Organelle-targeting ratiometric fluorescent probes: design principles, detection mechanisms, bio-applications, and challenges

Biological species, including reactive oxygen species (ROS), reactive sulfur species (RSS), reactive nitrogen species (RNS), F−, Pd2+, Cu2+, Hg2+, and others, are crucial for the healthy functioning of cells in living organisms. However, their aberrant concentration can result in various serious diseases. Therefore, it is essential to monitor biological species in cellular organelles such as the cell membrane, mitochondria, lysosome, endoplasmic reticulum, Golgi apparatus, and nucleus. Among various fluorescent probes for species detection within the organelles, ratiometric fluorescent probes have drawn special attention as a potential way to get beyond the drawbacks of intensity-based probes. This method depends on measuring the intensity change of two emission bands (caused by an analyte), which produces an efficient internal referencing that increases the detection's sensitivity. This review article discusses the literature publications (from 2015 to 2022) on organelle-targeting ratiometric fluorescent probes, the general strategies, the detecting mechanisms, the broad scope, and the challenges currently faced by fluorescent probes.

Dr Neetu Tripathi obtained her PhD from Guru Nanak Dev University, Amritsar, India. She completed her masters with 1st rank (Gold medal) in MSc Chemistry (Instrumental Analysis). She has been a recipient of the prestigious INSPIRE fellowship of the Government of India during her PhD She has also cleared the National Eligibility Test for Assistant Professor (CSIR-UGC NET) and the Graduate Aptitude test (GATE) in the subject of chemical sciences. Her research interest includes organic chemistry, materials chemistry, supramolecular chemistry, and nanomaterials. She has published various research papers in international peer-reviewed journals (Royal Society of Chemistry, Elsevier journals and Springer journal). She is also an author of a book published by CRC press (an imprint of the Taylor & Francis group).

Introduction
In this modern era, everyone is interconnected. Similarly, in living beings, cells, organelles and biological species are interconnected and act cooperatively for the normal functioning of living systems. 1,2 For instance, biological species (anions, cations, thiols, nitrogen and oxygen) perform a variety of role in organelles such as signaling molecules, enzyme cofactors, etc. However, their chemical imbalance can cause cellular malfunction. 3 Therefore, smart research work on organelles and biological species is essential, which is challenging.
Over the years, signicant studies and research work have been done on organelles and biological species present within the organelles. For example, organelle-targeting medicine has been developed for curing various diaeases. 4,5 Bioimaging technique has been developed for examining biomolecules in living cells and tissues. 6 Choi et al. studied uorescent probes for organelles with respect to recent advances and bioapplications. 7 Nowadays, uorescent probes for targeting organelles have gained more attention due to their excellent photo-physical characteristics, high sensitivity, rapid response, low cost, non-invasiveness, and real-time imaging. 8 Notably, many single emission probes are unable to withstand environmental interference. Through a built-in correction of two emissions at different wavelengths, ratiometric uorescence probes could eliminate background interference. 9 Additionally, ratiometric uorescence imaging has an even better resolution due to the two clearly dened emission peaks. 10 Therefore, the development of ratiometric uorescent probes is a winning strategy for the sensitive detection of small molecules, thanks to its reduced environmental effect.
Although there are several outstanding reviews in the literature based on ratiometric probes with sensing applications, each one has concentrated on a single aspect, such as the optical processes, a particular class of uorophores, or a specic subset of target analytes. In 2018, Huang et al. reported ratiometric optical nanoprobes for molecular detection and imaging. 10 In an excellent review, the design principles and applications of uorescent probes in the ratiometric detection of anions, cations, and biological molecules has been beautifully demonstrated. 11 Another review article highlighted various chemo/probe-based semiconductor quantum dots (QDs). 12 Prof. Goutam Kumar Patra, Head of the Department of Chemistry, Guru Ghasidas Central University, Bilaspur did PhD from Jadavpur University, under the supervision of Prof. Dipankar Datta at Indian Association for the Cultivation of Science, Kolkata. Then he joined the Tel Aviv University, Israel, as a postdoctoral research fellow with Prof. Israel Goldberg (2000)(2001)(2002). Subsequently he moved to the Carnegie Mellon University, USA where he worked with Prof. Catalina Achim. Successively he joined as Asst. Prof. in Vijoygarh Jyotish Ray College, Kolkata in December 2003. He visited Max Planck Institute of Bioinorganic Chemistry, Mülheim, Germany as a BOYSCAST fellow during 2006-07 and worked in the group of the then Director, Prof. Karl Wieghardt. He has been a Professor in Guru Ghasidas Central University, Bilaspur since 2012 and former Dean, School of Physical Science. His research interests include chemosensors, redox activity, aza macrocyclic chemistry, crystal engineering, porphyrin and supramolecular chemistry, peptide nucleic acids (PNAs) and free radical chemistry. So far, he has published more than hundred research papers in journals of national and international repute and guided eight doctoral students and one post-doctoral student.
Dr Manohar Chaskar is the Dean of the Science and Technology Faulty of Savitribai Phule Pune University. Dr Chaskar completed his PhD in materials science, and his post-doc from Nagoya University, Japan in photocatalysis. Dr Chaskar's research interests include the use of nanomaterials in catalyzing organic reactions. Dr Chaskar has published several high impact research articles in reputed national and international journals and has one patent to his credit while two are in the process. He is also a visiting scientist in Germany and Japan.
In 2020, Wu et al. summarized recent advancements in the metal-organic framework (MOF)-based ratiometric uorescent probes. 13 Recently, Bigdeli et al. published a review article on uorescent nanoprobes for visual detection. 8 We illustrate the design principles, fundamental detection mechanisms, and applications of organelle-targeting ratiometric uorescent probes. The current limitations and prospective future directions, which would spur additional research interest and bring up fresh opportunities for biological analysis, are also covered.

