Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in

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Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells Jing Shi, Ming Zhou, Aihua Gong, Qijun Li, Qian Wu, Gary J. Cheng, Mingyang Yang, and Yaocheng Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03689 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Analytical Chemistry

Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells

Jing Shia, Ming Zhou*a,b, Aihua Gongc, Qijun Lia, Qian Wua, Gary J. Chengb, Mingyang Yanga, Yaocheng Sunc

[*] Ming Zhou* State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, People’s Republic of China Email: Ming Zhou ([email protected] )

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Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells Jing Shi†, Ming Zhou*†, ‡, Aihua Gong§, Qijun Li†, Qian Wu†, Gary J. Cheng‡, Mingyang Yang†, Yaocheng Sun§ †

State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, People’s Republic of China

‡

Department of Industrial Engineering, Purdue University, 225 South University Street, West Lafayette, Indiana 47907, USA §

School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, People’s Republic of China

ABSTRACT: The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biological and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepared nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomolecules.

Cancer is the leading cause of mortality worldwide1. It poses a great challenge to diagnose cancer early and provide treatment for cancer patients. Researchers have proved that endogenous tumor-related mRNA is a specific indicator of cancer and its expression level can help to illustrate the disease progression and prognosis2-4. Thus, the identification and characterization of tumor-related mRNA in cells hold great promise for the early diagnosis and treatment of cancer. Various methods have provided insight into identifying and characterizing mRNA, such as Northern-blots5, 6, microarray analysis7, 8 and reverse transcription polymerase chain reaction (RT-PCR) 9, 10. However, these approaches are laborious and require a large number of cells, making them unsuitable for identification of single cells in real time11. Fluorescence-intensity based measurements have also been developed for mRNA detection in live cells12-16. One of the alternatives is nanoflare, a nanoprobe first brought forward in 2007 for intracellular mRNA detection and now widely used for characterizing particular intracellular molecules as well as labeling and isolating specific cells17-20. The nanostructure consists of a gold nanoparticle coated with a dense layer of recognition oligonucleotides whose sequences are complementary to the target mRNA but hybridized to so-called flare oligonucleotides with fluorophore modification. Because of the close proximity between the particle and the fluorophore, the fluorescence is quenched by the gold nanoparticle21-25. Encountering target mRNA, the recognition sequence hybridizes to it, forming longer and more stable duplex structure, and thus liberate the flare oligonucleotides, with their quenched fluorescence restored correspondingly. By examining the amplified fluorescence signal, specific mRNA in cells can be detected. Besides quenching fluorescence, the AuNP also plays the part of transfection agents with low toxicity26.

Furthermore, attaching several kinds of antisense oligonucleotides targeting at different mRNA to the surface of AuNPs and using flares modified with fluorophores of different colors, multiple kinds of biomolecules can be determined simultaneously with a single nanoconjugate through different fluorescence responses. With quencherlabeled aptamers, nanoflares can also be used for sensitive and selective detection of other biomarkers, such as adenosine triphosphate (ATP) 27. However, detection based on fluorescence intensity has such disadvantages as the influence of intensity-signal artifacts, high background signal and may exhibit false-positive results. Therefore, there is an urgent need for new techniques to precisely detect specific mRNA in cells Fluorescence lifetime imaging microscopy (FLIM) is a technique that provides spatially resolved images of the distribution of fluorophore lifetime28, 29. Since lifetime is an intrinsic property of fluorophore and is sensitive to the fluorophore’s microenvironment but independent of the multiple intensity artifacts30, it has been used as an important indicator of the dynamic interactions between fluorophore-labeled molecules and micro changes in the environment of a biological system. At present, this technique has been exploited to provide insight into molecular interactions as well as to reveal various biological information in cells, such as the differentiation of NADH and NADPH in live cells31, and intracellular dynamic processes32. And it has also been performed with FRET to investigate protein interactions33-35, providing an additional source of contrast for fluorescence-associated study. Thus, compared with the routinely employed fluorescence intensity-based steady state fluorescence microscopy, FLIM provides a different approach to manifesting the same fluorescence-associated phenomenon with supplementary information and more reliable results.


