Systematic Screening of Trigger Moieties for Designing Formaldehyde Fluorescent Probes and Application in Live Cell Imaging

Formaldehyde (FA) is involved in multiple physiological regulatory processes and plays a crucial role in memory storage. Meanwhile, FA has a notorious reputation as a toxic compound, and it will cause a variety of diseases if its level is unbalanced in the human body. To date, there have been numerous fluorescent probes for FA imaging reported. Among them, the probes based on the 2−aza−Cope rearrangement have attracted the most attention, and their applications in cell imaging have been greatly expanded. Herein, we screened the various trigger moieties of FA fluorescent probes based on the mechanism of 2−aza−Cope rearrangement. FA−2, in which a fluorophore is connected to a 4−nitrobenzylamine group and an allyl group, demonstrated the highest sensitivity, selectivity, and reaction kinetics. Furthermore, FA−Lyso, derived from FA−2, has been successfully designed and applied to monitor exogenous and endogenous FA fluctuations in lysosomes of living cells.


Introduction
As the simplest aldehyde molecule, formaldehyde (FA) is used in many fields, including building materials, wood furniture, paint pigments, cosmetics, preservatives, and disinfectants in medical laboratories [1]. FA is also known as a hazardous substance because it can cause serious health problems. Nonetheless, FA can be generated endogenously via the demethylation of N−methylated amino acid residues by demethylase and oxidase enzymes [2]. As a result, FA maintains a concentration between 0.2-0.4 mM and acts as a metabolic intermediate in the normal physiological environment of the brain, which is essential for memory acquisition through the DNA demethylation cycle and cognitive function [3][4][5][6]. When FA is overloaded, it can cause Alzheimer's disease (AD), diabetes, respiratory illness, cancer, and other diseases [7]. Consequently, developing robust tools to monitor FA concentration fluctuations in living cells and tissues is critical.
Due to its high sensitivity, selectivity, and spatial and temporal resolution, fluorescent techniques have attracted continuous attention and have been applied in the detection of many types of biomolecules in living organisms [8][9][10][11][12][13][14][15][16]. To date, two primary strategies have been reported for FA fluorescent probe design: one employs the 2−aza−Cope mechanism of rearrangement [17,18], and the other utilizes the condensation reaction mechanism between amines or hydrazones [19,20]. Among these design strategies, the method based on 2−aza−Cope mechanism has attracted much interest due to its high selectivity for FA. However, up to now, there has been a lack of systematic screening of trigger moieties based on the performance of selectivity, sensitivity, kinetics, etc. [21][22][23][24][25][26][27].
Herein, we developed a number of fluorescent probes (FA−1, FA−2, FA−3, FA−4, FA−5) with different FA reaction units based on the same fluorescent dye to evaluate their overall efficacy of FA detection (Scheme 1). Under the same conditions, the detailed comparison study revealed that FA−2, in which the fluorophore is connected with a 4−nitrobenzylamine group and an allyl group on the carbonyl carbon atom, demonstrated the highest sensitivity, selectivity, and reaction kinetics. On the basis of FA−2, FA−Lyso has been further designed and applied to monitor exogenous and endogenous FA fluctuations in lysosomes in living cells successfully.
Herein, we developed a number of fluorescent probes (FA−1, FA−2, FA−3, FA−4, FA−5) with different FA reaction units based on the same fluorescent dye to evaluate their overall efficacy of FA detection (Scheme 1). Under the same conditions, the detailed comparison study revealed that FA−2, in which the fluorophore is connected with a 4−nitrobenzylamine group and an allyl group on the carbonyl carbon atom, demonstrated the highest sensitivity, selectivity, and reaction kinetics. On the basis of FA−2, FA−Lyso has been further designed and applied to monitor exogenous and endogenous FA fluctuations in lysosomes in living cells successfully. Scheme 1. FA fluorescent probes with different reaction units.

Materials and Instruments
2.1.1. Apparatus 1 H NMR and 13 C NMR spectra were obtained on a Bruker 400 MHz spectrometer (Bruker BioSpin, Fällanden, Switzerland). Mass spectrometry was performed with Thermo TSQ Endura Triple Quadrupole Mass Spectrometer (Thermo Fisher, Frederick, MD, USA). UV absorption spectra were recorded on Shimadzu UV−3600 Plus UV−VIS−NIR Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Fluorescence spectra were acquired with a FluoroMax−4 fluorescence photometer (Horiba Scientific, Kyoto, Japan). Cell imaging was performed by a ZEISS LSM 800 Confocal Laser Scanning (Carl Zeiss AG, Oberkochen, Germany) Microscope. Milli−Q water was applied in all experiments.

