Ion Monitoring at Nanoscale Sites of Interorganelle Membrane Contact in Living Cells

Nanostructural contact sites formed by interorganelle membrane contacts, including mitochondria and lysosome contacts (MLC), facilitate the exchange of substances during various life processes. However, existing bioanalytical technologies have yet to provide accurate information on the exchange of substances, as these techniques exhibit limited spatial and temporal resolution. To address this limitation, a strategy is proposed that combines fluorescence resonance energy transfer (FRET) probes with high spatial resolution detection and super‐resolution microscopes (SRM) that feature time‐resolved imaging. Specifically, a proof‐of‐concept approach is presented for monitoring H+ fluctuations during MLC with a spatial H+ biosensor targeting lysosomes, BDP‐RhB. The biosensor comprises H+‐sensitive rhodamine B as a FRET acceptor connected by a flexible chain to a BODIPY derivative as a donor. The acidity of MLC sites may vary, influencing the spatial distance of the flexible chain and causing a fluorescence transition in BDP‐RhB. Consequently, the spatial distribution of H+ can be identified using SRM. Furthermore, an algorithm has been developed to screen and identify potential compounds that control substance exchange in the MLC. Collectively, this work presents the dynamic of H+ in lysosomes within living cells, which provides a drug screening tool for studying substance exchange through interorganelle membrane contacts.

[13][14][15] Successful implementation of FRET technology requires a suitable pair of fluorescent systems with overlapping emission and absorption spectra, as well as the capacity to be excited by specified conditions.[18][19][20] While FRET probes with conventional fluorescent microscopy provide sufficient spatial resolution for subcellular analysis, the low temporal resolution poses challenges for accurate analysis and measurement.
The use of FRET probes requires suitable imaging equipment with adequate sensitivity and resolution to detect signals, enabling precise quantitative analysis and rapid imaging. [21,22]raditional fluorescence microscopes employ continuous illumination and camera-based image acquisition, resulting in relatively slow imaging speeds (typically between 10 and 24 fps); this factor impedes the timely acquisition of high-quality images during dynamic processes. [23,24]Although confocal microscopy employs a point scanning method, it still falls short of fulfilling the real-time imaging requirements for certain fast dynamic processes. [25]Moreover, the resolution of conventional imaging equipment (above 200 nm) is limited by the Abbe diffraction limit, thereby hindering accurate observation of nanoscale structures, particularly interorganelle membrane contacts.Recent advancements in super-resolution fluorescence microscopy techniques like the structured illumination microscope (SIM), which can overcome technical challenges with high spatial resolution (80-120 nm) and high time resolution (approximately 100 fps), have provided technical options for nanoscale FRET detection and measurement in living cells. [26,27]o address the need for analysis spatiotemporal structures at the nanoscale in living systems, we combined the use of a BODIPY-rhodamine dyad FRET probe, BDP-RhB, with SIM imaging to develop a system with high spatial and temporal resolution to detect H þ exchange at MLC sites.By monitoring the fluorescence switching of FRET when lysosomes carrying the probe interacted with mitochondria, real-time changes in the H þ microenvironment during MLC formation and dissociation were found to be detectable.Furthermore, we developed an image algorithm to identify drug candidates that control H þ transport during MLC, facilitating drug screening.This work provides the first combination of two systems with high spatial and temporal resolution to trace the spatiotemporal dynamic movement of H þ in living cells and to record changes in H þ levels within lysosomes during MLC.