Design principles
In designing uorescent probes, photo-physical parameters such as photo-induced electron transfer (PET), internal charge transfer (ICT), monomer-excimer formation, Förster resonance energy transfer (FRET), and excited state intramolecular proton transfer (ESIPT) are frequently used. FRET is a relatively excellent strategy for designing ratiometric uorescent probes and increasing the Stokes shi. 14 Generally, the design of ratiometric probes involves the combination of two uorophores, one reference uorophore (may or may not show a change in emission intensity upon interaction with the analyte) and another dynamic uorophore (always shows a change in emission intensity upon interaction with the analyte). 8 Generally, ratiometric changes in the emission spectrum are of the following type: (I) Static + dynamic change: herein, upon the addition of an analyte, the emission intensity of one uorophore is almost kept unchanged, whereas other uorophores may undergo an increase/decrease/shi in emission intensity (Fig. 1A). 15,16 (II) Dynamic + dynamic change: herein, both the uorophores change (increase/decrease/or shi in emission intensity), but in opposite directions (Fig. 1B). 17 Generally, organelle-targeting ratiometric uorescent probes contain a uorophore, recognition units, and targeting moieties (Fig. 2). The organelle-targeting groups are selective to specic organelles. For example, due to its alkalinity, the morpholine group is a conventional lysosome-targeting moiety. 18 In addition, the polarity-dependent approach helps create various biological probes, which are helpful for imaging multiple organelles. 19 2.1 Designing mitochondria-targeting ratiometric uorescent probes Since the mitochondrial membrane typically has a potential of −180 mV, this property is employed to direct lipophilic positively charged probes into the mitochondria. 20 The common mitochondrial-targeting units include the quaternized pyridine moiety, 21 triphenylphosphonium (TPP), 22,23 indole, 24 cyanine, 25,26 pyridinium, 27,28 and rhodamine 29 (Fig. 2a). [30][31][32][33] Furthermore, to ascertain the mitochondria localization of probes in cells, the co-localization experiment was performed using commercially available mitochondrion-specic dyes such as MitoTracker Green FM, MitoTracker Red, and MitoTracker Orange. 30 Generally, mitochondrion-targeting uorescent probes consist of a uorophore linked to the mitochondriontargeting moiety and an activation unit.

Designing lysosome-targeting ratiometric uorescent probes
Modifying them with lipophilic amines is the most popular method for directing probes into lysosomes. [34][35][36] Due to the membrane-impermeable protonated amines in lysosomes, selective probe trapping occurs. The pyridine group, 37 monothio-bishydrazide moiety, 14 and morpholine 15 are the common lysosome-targeting groups (Fig. 2b). Typically, the design of the probe for lysosome requires linking of the uorophore with the lysosome-targeting group and an activation unit.

