Peer review of: Single-molecule photoreaction quantitation through intraparticle-surface energy transfer (i-SET) s  (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

This manuscript addresses experimental realization of highly efficient Forster resonant energy 
transfer from nanoparticles to molecules at the surface by doping Tb3+ inside the 
nanocrystals/nanoparticles, as well as resultant great enhancement of photoluminescence from the 
surface molecules. Tb has advantages compared to other lanthanide elements because vibrational 
levels of the surface molecules are well isolated from the electronic levels of the Tb3+ (that are 
relevant to the excitations by NIR) and the electronic levels of the surface molecules, which 
prevents fast non-radiative decay. The authors unambiguously present their key results through 
many different types of measurements and control experiments by varying the dopant types, Tb 
concentration, the number of dye molecules, the number of BDP molecules (surface molecules), 
and local environment. In addition, a phenomenological model adapted from Ref.36 supports the 
experimental data with several fitting parameters. The manuscript is very well written and it is 
recommended for publication with the following minor revision. 

There are two typos: 
1) On page 3, right hand side, 2nd line: 4D5 --> 5D4 in “From the 5D4 state of a nearby Tb3+....” 
2) In Ref.36: The page number is incorrect. 

There are a few scientific questions on the manuscript. 
1) On page 3, the authors mentioned that “the calculation shows the presence of intraparticle 
Tb3+-Tb3+ interaction increases energy transfer efficiency by a factor of 4-25, depending on the 
coverage (20-1 BDP per nanoparticle) of dye molecules (Fig. 2d)”. But if this is from a simple 
model with fitting parameters that the authors presented in the supporting information, it would 
be good that the authors explain a bit more about this statement. Experimentally it is not possible 
to measure the distance between the neighboring Tb3+ dopants inside the nanoparticles. Perhaps 
the authors have drawn this conclusion from the concentration of the Tb3+ for a given 
nanoparticle size. 
2) Interestingly, in Fig.S5, the peak from 5D4 to 7F5 is higher than that from 5D4 to 7F6, although 
7F6 is the ground electronic level of Tb3+. Is there any chemical or physical reason for this? 
3) From the experimental data, it is clear that the energy is transferred from the Tb3+ to the 
surface molecules. However, how would the authors know that the excited energy is transferred 
from one Tb3+ to nearest neighboring Tb3+ site? Isn’t just a higher concentration of Tb3+ enough 
to explain the experimental data? 



Reviewer #2 (Remarks to the Author): 

The article "Single-molecule photoreaction quantitation through intraparticle-surface energy 
transfer (i-SET) spectroscopy" by J.Zhou et al. presents intraparticle-surface energy transfer using 
Tb3+ ions aiming to study single fluorophores tethered on a single NP, in opposite to existing 
studies, where number of acceptor fluorophores is increased. The idea of removing Yb3+ ions 
(highly susceptible to surface quenching by overtones of O-H vibrations) and exploiting high 
energy gap Tb3+ as donor) is smart, original and novel and it is convincingly and nicely presented. 
The article is very well written/prepared, experiments are convincing and sufficiently well planned 
to demonstrate the hypotheses. However some points require correction and more in depth 
explanations. 

1. What is the power density of CW 980 nm excitation in Fig.S5? How does it compare to excitation 
intensities required for conventional YbEr UC ? The question arises from a fact, the CSU is typically 
100-times weaker than e.g. YbEr UC. In the same time, stronger pumping at 980 nm (required for 
CSU) could lead to increased and undesired heating in water environments - did authors 
measure/notice such effects ? 

2. why 5%Tb@... sample (Fig.S11) shows double exponential behavior while the samples with 
higher Tb content do not? 

3. Why infinitely small YbEr@Y NPs show FRET efficiency, which is dependent on number of BDP 
acceptors, unlike similarly small YbTb@Tb NPs ? I cannot see a reason for that, which somehow 
questions the modelling results in its current form. 

4. The history of Yb-Tb upconversion is slightly longer and rich. 

5. Fig.2f - it is not clear and not explained why such behavior is observed and what it really means. 
More in depth discussion is needed, including the discussion about the radiative rates of A and D 

6. Fig.4 - what are the expected reasons for >60 minutes photo stability when most A molecules 
photo bleach faster. I know this is statistics, but can the authors draw deeper conclusions about 
this interesting observation. Does this behavior change (and how) with excitation intensity, 
number of A molecules per NP? What is the environment the measurements were made in ? Most 
biosensors require water based conditions. 