An H þ -Responsive Probe, BDP-RhB, for Tracking pH Dynamics in Lysosomes
To generate a FRET-based strategy for the recognition of H þ ions, rhodamine B, which is responsive to H þ changes, was designated as the fluorescent acceptor and was linked by a flexible chain to a BODIPY derivative as the fluorescent donor.To deliver the donor and acceptor fluorophores to lysosomes, a lysosometargeting tertiary amine group was added to rhodamine B to generate the FRET-based H þ responsive biosensor BDP-RhB (Figure S1, Supporting Information).According to the above design, in the presence of high concentrations of H þ , the lactam ring of rhodamine B is disrupted, and the resulting alteration to the orientation of the rhodamine B moiety facilitates FRET signal and increased red fluorescence of the acceptor.In contrast, in the presence of low H þ or higher OH À , the closed lactam ring is restored, resulting in decreased FRET signal and attenuated red fluorescence (Figure 1a).
To validate this process, we assessed the optical properties of BDP-RhB in vitro (Figure S2, Supporting Information) in the presence of H þ environmental changes.BDP-RhB exhibited only the fluorescence emission peak at 520 nm that is characteristic of BODIPY derivatives under neutral or alkaline conditions.However, when the closed lactam ring of rhodamine B was opened under acidic conditions, a new fluorescence peak could be observed near 585 nm (Figure 1b).Accordingly, the ratio of the two fluorescence intensities of BDP-RhB (I 585 /I 520 ) in the solutions with different pH values was stable between 0.1 and 1.8, and the responsive pH range was between 4 and 9 (Figure 1c).These results indicate that the BDP-RhB dual-fluorescent molecule is capable of H þ -responsive FRET-based fluorescence changes, which makes it useful for the detection and quantitative analysis of H þ in the microenvironment.

Identification of H þ Changes in Lysosomes via BDP-RhB in Different Physiological Environments
After assessing the cytotoxicity of BDP-RhB, we chose a cellcompatible concentration of 2.0 μM for cell imaging (Figure S3, Supporting Information).We explored the imaging capabilities of BDP-RhB in living cells using both traditional confocal microscopy and SIM (Figure S4, Supporting Information).As expected, conventional confocal microscopy was unable to clearly resolve the borders of fluorescently labeled lysosomes (Figure S4b,c, Supporting Information).However, the higher resolution of SIM allowed it to distinguish two fluorescent particles in close proximity (Figure S4e,f, Supporting Information).
Additionally, we evaluated the imaging properties of BDP-RhB in two different cell lines, HepG2 and H9C2, and we discovered that the probe was able to enter both types of cells leading to the development of red-green fused round dots (Figure S5, Supporting Information).To confirm the lysosome targeting ability of BDP-RhB, we costained the cells with LysoTracker Blue (LTB) for 30 min, followed by SIM imaging using a triple channel mode (BDP-RhB: λ ex = 488 nm, λ em = 520 nm/585 nm; LTB: λ ex = 405 nm, λ em = 425 nm), which revealed that both green and red fluorescent particles were localized in the LTBlabeled lysosomes (Figure 2a,b, and S6, Supporting Information), with a high Pearson colocalization coefficient (Figure 2c).Because the normal lysosome is in an acidic environment, BDP-RhB is in a state of high FRET, and the fluorescence of RhB is stronger than that of BDP, so the colocalization value is slightly higher than that of BDP.We also excluded the possibility that BDP-RhB localized at lipid droplets or nuclei (Figure S7, Supporting Information).
Next, we observed the pattern of uptake of BDP-RhB in HeLa cells by imaging cells after incubation with the dye at different temperatures and in the presence of the endocytosis inhibitor chlorpromazine (CPZ) (Figure S8, Supporting Information), and we performed statistical analyses of the fluorescence intensity of BDP-RhB in cells under different conditions (Figure S9, Supporting Information).The results indicated that the fluorescence uptake of BDP-RhB was significantly reduced with the treatment of low temperature and endocytosis inhibitors, that is, BDP-RhB entered the cells via energy-dependent endocytosis (Figure 2d).
The high-power laser used in SIM may cause photobleaching of the fluorophore. [28]Therefore, to characterize the propensity of BDP-RhB to undergo photobleaching, cells incubated with the novel fluorophore were exposed to continuous laser irradiation.
After 200 s of continuous lasers intensity of 100% at 488 nm, the loss of fluorescence intensity of BDP-RhB was less than that observed for previously reported imaging probes (Figure 2e), which suggests that BDP-RhB is resistant to photobleaching and is suitable for long-term tracking of lysosomes in living cells. [29]e then sought to determine the capacity of BDP-RhB to identify the changes of H þ within lysosomes under different physiological environments.It is generally accepted that starvation treatment with Earle's Balanced Salt Solution (EBSS) reduces the pH in lysosomes and induces autophagy, whereas treatment of cells with H 2 O 2 causes apoptosis and raises the pH in lysosomes. [30,31]Therefore, we treated HeLa cells with EBSS and H 2 O 2 separately to maintain lysosomes at different pH levels.When we stained EBSS-treated cells with BDP-RhB and performed SIM imaging, the red fluorescence of BDP-RhB in the lysosomes was found to be brighter than the green fluorescence (Figure 2f ), consistent with a relatively high FRET signal under conditions of low pH.Conversely, in cells treated with H 2 O 2 , the green fluorescence signal was higher than that of red fluorescence (Figure 2g).Quantitative analyses of the fluorescence intensity of BDP-RhB in lysosomes under the two treatments indicated that the FRET signal of BDP-RhB responded quantitatively to pH changes in lysosomes under different physiological environments (Figure 2i).Therefore, changes in H þ and OH À levels in lysosomes can be represented by the red and green fluorescence of BDP-RhB, respectively.