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Herein, we made use of fluorescence lifetime imaging of nanoflares to detect endogenous mRNA selectively. We designed and prepared the AuNP-DNA complex termed nanoflares. The gold nanoparticle is coated with a monolayer of recognition oligonucleotides hybridized to flare oligonucleotides with cyanine dye modification through thiol-gold bond. The localized field induced by the gold nanoparticle can greatly enhance the non-radiative decay rate of the fluorophore due to energy transfer and shortens its lifetime consequently. In the presence of mRNA of interest, recognition sequences attached to AuNP will hybridize to the targets and the fluorophore-modified oligonucelotides will be released from gold nanoparticle as previously described and thus get free from the lifetime modification induced by gold. By examining the fluorescence decay trace in cells, the fluorescence lifetime can be calculated, providing an accurate way to detect target mRNA in cells. In addition to preparing and characterizing the lifetime change of the nanostructure in response to the target molecules in extracelluar environment, we transferred nanoflares into cells and recorded the fluorescence lifetime map. Fluorescence intensity-based assays have also been carried out as demonstration and the outcome of which was in agreement with that of fluorescence lifetime-based experiments, verifying the feasibility of detecting and signifying mRNA in cells using fluorescence lifetime imaging as well as its potential usage in other biological investigations. For proof-of-concept study, we fabricated the complex as a probe targeting at exon8 of BRCA1 mRNA. BRCA1 is a human tumor suppressor gene that plays an important role in repairing damaged DNA whose mutation will increase the risk of breast cancer. As illustrated in figure 1, ex8recognition antisense oligonucleotides form a dense monolayer on the ~13nm self-made gold nanoparticle through a thiol linker at 3’ end and are hybridized with ex8-flares in a sequence specific manner. The three components make up the nanostructure termed nanoflare. Since cy5 is at a distance of ~2nm from the surface of gold, the nanoparticle induces a fluorescence quenching effect through energy transfer mechanism and shortens its fluorescence lifetime. When the nanoflares encounter target sequences, ex8-flares are displaced from the surface of the nanoparticle, with their fluorescence as well as lifetime getting restored. When the nanoflares are taken up by breast cancer cells, ex8-recognition DNA hybridizes to part of BRCA1 mRNA sequences and the ex8-flares displace, exhibiting longer fluorescence lifetime.

Figure 1. Principle of the experiment.

Fluorescence study indicated that 54±3 flare sequences were bound to each gold nanoparticle (Fig S2) and binding study was conducted with perfectly matched single stranded DNA termed ex8-target to demonstrate the feasibility of recognizing specific mRNA through lifetime change. A non-complementary nanoflare whose recognition sequences are complementary to ex8-flare but is several bases mismatched with target mRNA was also prepared as a control. Lifetime measurements were performed with Picoquant PicoHarp 300 TCSPC module exciting at 640nm and recorded at 690±35nm channel. Representative lifetime decay curves and corresponding fitting values were shown in Table 1 and Figure 2. Compared with that of free ex8-flares, the lifetime of nanoflares decays rapidly with a small peak which is attributed to the quenching effect induced by the gold nanoparticle in close proximity. When 200 fold excess target sequences were added to nanoflares, the peak disappeared. This phenomenon can be intuitively understood as the result of target binding and flare releasing. To further disentangle the effect of gold nanoparticle on the fluorescence lifetime, the recorded lifetime trace data was fitted by exponential functions. The measured fluorescence decay traces of ex8-flares and nanoflares with excessive target oligonucleotides were fitted to a double-exponential function as

−𝑡 −𝑡 I(t) = A1 exp ( ) + A2 exp ( ) 𝜏1 𝜏2 However, the fluorescence decay curve of nanoflares was fitted to a triple one −𝑡

−𝑡

−𝑡

I(t) = A1 exp ( 𝜏1 ) + A2 exp ( 𝜏2 ) + A3exp⁡( 𝜏3 ) Where τ1, τ2, τ3 are the three components of lifetime decay, τ3 is the ultra-short one induced by the gold nanoparticle and the A1, A2, A3 are their amplitudes correspondingly. As shown in table 1, the lifetime of ex8-flares exhibits a two-exponential character and an average lifetime of ~1.66ns. It is obvious that the presence of gold nanoparticle induces an ultrashort lifetimeτ3 (0.08~0.11ns) of cy5 and greatly shortens the nanoflares’ fluorescence lifetime to ~0.466ns. With 200 fold ex8-targets, the lifetime of



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nanoflares lengthened to ~1.232ns again. As a control, we quenched lifetime remained (~0.513ns) nearly the same, added the same amount of non-targets of random sedemonstrating the detection selectivity of the nanoflares. quences as that of ex8-targets to the nanoflares, and the Table 1 Fluorescence lifetimes of prepared nanoflares and the control in 1×PBS solvents