Designing endoplasmic reticulum-targeting ratiometric uorescent probes
The commonly used ER-targeting moieties include glibenclamide, 38 methyl sulphonamide, 39 and the p-toluenesulfonamide group (Fig. 2c). 40,41 ER-targeting uorescent probes generally have (1) a moderate size (conjugated band numbers (CBN < 40)), (2) a cationic character, and (3) an appropriate lipophilicity (+6 > log P oct > 0). 42 The ER-targeting uorescent probes mainly track cellular concentrations of stress-responsive substances like NO, H 2 S, H 2 O 2 , and HOCl. 43,44 2.4 Designing Golgi-apparatus-targeting ratiometric uorescent probes Motivated by the abundance of cysteine residues in the Golgi apparatus, Huang and coworkers proved L-cysteine as an effective Golgi apparatus targeting ligand. They created various probes using this technique (Fig. 2d). [45][46][47][48] 2.5 Designing nucleus-targeting ratiometric uorescent probes The nuclear envelope is a highly controlled membrane barrier. Therefore, passive diffusion or active transport uses the nuclear pore complex (NPC) to target the nucleus. 49 Small uorescent probes with cationic centers and hydrophobic planar aromatic structures can selectively label DNA molecules by focusing on the minor grooves in DNA (negatively charged double strands). Some of them have been made available for purchase. 50  electrostatic binding between a positively charged probe and negatively charged nucleus RNA major groove (affinity energy = −5.78 kcal mol −1 ). 52 Nucleus-targeting unit functionalization is an excellent strategy for delivering uorescent functional probes into the nucleus of living cells (Fig. 2e). 53,54 2.6 Designing membrane-targeting ratiometric uorescent probes Currently, the available probes for the membrane share a common approach: the conjugation of an environmentsensitive uorophore to generate membrane-specic signals and a membrane-anchoring moiety to minimize the diffusion of the probe (Fig. 2f). 7

Detection mechanism
The various uorescence-based sensing mechanisms include photo-induced electron transfer (PET), internal charge transfer (ICT), monomer-excimer formation, Förster resonance energy transfer (FRET), and excited state intra-molecular proton transfer (ESIPT). These mechanisms are available in detail in our recent publications [55][56][57] and some excellent reviews. 9,58 In this section, we explain the uorescence-based sensing mechanisms briey. The HOMO localizes on the donor moieties in the ICT-based uorescent probe. The LUMO is centered on acceptor moieties, thus creating a solid dipole with a charge transfer phenomenon upon excitation. The preferential interaction of the analyte at either the donor or acceptor results in a change in dipole strengths and, consequently, spectral shis (Fig. 3). To effectively measure ratiometrically, an ICT-based probe should exhibit a visible difference in uorescence intensity as well as a signicant change in emission wavelength. In the PET process (Fig. 4), upon excitation, an electron is transferred from the HOMO (highest occupied molecular orbital) of the receptor (donor) to the LUMO (lowest unoccupied molecular orbital) of the uorophore (acceptor). However, in the case of the guest-bounded receptor, the HOMO energy levels become lower than those of the uorophore, inhibiting the PET process and uorescence change. In the monomer-excimer-based sensing mechanism, upon the addition of an analyte, generally excited state complex formation (excimer) occurs by the interaction between the excited states of one uorophore and the ground state of another molecule (Fig. 5). The probes that include polyaromatic hydrocarbon (PAH) moieties, such as pyrene, anthracene, etc., typically display this type of sensing mechanism. In the FRET process, energy is transferred from the excited donor molecule to the ground-state acceptor molecule. The critical parameter which governs the FRET phenomenon is spectral overlaps (donor emission spectrum and acceptor absorption spectrum) (Fig. 6). FRET-based probes prove to be an excellent tool for ratiometric imaging due to the stoichiometric relationship between the donor (D) and acceptor (A). In the ESIPT process, proton transfer occurs from the preferred enol-form to the excited state ketoform upon excitation. During relaxation, the excited state ketoform converts back to the enol form by reverse proton transfer. Interestingly, intense uorescence, large Stokes-shi, and photostability are the various unique features of ESIPT-based probes (Fig. 7).    Mitochondria are organelles that play a vital role in cell physiology, including oxidative respiration, ATP production, and signal transduction. Mitochondria are widely known as the main generators of various ROS, RSS and RNS. Thus, probes that can specically target mitochondria play a key role in monitoring multiple functions of mitochondria and mitochondrion-related illnesses. 59-62