H þ Fluctuations in Lysosomes During MLC
[34] The acidity of lysosomes is a key molecular characteristic of lysosomes because this organelle contains more than 50 various types of acid hydrolases. [35]itochondria, which serve as the primary site of cellular energy production, are also central to cellular physiology.Studies have shown that in Parkinson's disease, the acidification of lysosomes is abnormal and characterized by a significant prolongation of the contact time between mitochondria and lysosomes in the form of the MLC. [36]herefore, to further explore the interaction between MLC and lysosomal acidification, cells were costained with BDP-RhB and the commercial mitochondrial probe MitoTracker Deep Red (MTDR).The subsequent imaging revealed that the red/green fluorescence ratio differed between various lysosomes, indicating that BDP within lysosomes exhibits different FRET signals (Figure 3a).In consideration of our data showing that BDP-RhB responds to the pH of lysosomes (Figure 2f,g), we conclude that the distribution of H þ in lysosomes could be illustrated with the red fluorescence of BDP-RhB, while the distribution of OH À in lysosomes could be illustrated by the green fluorescence.Notably, the red fluorescence of BDP-RhB was predominant in lysosomes that were in contact with mitochondria (Figure 3b, panel 1); conversely, the ratio of red to green fluorescence of BDP-RhB was not different in lysosomes that were not in contact with mitochondria (Figure 3b, panel 2).As the interaction of mitochondria and lysosomes can occur due to traditional mitophagy, [37,38] we used cells genetically lacking ATG13 (ATG13 KO) and thus unable to undergo autophagy to exclude the influence of autophagy during MLC and found that BDP-RhB still mainly showed red fluorescence in lysosomes at the contact site (Figure S10, Supporting Information).Taken together, these imaging results indicate that lysosomes exhibit a preacidification process with an increase in H þ during the process of MLC.
To further support this claim, we selected BDP-RhB-labeled lysosomes that did and did not participate in MLC under physiological environments (Figure S11, Supporting Information) and compared the red-green fluorescence heat maps of each lysosome.Here, we found that BDP-RhB in lysosomes participating in MLC showed brighter red fluorescence relative to green fluorescence (Figure S12, Supporting Information).In contrast, BDP-RhB that was present in lysosomes not participating in MLC, the red-colored lysosomes were less fluorescent than green ones (Figure S13, Supporting Information).The levels of fluorescence of BDP-RhB in the two groups of lysosomes were quantified, and the red and green fluorescence ratios of the two groups were found to be significantly different (Figure 3c).The appearance of the red fluorescence oscillation peak during MLC suggested that there may be a process of crosstalk involving the transfer of H þ from mitochondria to lysosomes during contact.
Next, we analyzed the dynamic flow of H þ as indicated by red fluorescence of BDP-RhB during MLC.A heat map of the red fluorescence that permitted visualization of the H þ dynamics of lysosomes shows that there were several dynamic oscillatory peaks encompassing the entirety of the lysosomal structures (Figure 3d).The identification of the red fluorescence dynamics is consistent with the occurrence of a physiological process of flow of mitochondrial H þ to lysosomes during MLC (Figure 3e); this result suggests that functionally active H þ flow from mitochondria to lysosomes during MLC.The observation of H þ dynamic oscillations based on BDP-RhB prompted us to hypothesize that lysosomal acidification could serve as a prerequisite for the formation of MLC.This hypothesis was supported by evidence, demonstrating that lysosomes in contact with mitochondria were more acidic than were isolated lysosomes.To further support this hypothesis, cells were pretreated with two known pharmacological regulators of lysosome pH, bafilomycin A1 (Baf-A1), which is known to increase lysosomal pH, and rapamycin (RAPA), which is known to decrease lysosomal pH (Figure 4a). [39,40]Here, after treatment with these lysosomal modulators, HeLa cells were costained with BDP-RhB and MTDR.As expected, as Baf-A1 treatment increases the pH of lysosomes, cells treated with Baf-A and BDP-RhB primarily exhibited green fluorescence; we also discovered that most lysosomes in these cells were not in physical contact with mitochondria.In contrast, after RAPA treatment, BDP-RhB within cells mainly exhibited red fluorescence, and the majority of the stained lysosomes were in contact with mitochondria (Figure 4b,c).Moreover, the ratios of red to green fluorescence of both treatment groups were statistically different from that of the control group (Figure 4d).
To exclude the effect of autophagy-induced lysosomal acidification, ATG13 KO cells were also treated with Baf-A1 and RAPA, and the results showed that lysosomes in contact with mitochondria still showed predominantly red fluorescence of BDP-RhB.All these results indicate that lysosomal acidification exists commonly during MLC formation, and that this dynamic transformation is readily observed with BDP-RhB (Figure S14, Supporting Information).
[43] V-ATPase is a complex composed of multiple subunits, consisting mainly of two domains, the peripheral V 1 domain and the transmembrane V 0 domain (Figure S15, Supporting Information).The A subunit of the V 1 domain contains the site of catalysis of ATP hydrolysis, while the V 0 domain transports H þ . [44]The main function of V-ATPase is to maintain the lysosomal acidic environment by pumping H þ into lysosomes through coupling to the energy released upon ATP hydrolysis. [45,46]As mitochondria are the primary organelles supplying ATP within cells, it is conceivable that the V-ATPase promotes H þ flow from mitochondria to lysosomes at the MLC.
Accordingly, we constructed a plasmid directing the intracellular overexpression of subunit A of V-ATPase (ATP6V1A) fused to blue fluorescent protein (BFP; Figure 4e and S16, Supporting Information).When cells transfected with this plasmid and then costained with BDP-RhB and MTDR were analyzed, lysosomes containing ATP6V1A-BFP were found to mainly exhibit red fluorescence of BDP-RhB (Figure 4f ), indicating that these lysosomes were at a relatively low pH.Moreover, the lysosomal H þ oscillation at the MLC sites was abrogated; instead, the H þ levels were uniformly distributed (Figure 4g).These results demonstrated that V-ATPase can affect the transport of H þ from mitochondria to lysosomes during MLC.

