization (BISO), 8 – photobleaching (BLE); dSTORM-specific photophysical processes: 9 – photoinduced electron transfer from thiol (PET) to either S1 or T1 and ending up in either Cy5-SR-, Cy5-∙ or S0, 10 – OFF to ON via thermal elimination (TE) or photoinduced uncaging (PU), 11 – oxidation (OXI); Energy transfers (only acceptors vary, donor transitions always from S1 to S0): 12 – singlet-singlet annihilation (SSA), 13 – singlet-triplet annihilation (STA), 14 – FRET to cis-Cy5 (CET), 15 – FRET to OFF (OET), 16 – reverse ISC (RISC). Note the specific implementations described in the text.
Fig 2.
Photophysical statistics of a single Cy5 fluorophore with continuous excitation.
Photobleaching is disabled. (A – F) Statistics that are experimentally not available. They can also be predicted (crosses for A – E, line for F) using tools like the limiting distribution of the Markov chain. (A) The probability of occurrence of each photophysical state. (B) The probability of occurrence of each photophysical transition. (C) The lifetime of each photophysical state. (D) The transition lifetime of each photophysical transition. (E) The probability of occupation of each photophysical state. (F) The ePDF of durations of the photophysical state S1 overlaid with an exponential decay of corresponding relaxation time. (G – L) Analyses that are experimentally available. (G) The fluorescence trajectory showing photons per frame over time. (H) The eCDF of photon arrival times. (I) The ePDF of photon counts per frame. (J) The fluorescence correlation curve . (K) The ePDF of OFF periods (consecutive frames with photon count < 10). (L) The ePDF of ON periods (consecutive frames with photon count ≥ 10).
Fig 3.
Parameters that control the shape of PFA of single Cy5 fluorophores.
(A, B) Altering the photobleaching rate . (C, D) Altering the rates associated with entering the OFF state (
, see Table B in S1 Text). (E, F) Altering the rates associated with entering the ON state (
, see Table B in S1 Text). (A, C, E) The eCDF of photon arrival times. The markers depict simulated results. The analytical expression (solid lines) and fitting procedure are described in section 8 in S1 Text. (B) Probability histogram of total photon counts during 300 s. (D) Probability histogram of ON periods. (F) Probability histogram of OFF periods. A full set of all corresponding plots is provided in Fig H in S1 Text. For adjusting
via thiol concentration, see Fig I in S1 Text.
Fig 4.
PFA of multi-fluorophore systems undergoing OET.
(A) Two fluorophores, (B) three fluorophores, (C – H) four fluorophores. The photophysical model includes OET leading the OFF state to transition to S0 with a probability of 0.01%. (A – D) The eCDF of photon arrival times. In case of (D), the data is truncated such that only times ≥ 3 s are included to mimic acquisition in the beginning of an experiment. The markers depict simulated results. The analytical expression (solid lines) and fitting procedure are described in section 8 in S1 Text. (E) Probability histogram of photon counts per frame. (F) Probability histogram of ON periods. (G) Probability histogram of OFF periods. (H) Probability histogram of number of ON periods, i.e., ON events. A full set of all corresponding plots is provided in Fig L in S1 Text. Representative fluorescence time traces are provided in Fig M in S1 Text. Representative photophysical statistics of four fluorophores at 3 nm are provided in Fig N in S1 Text.
Table 1.
Fluorescence lifetimes in different simulated conditions. The fluorescence lifetimes are recorded using only S1 durations that deexcite via fluorescence. Each condition is simulated 10 times for 108 steps. N is the number of fluorophores, the distance is set to 3 nm, photobleaching is disabled. The adjustment numbers represent different changes to the photophysical model, either qualitatively or quantitatively: 1 – × 0.01 and
is 0,
× 0.1, i.e., CET and STA are set such that their impact on fluorescence lifetime is maximized (see Fig Q in S1 Text); 2 – energy transfer to R (
), where 2.1 only considers S1|R to S0|R, 2.2 also considers S1|R to S0|S0 with efficiency 10-4 and 2.3 with efficiency 10-3; 3 –
× 10; 4 –
× 100. A more detailed version of this table is provided in Table D in S1 Text.