Reactive oxygen species (ROS) detection in mitochondria
The reactive oxygen species H 2 O 2 and HOCl are potent oxidants with antibacterial capabilities. 63,64 However, the aberrant synthesis of ROS species in vivo has been linked to several illnesses, including lung damage, atherosclerosis, osteoarthritis, and rheumatoid arthritis. 27 Therefore, real-time and onsite detection of ROS is an exciting research topic.
Hu et al. constructed probe 1 with far-red emission by combining a pyrene unit (electron donor, high quantum yield, ability to form a complex in the excited state) with a benzo[e] indolium unit (electron acceptor, extended p-conjugation, mitochondrion-targeting group) linked by an ethylene bridge (Fig. 8, Table 1). 65 In EtOH/PBS solution, the free probe exhibited emission at 632 nm (l ex = 525 nm). Upon the addition of ClO − , emission at 632 nm gradually fades away with a concomitant increase in uorescence intensity at 455 nm (blue emission). Probe 1 with excellent mitochondrial targeting features, such as high selectivity (detection limit = 182 nM), fast response time, signicant Stokes-Stokes shi (107 nm), photostability, and live cell membrane permeability, displayed potential for detecting ClO − in mitochondria.
An important ROS, endogenous H 2 O 2 , functions as a signaling molecule to control various cellular processes, such as cell division, proliferation, and migration. 66,67 However, high H 2 O 2 concentrations can harm proteins and nucleic acids, which are strongly related to many disorders such as malignancies, diabetes, and Alzheimer's disease. 68,69 Therefore, monitoring of H 2 O 2 concentration is essential. He et al. designed benzothiazole dye (adequate stability, large Stokes-shi, large quantum yield, tunable emission) based probe 2 containing an aromatic boronic ester moiety (H 2 O 2 recognition group) (Fig. 8, Table 1). 70 The uorescence titration of probe 2 with H 2 O 2 revealed an increase in new emission maxima at 594 nm at the expense of emission at 666 nm. The limit of detection was 23.1 nM. The sensing mechanism was the H 2 O 2 triggered aromatic boronic ester moiety removal. Furthermore, probe 2, with excellent mitochondrial targeting properties, such as signicant Stokes shi (152 nm), photostability, and Pearson's colonization coefficient (0.94), displayed potential for detecting H 2 O 2 in mitochondria.
Shen et al. designed probe 3 based on the FRET platform for detecting OCl − (Fig. 8, Table 1). 71 In the presence of OCl − , probe 3 displayed ratiometric uorescence change, and the plot of intensity ratio (I 575 /I 467 ) with the concentration of OCl − was linear in the range of 0 to 5 mM. The limit of detection was 10.2 nM. From the uorescence imaging experiment, the applied probe 3 successfully examined endogenous OCl − in Murine RAW 264.7 cells.