An Image-Based Algorithm for Screening H þ Fluctuations in Lysosomes Following MLC
Our experimental results demonstrated that lysosomal changes can be evaluated by quantifying the H þ fluctuations using the fluorescence intensity ratio of BDP-RhB after SIM imaging.The use of SIM also permits the determination of the colocalization coefficient as an important index for assessing the degree of interaction between mitochondria and lysosomes.A key advantage of SIM as compared to conventional optical microscopy is that it can reduce the resolution limit by half and enable super-resolution imaging of living cells.The SIM technique involves the movement and rotation of the illumination pattern to cover the entire specimen followed by the combination of multiple images and reconstruction of a super-resolution image of the sample (Figure 5a).Therefore, the simultaneous evaluation of fluorescence intensity ratios and colocalization coefficients requires time-consuming manual processing and calculations.Another drawback is that the processing involves multiple subjective factors, and no appropriate standard has been developed.
To address these drawbacks, we developed an algorithm based on the advantages of BDP-RhB to evaluate the changes in fluorescence intensity and the occurrence of MLC under different drug treatment conditions.According to the flow of this algorithm (Figure 5b), we first preprocessed the input images to remove interfering pixels outside a single cell, and then used the median filtering algorithm to reduce the noise of the background color.As the noise reduction step was expected to result in the loss of useful information, we employed an image enhancement procedure to enhance the effect of this image.Next, the RGB color model of the image was converted into an Hue, Saturation, Valu (HSV) color model to better represent its hue, saturation, and brightness.Following image preprocessing, the images were split and categorized into four groups based on the fluorescence channels: mitochondria (M), green fluorescence of the probe (G), red fluorescence of the probe (R), and mitochondria and lysosome contact MLC group (C).
Because our results demonstrated that acidification of lysosomes is a prerequisite for MLC, we reasoned that we could use our algorithm to screen for compounds that increase lysosomal acidification.After treating cells with candidate drugs and generating microscopic images, we defined the FRET ratio of R to G (K f ) to quantify the lysosomal acidification and the ratio of C to the sum of M and R (K c ) to quantify the occurrence of MLC.K f 0 and K c 0 were assigned to represent the corresponding factors in untreated cells.The identification of a K f value that is greater than K f 0 would indicate enhanced lysosomal acidification, whereas the converse would indicate weakened lysosomal acidification.Drugs that altered the K f value would be evaluated further based on the K c value.A value of K c that was greater than K c 0 would indicate the ability of the drug to promote MLC; otherwise, the drug would be considered to not promote MLC.Thus, we used this algorithmic procedure to screen for drugs that potentially promote MLC.