Reagents
All reagents were purchased from commercial suppliers and used as received unless otherwise noted. 6−hydroxy−2−naph−thaldehyde, trifluoromethanesulfonic anhydride, allylboronic acid pinacol ester, 4−(2−aminoethyl) morpholine, BINAP, and sodium borohydride were bought from Bidepharm (Bide Pharmatech Co., Ltd., Shanghai, China). Pyrrolidine and NH 3 (7 M in methanol) were purchased from J&K Chemical (J&K Chemical Ltd., Beijing, China). Cs 2 CO 3 , palladium (II) acetate, 2,4−dinitrobenzaldehyde, 4−nitrobenzaldehyde, anhydrous DCM, and methanol were obtained from Adamas−beta ® (Titan Scitific Co. Ltd., Shanghai, China). Milli−Q water was applied for the experiment and the preparation of all the buffer. accordingly [24,[28][29][30]. All the characterizations of products were performed by 1 H NMR, 13 C NMR, and ESI−MS and shown in Supplementary Materials. Synthesis of compound E Palladium (II) acetate (23 mg, 0.102 mmol) and BINAP (54 mg, 0.87 mmol) were added into the mixture of compound D (500 mg, 1.7 mmol), 4−(2−aminoethyl) morpholine (1140 µL, 8.7 mmol) and cesium carbonate (848 mg, 2.6 mmol) in toluene under N 2 and stirred at 110 • C for 4 h. After that, the reaction was cooled to room temperature and water and ethyl acetate were added for extraction. Then, the combined organic phase was washed with saturated NaCl, dried over MgSO 4 , filtered, and concentrated. Compound E was purified by flash chromatography on silica (petroleum ether: ethyl acetate = 1:1) as light−yellow solid (243 mg, 49%). 1 13  Synthesis of the probe FA−2 and FA−3 FA−1 (1 eq) and 4−nitrobenzaldehyde/2,4−dinitrobenzaldehyde (1.2 eq) were dissolved in chloroform and stirred at room temperature for 3 h. Then, MeOH was added as solvent and NaBH 4 (10 eq) was added into the mixture in batches within 3 h at 0 • C. After stirring overnight at 0 • C, the solvent was removed by reduced pressure and the residue was separated by silica gel column to provide the products.  13  Synthesis of the probe FA−4 and FA−5 Compound A (1.2 eq) and compound B/C (1 eq) were dissolved in CHCl 3 and stirred at room temperature for 3 h. Then, MeOH was added as solvent and NaBH 4 (10 eq) was added into the mixture in batches within 3 h at 0 • C. After stirring overnight at 0 • C, the solvent was removed by reduced pressure, and the residue was separated by silica gel column to provide the products.  13 13  Synthesis of the probe FA−Lyso Compound F (90 mg, 0.3 mmol) and 4−nitrobenzaldehyde (55 mg, 0.36 mmol) were dissolved in chloroform and stirred at room temperature for 3 h. Then, MeOH was added as solvent and NaBH 4 (113.2 mg, 3 mmol) was added into the mixture in batches within 3 h at 0 • C. After stirring overnight at 0 • C, the solvent was removed by reduced pressure, and the residue was chromatographed on a silica gel column to provide the probe FA−Lyso (56 mg, 40%). 1 13
The stock solution of probe FA−1, FA−2, FA−3, FA−4, and FA−5 were added into a 5 mL volumetric flask of PBS solution (pH 5, 10 mM) and diluted to 5 µM as the final concentration. The different analytic species were then added, and the resulting solutions were incubated for 60 min at 37 • C. After that, the absorption and fluorescence experiments were conducted.

Cells Imaging
WI−38 cells were cultured in DMEM medium with 10% FBS and 1% antibiotics (penicillin and streptomycin) at 37 • C under 5% CO 2 atmosphere and seeded in a glass−bottomed confocal dish (35 mm). After 24 h, NaHSO 3 pretreated WI−38 cells were incubated with probe FA−2 for 30 min. Then, FA was added into the medium for 1 h incubation. After washing with DPBS, the dishes were placed on a confocal microscope and fluorescence images were taken.