A1

A2

A3

τ1

Ex8-flare

9.06(0.058)

3.789(0.071)

0

NF

2.545(0.086)

2.401(0.061)

NF+target

5.725(0.081)

NF+nontarget

3.68(0.18)

τ2

τ3

×2

τav

1.8996(0.003) 1.074(0.012)

--

1.038

1.656

5.0(0.061)

1.939(0.017)

0.0933(0.002) 1.003

0.466

6.61(0.08)

0

1.745(0.0085) 0.788(0.0079) --

3.30(0.1)

7.35(0.063)

1.877(0.024)

0.954(0.033)

0.874(0.037)

1.031

0.0917(0.002) 1.035

1.232 0.5129

NF is short for nanoflares. The numbers in brackets are deviations of fitting.

little for the control as expected. The consistency between fluorescence-intensity measurements and fluorescence lifetime results suggests that lifetime change is a good indicator of the presence of target DNA.

Figure 2. Representative fluorescence decay traces of nanoflares, ex8-flares and nanoflares treated with excessive target DNA.

Since the lifetime decay of ex8-flares and that of nanoflares are characterized by a double- and triple-exponential function respectively, it is reasonable to claim that the variation of amplitude ratio of A3/(A1+A2+A3), which we define as ε, is a qualitative indicator of the proportion of the two components, namely ex8-flares in bound state and in free state. In some published articles, the fluorescence intensity responds to the targets in a dose dependent manner implying the gradual displacement of flares with the increase of targets27, so it is thought that ε decreases with an increasing amount of target DNA. We prepared 1nM nanoflares with increasing concentration of ex8-targets and non-targets as a control to examine the fluorescence lifetime and fluorescence intensity. As indicated by figure 3, following the introduction of ex8-targets, the ε decreased nonlinearly, suggesting that an increasing amount of flares was released. It should be noted that when the concentration of the targets reached 80nM, the fluorescence decay trace can be fitted by a double-exponential curve perfectly, leading to the absence of A3 in the range of 100~200nM target DNA. This suggests that most of the bounded ex8-flares have been released into the solution. As for the control group, the fluorescence decay data is barely affected by the concentration of non-targets. To demonstrate the results, we also measured the fluorescence intensity of each sample. In correspondence to the results above, the fluorescence signal got intensified in response to variable concentration of target and reached nearly eight fold stronger signal than that of bare nanoflares, and finally maintained, suggesting that the limited number of ex8-flares coating gold nanoparticles have almost all been removed. The fluorescence signal changed

Figure 3. (a) Amplitude ratio ε and (b) fluorescence intensity of nanoflares in 1×PBS with different concentration of target DNA (red full circle) and non-target DNA (black squre). The error bars represent standard deviation from three independent replicates.

The stability of nanoflares against Dnase I and gluthione (GSH) as well as the kinetic study were performed by both fluorescence intensity and fluorescence lifetime measurements (Fig. S3 and Fig. S4). The results demonstrated that nanoflares were relatively stable in solution. They were not influenced by Dnase I since gold nanoparticles protect DNA from enzyme degradation and the little influence induced by GSH would not bring false positives in the detection process. The kinetic study showed that nanoflares interacted with target DNA rapidly within 5 minutes, which is favorable for sensitive molecular probes (Fig. S5). Having illustrated the feasibility and sensitivity of identifying target DNA in extracellular environment by measuring the lifetime change of nanoflares, we next tested the applicability of the method to detect endogenous mRNA using MDA-MB-231 cells. Cells were cultured for 24~48 hours before treatment and then incubated with nanoflares and non-complementary nanoflares for 2.5 hours respectively. Ex8-flares were transfected with commercial transfection reagents to cells and incubated for 2.5 hours. After the washing and fixing procedure, FLIM mapping was obtained and analyzed. During the photon collection process, it was noted that it took much longer time to collect the same number of photons from non-complementary nanoflares treated cells under the same laser excitation, which suggests that fluorescence exhibited by them was significantly weaker than that of nanoflares and ex8-