Fluctuations within MLC
By combining the algorithm developed in this work with the fluorescence conversion characteristics of BDP-RhB, we performed an SIM imaging-based drug screen in HeLa cells (Figure 6a).The K f and K c values obtained upon treatment with candidate drugs and application of the algorithm are displayed in Figure 6b,c.RAPA was used as a positive control because it was previously shown to induce autophagy by inhibiting mTOR activity. [47]ccordingly, the value of K f was found to increase in HeLa cells treated with rapamycin; further analyses demonstrated that the value of K c also increased, suggesting that rapamycin increases the incidence of MLC.Furthermore, both chloroquine (CQ) and bafilomycin A1 are also known to impact lysosomal acidity, [48,49] and our results also verified that both of these drugs lowered the K f and K c values, consistent with their increasing of lysosomal pH and lowering of the incidence of MLC.
GSK621, an agonist of adenosine 5 0 -monophosphate (AMP) dependent protein kinase, has also been shown to induce autophagy. [50]Similar to RAPA treatment, our analysis also indicated that the acidification of lysosomes and the incidence of MLC were increased after GSK621 treatment, as indicated by lower values of K f and K c (Figure 6b,c).Conversely, rotenone treatment led to higher values of K f and K c , which is consistent with its well-established apoptosis-inducing pharmacological effect.Conversely, we observed that lysosomes in cells treated with apocynin exhibited enhanced red fluorescence and higher K f and K c , so we speculated whether it could further reduce the occurrence of apoptosis by affecting the pH of lysosomes.
To further test the reliability of the algorithm-based method, we investigated the effects of drugs with high K f values on the expression of Rab7 protein, a molecular switch for intracellular vesicle trafficking.[53] Specifically, we treated HeLa cells with RAPA, GSK621, and apocynin under conditions that were shown to significantly promote lysosomal acidification as indicated by high K f values, and western blotting demonstrated that the expression of Rab7 was indeed increased in all three cases (Figure 6d,e).Thus, the feasibility of the screening of drugs based on lysosome acidification and MLC was further verified (Figure 6f ).This tool will enable the rapid quantitative analysis of lysosomal acidification, providing a new method for the screening of drugs that affect lysosomal acidification and MLC.