Reactive sulfur species (RSS) detection in mitochondria
Reactive sulfur species (RSS), such as cysteine (Cys), hydrogen sulde (H 2 S), and hydrogen polysuldes (H 2 S n ), are produced in large quantities in mitochondria and are associated with critical mitochondrial-related pathological and physiological processes. 7 Cys can act as an antioxidant in mitochondria by removing various ROS from mitochondria to stop oxidative damage. 72 Additionally, Cys is necessary for the mitochondrial  process of protein turnover. [73][74][75] Therefore, it is essential to identify and measure Cys in real-time within cells, particularly in the mitochondria, to understand the pathological and physiological processes properly.
Yang et al. designed xanthylene-based uorescent probe 4 for the ratiometric detection of cysteine levels in the mitochondria (Fig. 8, Table 1). 21 The probe 4 containing an acryloyl moiety (responsive site for cysteine) and a benzyl group (for easy distribution in the mitochondria) showed strong red uorescence at l em = 605 nm (l ex = 490 nm). In the presence of cysteine, probe 4 underwent ratiometric uorescence change with the formation of new emission maxima at 540 nm and the simultaneous decrease in emission intensity at 605 nm. The limit of detection was 33.7 nM for cysteine. Applied probe 4 examined endogenous cysteine levels in HeLa cells through bioimaging, demonstrating potential applications in natural areas.
Considering the advantage of two-photon uorescence microscopy and ratiometric detection, Niu et al. developed probe 5 for detecting cysteine over other biothiols (Fig. 8, Table  1). 76 In the presence of cysteine, the uorescence spectrum of probe 5 exhibited ratiometric change (I 518 /I 452 ), linear in the range between 0.5 and 40 mM. Interestingly, the merocyanine uorophore and probe 5 exhibited a large two-photon crosssection (F s max ) of 72.6 GM (l ex = 760 nm) and 65.2 GM (l ex = 740 nm), respectively, favorable in producing bright and high contrast images of living samples. In addition to detecting cysteine in live cells and mitochondria, probe 5 exhibited promising application for monitoring cysteine concentration in living tissues (down to 150 mm depth) using two-photon uorescence microscopy.
H 2 S plays essential roles in mitochondria, such as scavengers for reactive oxygen species, 77 and is associated with various mitochondrial-related pathological and physiological  processes. 78,79 Therefore, monitoring of H 2 S level in mitochondria is crucial. Liu et al. proposed probe 6 integrated with cyanine (mitochondria-targeting group) and naphthalimide group (responsive to H 2 S) for ratiometric detection of H 2 S in mitochondria (Fig. 8, Table 1). 24 In the CTAB solution, upon the addition of Na 2 S, probe 6 displayed ratiometric uorescence response, and the intensity ratio (I 530 /I 733 ) showed good linearity in the range of 1-9 mM. The detection limit was 1.31 mM for Na 2 S. In the presence of Na 2 S, reduction of the azide group to the amino group (electron donating) occurs, activating the ICT process and turn-on uorescence of the naphthalimide moiety. Probe 6 found applications for uorescence imaging and ratiometric detection of H 2 S in live cells.
Han et al. engineered probe 7 integrated with triphenylphosphonium (mitochondria-targeting group), 2-uoro-5nitrobenzoic unit, and 1,8-naphthalimide uorophore (wellknown ICT uorophore) 80 for the detection of H 2 S n (Fig. 8, Table 1). 81 In the phosphate-buffered saline (PBS) solution, uorescence titration of probe 7 with Na 2 S 2 revealed a decrease in emission intensity at 485 nm. A new emission maximum at 550 nm emerged and increased (Stokes-shi = 109 nm). Probe 7 successfully demonstrated its application in imaging intracellular H 2 S n with good selectivity and sensitivity. SO 2 is an essential endogenous signaling molecule that performs signicant roles in many physiological processes. However, increased SO 2 concentration is associated with severe lung cancer, nervous system diseases, and respiratory problems. [82][83][84][85] Therefore, detecting SO 2 and its derivatives in living systems becomes a high priority. Notably, a platform with uorophores attached to aromatic heterocycles through C-C bonds was frequently used as a Michael addition receptor. The Michael-addition principle allows nucleophiles, such as bisul-te, to attack the C-C double bond. 86,87 In this section, most probes for bisulte detection follow the nucleophilic addition reaction mechanism.