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flares treated cells, indicating the unsuccessful displacement of ex8-flares in non-complementary ones treated cells. The cy5 fluorescence lifetime images showed that the increased photons were mainly collected from cytoplasm, implying the location of target mRNA (Figure 4(a)). The colors exhibited by the FLIM images of cells treated with nanoflares and ex8-flares were homogenous, indicating a uniform distribution of lifetime. And the fluorescence lifetime of nanoflares treated cells were nearly the same as that of ex8-flares treated cells without an ultrashort component. The disappearance of the ultrashort lifetime component in the fluorescence lifetime of nanoflares treated cells can be explained as the releasing of ex8-flares from the nanoflares since the recognition sequences were hybridized to target mRNA in cells. However, the ultrashort lifetime component of that of cells treated with non-complementary nanoflares remained, indicating that ex8-flare oligonucleotides didn’t dislocate. Calculated overall lifetimes of each sample were listed in Table S1. The small difference between the cy5 lifetime in cells and that in buffer is attributed to the environment change in solution. To demonstrate the ability of nanoflares to detect a change in mRNA level in cells, a biological negative control was also studied. The BRCA1 mRNA in cells was knocked down by treatment with siRNA against BRCA1 using transfection reagent. After transfection, cells were incubated with nanoflares for 2.5h, and intracellular fluorescence lifetime was monitored. The relative level of BRCA1 expression in MDA-MB-231 cells was down-regulated by siRNA. As for the lack of target mRNA, ex8-flares on nanoflares remained binding to recognition sequences on the gold nanoparticles, so the FLIM images exhibited relatively short lifetime of fluorescence in cells. In order to demonstrate the outcome of FLIM experiments, scanning confocal microscopy and analytical flow cytometry were conducted as fluorescence-intensity demonstration. As shown in Figure 4, cells treated with nanoflares exhibited an amplified fluorescence signal but cells treated with non-complementary nanoflares as well as cells with BRCA1 knockdown showed much less intense fluorescence. Consistent with the results of confocal microscopy imaging, flow cytometry analysis also suggested that nanoflares-treated cells were much more fluorescent than the ones treated with non-complementary nanoflares since the target mRNA in cells liberated ex8-flares on the nanoconjugates, but ex8-flare sequences on the control cannot be replaced. The consistency between the results of the scanning confocal microscopy and the flow cytometry and those of FLIM further confirmed the ability of FLIM technique to detect intracellular mRNA.

Figure 4. (a)Fluorescence lifetime maps and confocal fluorescence microscopy images of MDA-MB-231 cells incubated with nanoflares, bare ex8-flares and non-complementary nanoflares as well as nanoflares incubated with BRCA1 knockdown cells. Scale bar, 20μm. (b) Fluorescence intensity corresponding to cells treated with nanflares and non-complementary nanoflares and BRCA1 knockdown cells treated with nanoflares determined by flow cytometry.

In conclusion, fluorescence lifetime imaging of nanoflares in living cells has been demonstrated for the first time to be a suitable method for intracellular mRNA detection. The experiments indicate a significant reduction of fluorescence lifetime upon the attachment of cy5-modified oligonucleotides to the gold nanoparticles and a restoration of lifetime after the introduction of target which could be used for characterizing the mRNA of interest by measuring the fluorescence lifetime change. Fluorescence lifetime results obtained were demonstrated to be in good agreement with those of fluorescence intensity changes in extra- and intracellular environment, and avoid the false positives brought by the fluorescence intensity artifacts. Moreover, it is assumed that the method can also be used for other biomarkers’ detection and identify multiple targets of interest with different kinds of recognition sequences and flares’ fluorophore modification.



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ASSOCIATED CONTENT Supporting Information Experimental sections and supporting tables and figures. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB013004) of the Ministry of Science and Technology of China and Major Project of Chinese State Key Laboratory of Tribology (Grant No. SKLT2014A01).

REFERENCES (1) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J. Clin. 2011, 61, 69-90. (2) Brabender, J.; Danenberg, K. D.; Metzger, R.; Schneider, P. M.; Park, J.; Salonga, D.; Hölscher, A. H.; Danenberg, P. V. Clin. Cancer Res. 2001, 7, 1850-1855. (3) Taron, M.; Rosell, R.; Felip, E.; Mendez, P.; Souglakos, J.; Ronco, M. S.; Queralt, C.; Majo, J.; Sanchez, J. M.; Sanchez, J. J. Hum. Mol. Genet. 2004, 13, 2443-2449. (4) Lehner, R.; Enomoto, T.; Mcgregor, J. A.; Shroyer, L.; Haugen, B. R.; Pugazhenthi, U.; Shroyer, K. R. Acta Obstet. Gyn. Scan. 2002, 81, 162-167. (5) Zhang, L.; Zhou, W.; Velculescu, V. E.; Kern, S. E.; Hruban, R. H.; Hamilton, S. R.; Vogelstein, B.; Kinzler, K. W. Science 1997, 276, 1268-1272. (6) Beer, D. G.; Kardia, S. L.; Huang, C.-C.; Giordano, T. J.; Levin, A. M.; Misek, D. E.; Lin, L.; Chen, G.; Gharib, T. G.; Thomas, D. G. Nat. Med. 2002, 8, 816-824. (7) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (8) Chen, J. J. Pharmacogenomics J 2007, 8, 473-482. (9) Bieligk, S. C.; Ghossein, R.; Bhattacharya, S.; Coit, D. G. Ann. Surg. Oncol. 1999, 6, 232-240.