For colocalization imaging, the cells were treated with FA−Lyso for 30 min incubation firstly. Then, FA was added into the medium for another 30 min incubation. After that, cells were incubated with Lyso−Tracker Red and Mito−Tracker Deep Red for 15 min respectively. A confocal fluorescence image was taken of the dishes in the end.
The WI38 cell line and HeLa cell line were obtained from Dr. Yongguang Jia in SCUT.

Result and Discussion
To begin with, the fluorophore of 6−(pyrrolidine−1−yl)−2−naphthaldehyde (FA-A) was chosen as the basic building block due to its extraordinary features, including high quantum yield, facile modification, and moderate stability towards ROS, RNS, and RSS. Meanwhile, five recognition groups for FA were introduced into FA-A. All of the probes were synthesized successfully with satisfied yields. The synthesis routes and chemical structure characterizations ( 1 H NMR, 13 C NMR, and ESI−MS) were shown in Scheme S1 (Supplementary Materials).
After synthesis and characterization, initial absorption and fluorescence emission experiments were performed. The absorbance variation of all probes in the presence and absence of FA can be observed via UV−vis absorption spectrum ( Figure S1 in Supplementary Materials). As a result of reaction with FA, FA−1, FA−2, and FA−3 showed a notable enhancement and slight red−shift from 360 nm to 395 nm. However, FA−4 exhibited just a mild increase in the presence of FA. As to FA−5, a remarkable and broad enhanced absorbance has been observed after the reaction with FA. Using the absorption results, we can conclude that all the probes showed the feasibility to react with FA in PBS buffer. This result strengthened our confidence in subsequent studies of the fluorescence performance.
Next, we systematically investigated the reaction behavior of all probes towards FA by time−dependent fluorescence profiles under the different pH values. As shown in Figure 1 and Figure S2, FA−1 itself possessed remarkable fluorescence and the maximum emission wavelength at 442 nm. After treatment with FA, the fluorescence signal at 442 nm declined, and a new emission at 530 nm occurred simultaneously. Despite that FA−1 exhibited an increased ratio with the reaction of FA and a relatively optimal value at pH 7, the increased ratio was relatively low (<6−fold), which limited its application as the fluorescent probe (Figure 1 and Figure S2  fluorescent probe (Figure 1 and Figure S2 in Supplementary Materials). For FA−2, the reaction kinetics were significantly fast, and the fluorescence at 530 nm was sharply increased (ca. 280−fold) in 1 h at pH 4 and 5 (Figure 1). The fluorescence response of FA−2 with FA increased when pH value decreased, whereas no fluorescence enhancement was observed between pH 8-10 ( Figure S3 in Supplementary Materials). FA−3 also showed a similar response at 530 nm from pH 4 to 10 ( Figure S4   The obvious difference on maximum emission wavelength between FA−2 and FA−4 is due to the difference of reaction products. The reaction of FA−2 and FA produces FA-A, which contains aldehyde compound as the electron−withdrawing group in the fluorophore (Scheme S2 in Supplementary Materials), while FA−4 produces 4−nitrobenzaldehyde and FA-P, which is an alkylamine compound (Scheme S3 in Supplementary Materials) [17,24]. As the literature reported, higher electron−withdrawing effect in ICT fluorophore induces the redshift of emission wavelength, as well as the increase of fluorescence quantum yield [28,33]. To confirm the above theory, we synthesized fluorophore FA-A and FA-P and tested the fluorescence intensity in PBS buffer (pH 5, 10 mM). The result showed that FA-P, the reaction product of FA−4/FA−5 with FA displayed the maximum emission wavelength at 442 nm with the fluorescent quantum yield of 0.11. However, the maximum emission wavelength of FA-A, which is the product of FA−2/FA−3 and FA, is red−shifted to 530 nm with the fluorescent quantum yield of 0.25 ( Figure S7 in Supplementary Materials) [22,28]. These results confirmed that FA−2′s fluorescence response is extraordinary for FA probe design based on the mechanism of 2−aza−Cope rearrangement.