Taking advantage of the merits of FRET-based systems such as a large Stokes-shi, Huang et al. developed nearinfrared (NIR) uorescent probe 8 by combining a coumarin carboxylic acid group with a piperazine substituted benzopyrylium salt (mitochondrion-targeting group) (Fig. 8, Table  1). 88 In PBS solution, the uorescence spectrum of probe 8 exhibited emission maxima at 635 nm and a large Stokes-shi (230 nm). The nucleophilic addition reaction of SO 3 2− at the double bond of the benzopyrylium unit resulted in the interruption of the conjugated p-electron cloud, and the uorescence at 635 nm decreased, accompanied by the simultaneous increase in the new emission peak at 455 nm. From the co-localization experiment, probe 8 efficiently targeted mitochondria (Pearson's co-localization coefficient = 0.84), and further investigations in HeLa cells and the nude mice experiment showed the application of probe 8 in biological systems for detecting SO 3 2− (Fig. 9).
Liu et al. exploited probe 9, prepared by the condensation reaction between indolium or pyridinium and 3-formyl-9methyl carbazole unit (Fig. 8). 89 In the presence of HSO 3 − , probe 9 displayed ratiometric uorescence change and the ratio of emission intensity (I 490 /I 590 ) varied from 0.0383 to 3.8769 (101-fold enhancement). The in vitro imaging experiment proved the application of probe 9 for the quantication of SO 2 derivatives in the mitochondria.
The same group presented another probe, probe 10, containing a carbazole and an alkyl sulfonated benzoindole (water soluble, mitochondria-targeting group) as the basic skeleton ( Fig. 8, Table 1). 90 In the presence of HSO 3 − , the emission spectrum of probe 10 revealed a blue shi of 162 nm, and the emission intensity ratio (I 463 /I 625 ) was 56 (  Wang et al. applied the strategy of combining two classical dyes to construct long-wavelength probe 12, prepared from benzopyrylium and chromenoquinoline dyes (Fig. 8, Table 1). 92 The emission spectrum of the two-photon uorescent probe showed a gradual decrease in red uorescence at 613 nm (l ex = 580 nm) and an increase in blue uorescence at 514 nm (l ex = 405 nm) upon the addition of HSO 3 − . The detection limit was 103 nM and 17 nM for the red and green channels, respectively. The nucleophilic addition reaction of HSO 3 − at the C]C bond of probe 12 resulted in the interruption of p-conjugation/ inhibition of the ICT process from the chromenoquinoline to the benzopyrylium group. Thus, a signicant blue shi (99 nm) in the emission spectrum was observed. Furthermore, probe 12 displayed application for detecting SO 2 derivatives in the solid state. Wang et al. developed probe 13 based on pyrazoline (high quantum yield, cell permeability, and low cytotoxicity) and hemicyanine dyes (water soluble, mitochondrion-targeting group) (Fig. 8, Table 1). 93 The uorescence titration of probe 13 with SO 3 2− revealed ratiometric uorescence change, and the intensity ratio (I 480 /I 640 ) changed from 0.45 to 445 (989 times). Intriguingly, probe 13 demonstrated an application for the ratiometric imaging of mitochondrial SO 2 derivatives in living cells.
Keeping in mind Michael's addition principle and the FRET process, Wu et al. designed probe 14 based on the conjugated platform of dansyl, piperazine, and benzothiazolium, in which the benzothiazole moiety acts as a recognition unit and mitochondrion-targeting group (Fig. 8, Table 1). 94 Upon the incremental addition of HSO 3 − , the uorescence intensity ratio (I 540 /I 590 ) of probe 14 showed a change from 0.3 to 1.5 (5-fold), and the limit of detection was 69 nM. Furthermore, probe 14 demonstrated a successful application for detecting HSO 3 − in the mitochondria of living cells through uorescence imaging (Fig. 11). a,b-Unsaturated compounds are prone to a nucleophilic addition reaction by HSO 3 − . With this in mind, Xu et al. prepared probe 15 by the condensation of 1H-benzo[e]indolium (water soluble, high quantum yield, signicant Stokes shi (>100 nm), and mitochondrion-targeting group) with carbazole-3-aldehyde (Fig. 8, Table 1). 95 Upon the addition of HSO 3 − , the uorescence spectrum of probe 15 underwent ratiometric uorescence change with a decrease in the emission band at 588 nm and an increase in the new emission band at 462 nm, simultaneously. Interestingly, the cell staining experiment  revealed probe 15 as cell-permeable and mitochondriatargetable, and it can monitor the intracellular SO 2 derivatives in live cells (HeLa cells).
Zhang et al. constructed probe 16 composed of the coumarin-hemicyanine skeleton, in which the FRET process occurs from coumarin to hemicyanine and the ICT process occurs from the styryl to the indolium group (mitochondriontargeting) (Fig. 8, Table 1). 96 The nucleophilic approach of Zheng et al. constructed probe 19 by the condensation reaction between trimethylbenzoindolium (mitochondriontargeting moiety) and p-diphthalaldehyde (Fig. 8, Table 1). 98 The uorescence response of probe 19 toward HSO 3 − was initially ratiometric (at low concentrations) and then turn-on emission (at high concentrations). The sensing mechanism was a twice nucleophilic addition reaction. The cell imaging revealed that the probe 19 could specically detect HSO 3 − in the mitochondria of living cells (HepG2 cells).