Conclusion
We present here a FRET-based color-switchable fluorescent probe, BDP-RhB, featuring an acid-sensitive FRET acceptor.By specifically targeting the lysosome, BDP-RhB converts biological signals into color signals through the fluorescence switching of FRET.Observing of these signals by SIM enables real-time monitoring of H þ flow in the microenvironment when lysosomes interact with mitochondria.BDP-RhB not only facilitates the identification of H þ fluctuations in lysosomes under diverse physiological conditions but also enables the screening candidate drugs to modulate MLC.To expedite the identification of images and enable transient quantitative analysis of dynamic ion flow between subcellular membranes, we have developed an automatic image analysis program that is compatible with BDP-RhB.Collectively, we provide an approach to transform dynamic biological processes into easily trackable and accurately quantifiable transient signals, which leads to a better understanding of the complex communication between organelles.

Experimental Section
Materials: All materials and solvents were purchased from commercial suppliers and used without further purification.BDP-RhB was synthesized in accordance with our previously described procedure. [54]LTB and MTDR were purchased from Invitrogen (Eugene, OR, USA).Bafilomycin A1, rapamycin, CQ, erastin, and oligomycin were purchased from MedChemExpress (NJ, USA).The primary antibodies of rabbit anti-LC3B and rabbit anti-ATG13 were purchased from ABclonal (Wuhan, China).The secondary antibody of Goat anti-Rabbit IgG was purchased from Abcam (Cambridge, UK).Fetal Bovine Serum, Dulbecco's modified eagle medium (DMEM), and other cell culture reagents were obtained from VivaCell (Shanghai, China).HeLa cells, HepG2 cells, and H9C2 cells were gifted from Dr. Fei Liu lab (Shandong Academy of Pharmaceutical Science, Jinan, China).
Cell Culture and Transfection: Cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units mL À1 ), and streptomycin (100 μg mL À1 ) in a 5% CO 2 humidified incubator at 37 °C.The cells were then transfected using Lipofectamine 3000 (Invitrogen, USA) with plasmid pEBFP-N1-ATP6V1A.Specifically, 5 μg of plasmid and 10 μL of P3000 were gently added to 250 μL of Opti-MEM medium.3.75 μL of Lipo3000 were gently added to 125 μL of Opti-MEM medium.The two mixtures were then gently mixed in 1:1 ratio and incubated for 15 min.The transfection mixture was added into a cell culture dish, and the cells were further incubated at 37 °C for 6-8 h.After the medium was changed, the cells were cultured for another 30 h to express the ATP6V1A fusion protein.
Spectroscopic Study: The stock solution of BDP-RhB (10.0 mM) was prepared with DMSO, and stored at À20 °C.To assess the sensitivity of BDP-RhB toward pH, we examined the fluorescence spectra of BDP-RhB at different pH levels.The work solutions of this BDP-RhB for spectroscopic study were prepared by diluting this stock solution to the final concentration of 10.0 μM in PBS with pH ranging from 3 to 10.All fluorescent spectra were recorded upon excitation at 480 nm.Fluorescent spectra were obtained on a F-7100 (Hitachi).
Fluorescence Imaging of Cells: All fluorescence images were obtained by OMX 3D-SIM super-resolution microscope (DeltaVision) or LSM-980 confocal laser scanning microscope (Carl Zeis).The OMX 3D-SIM super-resolution microscope is equipped with a 60Â/1.42numerical aperture oil-immersion objective lens and solid-state lasers.3D-SIM images were obtained at 512 Â 512 using Z-stacks with a step size of 0.125 μm.The LSM-980 confocal laser scanning microscope equipped with a 63Â/1.49numerical aperture oil immersion objective lens.All fluorescence images were analyzed, and their backgrounds were subtracted with ImageJ software (National Institutes of Health).