To further compare the integrated capability towards FA detection, the selectivity experiment was carried out in PBS buffer (pH 5.0, 10 mM). The probes were treated with different biological species such as H2O2, HOCl, GSH, Hcy, Cys, Ca 2+ , Fe 2+ , Fe 3+ , etc. In addition, we also included several aldehyde compounds, crotonaldehyde, acraldehyde, butyraldehyde, propionaldehyde, benzaldehyde, p−hydroxybenzaldehyde, and 4−nitrobenzaldehyde, to examine whether these probes show the reactivity to other aldehyde The obvious difference on maximum emission wavelength between FA−2 and FA−4 is due to the difference of reaction products. The reaction of FA−2 and FA produces FA-A, which contains aldehyde compound as the electron−withdrawing group in the fluorophore (Scheme S2 in Supplementary Materials), while FA−4 produces 4−nitrobenzaldehyde and FA-P, which is an alkylamine compound (Scheme S3 in Supplementary Materials) [17,24]. As the literature reported, higher electron−withdrawing effect in ICT fluorophore induces the redshift of emission wavelength, as well as the increase of fluorescence quantum yield [28,33]. To confirm the above theory, we synthesized fluorophore FA-A and FA-P and tested the fluorescence intensity in PBS buffer (pH 5, 10 mM). The result showed that FA-P, the reaction product of FA−4/FA−5 with FA displayed the maximum emission wavelength at 442 nm with the fluorescent quantum yield of 0.11. However, the maximum emission wavelength of FA-A, which is the product of FA−2/FA−3 and FA, is red−shifted to 530 nm with the fluorescent quantum yield of 0.25 ( Figure S7 in Supplementary Materials) [22,28]. These results confirmed that FA−2 s fluorescence response is extraordinary for FA probe design based on the mechanism of 2−aza−Cope rearrangement.
To further compare the integrated capability towards FA detection, the selectivity experiment was carried out in PBS buffer (pH 5.0, 10 mM). The probes were treated with different biological species such as H 2 O 2 , HOCl, GSH, Hcy, Cys, Ca 2+ , Fe 2+ , Fe 3+ , etc. In addition, we also included several aldehyde compounds, crotonaldehyde, acraldehyde, butyraldehyde, propionaldehyde, benzaldehyde, p−hydroxybenzaldehyde, and 4−nitrobenzaldehyde, to examine whether these probes show the reactivity to other aldehyde groups or not. In general, all of the probes (5 µM)) were treated with different analytes (500 µM) and FA (500 µM) for one hour, and then the fluorescence spectrum was measured. As shown in Figures S8 and S9, although FA−1 provided the ratiometric result, its reactivity and selectivity towards FA are less than satisfactory, especially for NO 2 − , which could form diazonium salt with the amine group in FA−1 ( Figure S8 in Supplementary Materials). For FA−5, there is also no acceptable result obtained because of its low kinetic and reactivity (Figures S16 and S17 in Supplementary Materials). In the reactions of FA−3 (Figures S12 and S13 in Supplementary Materials) and FA−4 ( Figures S14 and S15 in Supplementary Materials) with FA, although the tolerable selectivity has been achieved, the fluorescence intensity increment was relatively low, which was about 12 and 15−folds. With regards to FA−2, it exhibited the best selectivity for FA and other competitive species, which was more than 200−fold (Figure 2, Figures S10 and S11 in Supplementary Materials). Taking these results together, FA−2 demonstrated the outstanding performance in terms of reaction kinetic and selectivity upon the reaction with FA, suggesting that FA−2 could serve as an excellent candidate in cell imaging. groups or not. In general, all of the probes (5 μM)) were treated with different analytes (500 μM) and FA (500 μM) for one hour, and then the fluorescence spectrum was measured. As shown in Figure S8 and S9, although FA−1 provided the ratiometric result, its reactivity and selectivity towards FA are less than satisfactory, especially for NO2 − , which could form diazonium salt with the amine group in FA−1 ( Figure S8 in Supplementary Materials). For FA−5, there is also no acceptable result obtained because of its low kinetic and reactivity (Figures S16 and S17 in Supplementary Materials). In the reactions of FA−3 (Figures S12 and S13 in Supplementary Materials) and FA−4 (Figures S14 and S15 in Supplementary Materials) with FA, although the tolerable selectivity has been achieved, the fluorescence intensity increment was relatively low, which was about 12 and 15−folds. With regards to FA−2, it