Page 6 of 7

(10) Apostolaki, S.; Perraki, M.; Kallergi, G.; Kafousi, M.; Papadopoulos, S.; Kotsakis, A.; Pallis, A.; Xenidis, N.; Kalmanti, L.; Kalbakis, K. Breast Cancer Res. Tr. 2009, 117, 525-534. (11) Couzin, J. Science 2006, 313, 1559-1559. (12) Xu, H.; Li, Q.; Wang, L.; He, Y.; Shi, J.; Tang, B.; Fan, C. Chem. Soc. Rev. 2014, 43, 2650-2661. (13) Wei, W.; He, X.; Ma, N. Angew. Chem. 2014, 126, 5679-5683. (14) Chen, T.; Wu, C. S.; Jimenez, E.; Zhu, Z.; Dajac, J. G.; You, M.; Han, D.; Zhang, X.; Tan, W. Angew. Chem. 2013, 125, 2066-2070. (15) Jayagopal, A.; Halfpenny, K. C.; Perez, J. W.; Wright, D. W. J. Am. Chem. Soc. 2010, 132, 9789-9796. (16) Hejátko, J.; Blilou, I.; Brewer, P. B.; Friml, J.; Scheres, B.; Benková, E. Nat. protocols 2006, 1, 1939-1946. (17) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477-15479. (18) Prigodich, A. E.; Seferos, D. S.; Massich, M. D.; Giljohann, D. A.; Lane, B. C.; Mirkin, C. A. Acs Nano 2009, 3, 2147-2152. (19) Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C. Pro. of the Natl. Acad. Sci. 2014, 111, 17104-17109. (20) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. J. Am. Chem. Soc. 2015, 137, 8340-8343. (21) Dulkeith, E.; Ringler, M.; Klar, T.; Feldmann, J.; Munoz Javier, A.; Parak, W. Nano lett. 2005, 5, 585-589. (22) Swierczewska, M.; Lee, S.; Chen, X. Phys. Chem. Chem. Phys. 2011, 13, 9929-9941. (23) Cheng, Y.; Stakenborg, T.; Van Dorpe, P.; Lagae, L.; Wang, M.; Chen, H.; Borghs, G. Anal.Chem. 2011, 83, 1307-1314. (24) Acuna, G. P.; Bucher, M.; Stein, I. H.; Steinhauer, C.; Kuzyk, A.; Holzmeister, P.; Schreiber, R.; Moroz, A.; Stefani, F. D.; Liedl, T. ACS Nano 2012, 6, 3189-3195. (25) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365-370. (26) Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Anal. Chem. 2013, 85, 10581-10588. (27) Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano lett. 2009, 9, 3258-3261. (28) Chang, C.; Sud, D.; Mycek, M. Method Cell Bio. 2007, 81, 495. (29) Herman, B.; Wang, X.; Wodnicki, P.; Perisamy, A.; Mahajan, N.; Berry, G.; Gordon, G. Applied fluorescence in chemistry, biology and medicine 1999, 491-507. (30) Esposito, A.; Wouters, F. S. Cur. Protoc. Cell Biol. 2004, 4.14. 11-14.14. 30. (31) Blacker, T. S.; Mann, Z. F.; Gale, J. E.; Ziegler, M.; Bain, A. J.; Szabadkai, G.; Duchen, M. R. Nat. Commun. 2014, 5. (32) Chen, Y.; Saulnier, J. L.; Yellen, G.; Sabatini, B. L. Front. Pharmacol 2014, 5. (33) Padilla-Parra, S.; Auduge, N.; Coppey-Moisan, M.; Tramier, M. Biol. Rev. 2011, 3, 63-70. (34) Wallrabe, H.; Periasamy, A. Cur Opin Biotech 2005, 16, 19-27. (35) Richert, L.; Didier, P.; de Rocquigny, H.; Mély, Y. In Advanced Time-Correlated Single Photon Counting Applications; Springer, 2015, pp 277-307.


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Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in

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