Colocalization Assay: A total of 2 Â 10 5 cells were seeded on a 35 mm glass-bottom microwell dish and incubated with 2 mL of DMEM supplemented with 10% FBS for 24 h, and then stained with BDP-RhB (2.0 μM) for 30 min and with 200.0 nM LTB or 100 nM MTDR at 37 °C for another 30 min.After treatment, the cells were washed 5 times with prewarmed PBS and 3 times with complete medium.Finally, fluorescence images were acquired on an OMX 3D-SIM and Pearson's coefficient was quantified using the Colocalization Analysis plugin for ImageJ software.
Quantitative Detection of H þ Level in SIM Image: HeLa cells were stained with BDP-RhB and imaged with OMX 3D-SIM.The lysosomes within multiple cells were extracted, and the mean fluorescence intensity of BDP-RhB in lysosomes was calculated to quantify H þ level.The fluorescence intensity of lysosomes stained with BDP-RhB in OMX 3D-SIM images was calculated to quantify H þ .Quantitative analysis of fluorescence intensities and the quantity of H þ were performed with ImageJ and Origin 8.5.
Cytotoxicity Assay: The cytotoxicity assay of BDP-RhB was determined with the Cell Counting Kit-8 (CCK-8) Assay (Dojindo, Japan).HeLa cells were seeded with complete DMEM in a 96-well plate at a density of 8000 cells/well and then HeLa cells were cultured in DMEM with 10% FBS at 37 °C for 24 h.Then the culture medium was replaced with 100 μL fresh medium containing 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 5.0 μM of BDP-RhB and cultured at 37 °C for 2, 6, and 12 h, respectively.Next, cells were incubated with 10.0 μL CCK-8 solution for 1 h at 37 °C.Finally, the absorption wavelength was measured by enzyme-linked immunosorbent assay (ELISA, Tecan) at450 nm.
Cellular Uptake Assay: HeLa cells were incubated by 2.0 μM BDP-RhB under different conditions.37 °C: the cells were incubated with BDP-RhB at 37 °C for 30 min.4 °C: the cells were incubated with BDP-RhB at 4 °C for 30 min.CPZ: the cells were preincubated with CPZ (20.0 μM) in FBS-free DMEM at 37 °C for 0.5 h and then incubated with BDP-RhB at 37 °C for 30 min. [55]Finally, fluorescence images were acquired on a OMX 3D-SIM and the fluorescence intensities of BDP-RhB were calculated with ImageJ software.
Western Blot: Protease inhibitor cocktail and 2Â RIPA lysis and extraction buffer were added to the centrifuged cell pellets, and were then sonicated for 5 min each time, 3 times in total.Purified lysates were normalized using Bradford reagent.Normalized samples were mixed with loading buffer and loaded onto 12% polyacrylamide gels.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at room temperature.Samples were transferred to polyvinylidene fluoride (PVDF membranes at 200 V, 4 °C.Membranes were blocked in 5% BSA/ TBST for 1 h at room temperature and probed with the primary and secondary antibodies according to company guidelines.Membranes were incubated with enhanced chemiluminescence (ECL) substrate and imaged using a ChemiDoc XRS chemiluminescence detector (Bio-Rad).Signal analysis was performed using ImageJ software.
Data Analysis: Statistical analysis was performed with Prism 8 (GraphPad).Normality tests are performed to check the normal distribution.In the case of normal distribution, the statistical analysis was conducted with Student's t-test.In the case of non-normal distribution, the statistical comparison of results was test with a Mann-Whitney test, with levels of significance set at n.s.(no significant difference), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Data are presented as mean AE SEM.Analyzed cells were obtained from three replicates.Statistical significances and sample sizes in all graphs are indicated in the corresponding figure legends.