exhibited the best selectivity for FA and other competitive species, which was more than 200−fold (Figure 2, Figures S10 and S11 in Supplementary Materials). Taking these results together, FA−2 demonstrated the outstanding performance in terms of reaction kinetic and selectivity upon the reaction with FA, suggesting that FA−2 could serve as an excellent candidate in cell imaging. To evaluate the sensitivity of FA−2 towards FA, the titration experiments were conducted in PBS buffer and monitored by fluorescence spectrum. As shown in Figures 3a and S18, the free probe FA−2 (5 μM) showed negligible fluorescence due to the PET effect in PBS buffer (pH 5, 10 mM), while the fluorescence was increased gradually upon the addition of FA. The fluorescence intensity at 530 nm matched well with added FA concentration from 0 to 1 mM, and the maximum intensity was reached when FA concentration was up to 2 mM. Specifically, FA−2 also displayed a good linear relationship (R 2 = 0.997) with 0.65 μM of the limit of detection of and 2.56 μM of the limit of quantification. However, for FA−1, FA−3, FA−4, FA−5, the detection limit was 28.64 μM, 15.58 μM, 3.75 μM, and 89.55 μM, respectively (Figures S19-S22 in Supplementary Materials). FA−2 also displayed good recovery efficiency with different FA concentration using the standard curves (Table S1 in Supplementary Materials). The above result suggested that FA−2 is sensitive enough to detect low concentration of FA in aqueous solution.
To further validate the pH effect on FA−2, the fluorescence intensity at 530 nm was recorded when the pH value was switched between 5.0 and 7.4. As shown in Figure 3b, the fluorescence signal at 530 nm gradually increased at pH 5.0. The reaction kinetics, however, was almost stopped when the pH was adjusted to 7.4 in the same reaction solution. Upon adjusting the pH back to 5.0, the fluorescence continued to increase again. By converting pH three times, the same result was obtained. Based on these results, FA−2 performed better in recognizing FA in acidic media than in neutral and basic media, To evaluate the sensitivity of FA−2 towards FA, the titration experiments were conducted in PBS buffer and monitored by fluorescence spectrum. As shown in Figure 3a and Figure S18, the free probe FA−2 (5 µM) showed negligible fluorescence due to the PET effect in PBS buffer (pH 5, 10 mM), while the fluorescence was increased gradually upon the addition of FA. The fluorescence intensity at 530 nm matched well with added FA concentration from 0 to 1 mM, and the maximum intensity was reached when FA concentration was up to 2 mM. Specifically, FA−2 also displayed a good linear relationship (R 2 = 0.997) with 0.65 µM of the limit of detection of and 2.56 µM of the limit of quantification. However, for FA−1, FA−3, FA−4, FA−5, the detection limit was 28.64 µM, 15.58 µM, 3.75 µM, and 89.55 µM, respectively (Figures S19-S22 in Supplementary Materials). FA−2 also displayed good recovery efficiency with different FA concentration using the standard curves (Table S1 in Supplementary Materials). The above result suggested that FA−2 is sensitive enough to detect low concentration of FA in aqueous solution.
Biosensors 2022, 12, x 8 of 12 indicating that FA−2 or its modified probe can be used to monitor FA fluctuation in lysosomes in living cells since the pH of the lysosome ranges from 4.0 to 6.0. Having established that FA−2 can sense FA in vitro, we next evaluated its ability to monitor FA concentration in living cells. Firstly, we modified probe FA−2 to FA−Lyso, in which we rationally introduce a morpholine group for lysosomal targeting purposes. After synthesis and structure characterization, the fluorescence response properties of FA To further validate the pH effect on FA−2, the fluorescence intensity at 530 nm was recorded when the pH value was switched between 5.0 and 7.4. As shown in Figure 3b, the fluorescence signal at 530 nm gradually increased at pH 5.0. The reaction kinetics, however, was almost stopped when the pH was adjusted to 7.4 in the same reaction solution. Upon adjusting the pH back to 5.0, the fluorescence continued to increase again. By converting pH three times, the same result was obtained. Based on these results, FA−2 performed better in recognizing FA in acidic media than in neutral and basic media, indicating that FA−2 or its modified probe can be used to monitor FA fluctuation in lysosomes in living cells since the pH of the lysosome ranges from 4.0 to 6.0.