7. I found numerous figures and tables in SI, which are not commented in main article (which 
would be desirable, so don't skip these figures, but add comments). For example - how the 
number of A dyes per NP has been calculated ? Some more detailed information about statistics of 
number of A molecules per NPs should be provided and discussed. 

8. The authors presented acceptor molecules directly anchored to the surface of NP. But actual 
biosensors require bio-specific molecules such as antibody-antigen or DNA molecules, which will 
inrease the distance between superficial Tb donors to the acceptor present a few nm away (due to 
intermediate bio-recognition molecules). What is the Forster distance for the given Tb-BDP 
molecules ? Did authors test any distance dependence for this interesting NPs ? 

Some language mistakes have been found and need correction (e.g. "intensity of single BDP 
molecule_S_", "reaches _to_ a maximum" and others). 

Overall, I liked the concept and the quality of the presented results. I find it novel and I am sure 
this will be inspiration for other scientists working in the field of bio sensing and lanthanide doped 
applications. 



Reviewer #3 (Remarks to the Author): 

This study by Deng and coworkers demonstrates single-particle energy transfer between 
lanthanides in upconversion nanoparticles (UCNPs) and surface-bound BODIPY fluorophores, 
optimizing Ln content and demonstrating wire-like Ln chains that mediate energy transfer from the 
core of the nanoparticle to surface fluorophores. This is an interesting and dense study (with about 
40 SI figures), and it includes some demanding single particle experiments. Overall, I would say 
the characterization of these nanoparticles is rigorous and interesting, particularly the impressive 
experiments in which the fluorophores are bleached one at a time to reveal stoichiometry. Some of 
the design work of how to optimize signal from the fluorophores is similar to previous studies from 
the past few years, and my sense is the paper should emphasize its key strength, which is the 
single particle work. I think a manuscript with some revisions would make a compelling addition to 
the UCNP and single particle imaging literature. 

Specific comments: 
1) Much of the nanoparticle design work here has been explored in detail before, including similar 
shell structures of NaLnF4 (JACS 141, 42 (2019)) and optimization of energy transfer to 
fluorescent reporters by varying Ln doping and surface dye attachment (Opt Mat 84, 345 (2018)). 
The latter paper also has data on enhanced fluorophore photostability, which is somewhat at odds 
with the current study. It would be useful to put the current findings in context of this prior work. 

2) “As shown in Figure 2c, even at a low BDP concentration of ~0.5 BDP per nanoparticle, the 
sensitized emission from BDP molecules is still detectable with a signal-to-noise ratio of up to 30.” 

Signal:noise is not the correct term for the calculation in 2c. For cuvette measurements, the noise 
of a fully background-subtracted spectrum is 0, so signal:noise would be infinite. More appropriate 
here would be a difference spectrum to see if the peak matches the free BODIPY dye emission, 
and to calculate the ET efficiency. 

This BODIPY has an unfortunately small Stokes shift, which may make calculations of ET efficiency 
difficult. 

3) The discussion of i-SET (a new term?) and cartoons in Fig 1 leave the impression of direct-
contact energy transfer, such as Dexter e- transfer. I don’t think this is likely, nor is there 
evidence for this. Even if an i-SET Ln wire ET process moves energy from the nanocrystal interior 
toward the surface, ET between Ln and fluorophore is still most likely a FRET process. The authors 
might consider clearer ways to distinguish FRET from the core vs FRET from the shell. If there is 
evidence of non-FRET processes such as Dexter, the authors should highlight it. 

4) Given the direct contact between BODIPY and heavy atom shell, as well as rapid O2-mediated 
quenching, do the authors believe triplets are forming in the BODIPY? There is evidence of this in 
NIR dye sensitization of Yb/Er UCNPs, although this remains somewhat controversial. 

5) “Nevertheless, for conventional Er3+, Tm3+, or Ho3+ activators co-doped with Yb3+ or 
Nd3ions as sensitizers21-23, a high content of activator and sensitizer combination would 
generally lead to low luminescence efficiencies of the nanoparticles due to concentration quenching 
effects24.” 

There is a growing literature at this point on high-Ln UCNPs, and it has become clear that 
concentration quenching is not a significant factor in core/shell UCNPs. The authors seem to 
recognize that adding Yb-free shells necessarily cuts off the prime energy loss pathway. See e.g., 
JACS 139, 3275 (2017) and Nat Comm 9, 3082 (2018) for previous, in-depth discussions of high 
Ln core/shell UCNPs and why the concept of concentration quenching is dated. I think the 
discussion in the current paper can take this prior work into account to substantially shorten this 
part of the manuscript.