Figure 1 .
Figure 1.Design and optical characterization of the H þ responsive probe BDP-RhB.a) Schematic diagram of the design of BDP-RhB for FRET-based fluorescence conversion in lysosomes.b) Fluorescence spectra of probe BDP-RhB (10.0 μM) in solutions with various pH values (3-10).c) The ratio between fluorescence intensity I 582 /I 518 versus pH value.

Figure 2 .
Figure 2. Measurement of H þ in lysosomes via BDP-RhB under different physiological conditions.a) SIM images of lysosomes in HeLa cells incubated with LTB (λ ex = 405 nm) and BDP-RhB (λ ex = 488 nm, λ em = 520 nm/585 nm) for 30 min at 37 °C.b) Higher-magnification images of the area indicated by a white rectangle in panel (a) and a heat map of the fluorescence intensity of BDP-RhB.c) Quantitative analysis of the colocalization between BDP or RhB and LTB according to the Pearson correlation coefficient.d) Schematic diagram of BDP-RhB uptake by a lysosome.e) Photostability of BDP-RhB was determined by imaging under continuous SIM laser-irradiation of HeLa cells incubated with the FRET dye.f,g) Cells stained with BDP-RhB and treated with EBSS (f ) or H 2 O 2 (g) were irradiated under a SIM laser (λ ex = 488 nm).h) Heat map of BDP-RhB fluorescence intensity of EBSS-or H 2 O 2 -treated cells.i) Fluorescence intensity of BDP-RhB in EBSS-treated or H 2 O 2 -treated cells.Data are presented as mean AE SEM (n = 6).Statistical differences between two groups were examined using t-tests (***P < 0.001).

Figure 3 .
Figure 3. Super-resolution imaging for mapping H þ /OH À spatiotemporal dynamics in lysosomes.a) HeLa cells costained with MTDR (λ ex = 635 nm) and BDP-RhB.b) Higher magnification images of areas denoted with white rectangles and heat maps of the BDP-RhB fluorescence intensity.c) Fluorescence intensity ratio of BDP-RhB in lysosomes in contact or not in contact with mitochondria.Data are presented as mean AE SEM (n = 10 from 10 images).Statistical differences between two groups were examined using t-tests (****P < 0.0001).d) SIM tracking of H þ distribution during mitochondria-lysosome interactions via BDP-RhB staining; white circles in the images indicate areas visualized as fluorescence intensity profiles in the lower panels.e) Schematic diagram of H þ delivery from mitochondria to lysosomes.

2. 4 .
Involvement of Vacuolar-Type ATPase Activity in the Lysosomal H þ Fluctuations During MLC

Figure 5 .
Figure 5.The automated super-resolution drug screening strategy based on differences of H þ fluctuations in lysosomes.a) Schematic illustration of the SIM imaging system.b) The process and classification indicators that allow the researchers to screen alternative drugs in further studies.

Figure 6 .
Figure 6.The application of drug screening based on H þ /OH À fluctuations within areas of mitochondria-lysosome contact.a) SIM Images after treatment of HeLa cells with candidate drugs and incubation with BDP-RhB.b,c) K f values (b) and K c values (c) determined from the experiments shown in panel (a).d) The expression of Rab7 in HeLa cells after treatment with RAPA, GSK621, or apocynin.e) The quantitative analysis of relative Rab7 expression.Statistical differences between two groups were examined using t-tests (*P < 0.05).f ) Schematic representation of screening for drugs that affect lysosomal acidification and mitochondrial-lysosomal contact.