Having established that FA−2 can sense FA in vitro, we next evaluated its ability to monitor FA concentration in living cells. Firstly, we modified probe FA−2 to FA−Lyso, in which we rationally introduce a morpholine group for lysosomal targeting purposes. After synthesis and structure characterization, the fluorescence response properties of FA detection were studied. The result indicates that FA−Lyso maintains fast kinetics and excellent sensitivity as FA−2 ( Figure S23  To further validate the fluorescence signal was generated from the specific reaction between FA−Lyso and FA, we performed inhibition experiment with NaHSO 3 , which could act as an excellent scavenger through reacting with FA at the central carbonyl group [20,34]. The fluorescence signal in WI−38 cells, which were treated with FA−Lyso with FA showed a statistically significant difference. In the cells pretreated with NaHSO 3 , however, the fluorescence intensity obviously decreased. In addition, the fluorescence intensity decreased accordingly with the increasing NaHSO 3 concentration, proving the capability of FA−Lyso to monitor FA fluctuation in living cells (Figure 4g-k).
Encouraged by the above results, we further evaluated the capability of FA−Lyso to detect endogenous FA in living cells. We chose HeLa cells for our experiments as this cell line has been reported to overexpress FA [35,36]. First, with the addition of FA−Lyso alone, significantly enhanced fluorescence was observed (Figure 5a,b). However, when cells were pretreated with NaHSO 3 and then incubated with FA−Lyso, the fluorescence intensity was dramatically decreased, indicating that FA in living cells has been depleted (Figure 5c,d). Subsequently, cells were sequentially preincubated with NaHSO 3 , FA, and FA−Lyso, the recovered fluorescence signal can be observed again (Figure 5e,f). Collectively, these studies demonstrate that FA−Lyso is capable of detecting endogenous FA fluctuation in living cells.
Subsequently, we further evaluated whether the targeted organelle of FA−Lyso is lysosomes. WI−38 cells were incubated with FA−Lyso and commercially available dye Lyso−Tracker Red (lysosome dye) or Mito−Tracker Deep Red (mitochondrial dye) in the presence of FA for 1 h. As shown in Figure S25 To further validate the fluorescence signal was generated from the specific reaction between FA−Lyso and FA, we performed inhibition experiment with NaHSO3, which could act as an excellent scavenger through reacting with FA at the central carbonyl group [20,34]. The fluorescence signal in WI−38 cells, which were treated with FA−Lyso with FA showed a statistically significant difference. In the cells pretreated with NaHSO3, however, the fluorescence intensity obviously decreased. In addition, the fluorescence intensity decreased accordingly with the increasing NaHSO3 concentration, proving the capability of FA−Lyso to monitor FA fluctuation in living cells (Figure 4g-k). alone, significantly enhanced fluorescence was observed (Figure 5a,b). However, when cells were pretreated with NaHSO3 and then incubated with FA−Lyso, the fluorescence intensity was dramatically decreased, indicating that FA in living cells has been depleted (Figure 5c,d). Subsequently, cells were sequentially preincubated with NaHSO3, FA, and FA−Lyso, the recovered fluorescence signal can be observed again (Figure 5e,f). Collectively, these studies demonstrate that FA−Lyso is capable of detecting endogenous FA fluctuation in living cells.

Conclusions
In summary, using 2−aza−Cope rearrangement strategy, five fluorescent probes, FA−1, FA−2, FA−3, FA−4, and FA−5, were designed from the same fluorophore to systemically screen their sensing ability of FA in aqueous buffer and living cells. Experiment

Conclusions
In summary, using 2−aza−Cope rearrangement strategy, five fluorescent probes, FA−1, FA−2, FA−3, FA−4, and FA−5, were designed from the same fluorophore to systemically screen their sensing ability of FA in aqueous buffer and living cells. Experiment results demonstrated that among all five probes, FA−2 achieved improved kinetics, selectivity, and sensitivity under weak acid condition. Furthermore, the probe FA−Lyso, derived from FA−2, exhibited excellent ability to visualize exogenous and endogenous FA fluctuations in lysosomes in living cells. Taken together, this study offers an attractive design strategy for FA probes based on 2−aza−Cope rearrangement. Moreover, this design strategy has been applied successfully for FA detection in living cells, demonstrating its robust utility.

Conflicts of Interest:
The authors declare no conflict of interest.