Fluoride detection in mitochondria
The uoride ion is one of the most signicant anions crucial to many biological and medicinal processes. According to literature reports, a high uoride ion concentration can damage mitochondria through oxidative stress and reduce the mitochondrial respiratory chain's efficiency, resulting in mitochondrial malfunction and the development of neurodegenerative disorders. 99 As a result, it is essential and benecial to monitor uoride ions in mitochondria. Shen et al. applied ICT-modulated strategies for constructing probe 20 based on a diethylaminocoumarin derivative (a wellknown ICT uorophore) in which a pyridinium salt behaves as a mitochondrion-targeting group (Fig. 8, Table 1). 100 Initially, probe 20 exhibited red uorescence at 639 nm (l ex = 490 nm) attributed to the ICT process between the pyridinium cation and 7-diethylamino-coumarin unit. Upon the addition of F − , a new emission band at 539 nm emerged and increased at the expense of a decrease in emission at 639 nm. The sensing mechanism was F − -induced cleavage of the Si-O bond between the phenyl and triisopropylsilyl groups. Furthermore, the applied probe 20 successfully examined mitochondrial F − in living cells.

Palladium ion (Pd 2+ ) detection in mitochondria
Due to its high stability and reliability characteristics, palladium has been extensively employed in various applications, including catalysis, synthesis of organic compounds, medicine, dental crowns, fuel cells, and electronics production. However, palladium ion enrichment in living organisms can cause severe health-related diseases. 101,102 Therefore, developing an efficient tool for palladium ion detection in a biological system is necessary.
Wang et al. developed probe 21 to detect Pd 2+ in living cells based on the FRET process and rhodamine ring-opening mechanisms (Fig. 8, Table 1). 29 In PBS buffer solution, free probe 21 exhibited emission maxima at 472 nm (l ex = 400 nm). Upon adding Pb 2+ , a decrease in emission at 472 nm and an increase in the new emission band at 594 nm were observed. Moreover, the uorescence imaging experiment revealed the application of probe 21 for the ratiometric visualization of Pd 2+ in the mitochondria of living cells.

Challenges
Most organelle-targeting methods discussed above focus on lipophilic cationic uorescent probes that selectively target mitochondria. However, problems with cationic probes, such as the effect on membrane potential and cellular toxicity, are yet to be overcome. Furthermore, to explain the probe's potential to target mitochondria, mostly co-localization experiments have been performed using commercially available mitochondria-specic dyes such as MitoTracker Green FM, MitoTracker Red, and MitoTracker Orange. However, there can be several other contributing factors and principles behind the probe's potential to target mitochondria, and these factors need a clear discussion.
In addition, mitochondria contain hundreds of biomolecules, such as anions, cations, enzymes, mitochondrial DNA, RNA, lipids, and so forth. Using uorescent probes, it is still challenging to selectively label bioactive compounds at low concentrations (oen nanomolar levels). No doubt, ratiometric uorescent probes hold promise for removing various background interferences. However, nonetheless, just a few mitochondrion-targeting ratiometric uorescent probes have been developed to date.

Lysosome-targeting ratiometric fluorescent probes
Lysosomes, an essential subcellular organelle, which can function as a digestive compartment in eukaryotic cells and include a variety of enzymes and proteins, are critical regulators in metabolic processes under acidic pH circumstances. 103 As key indicators of lysosome function and oxidative stress, reactive oxygen species (ROS) such as HClO and H 2 O 2 and reactive sulphur species (RSS) such as H 2 S have been the focus of numerous probe designs. Since the lysosome lumen is acidic, developing probes that can detect HOCl in an acidic medium is challenging. 4,30

Reactive oxygen species (ROS) detection in the lysosome
H 2 O 2 is a signicant reactive oxygen species (ROS) with distinct destructive oxidation characteristics. Lysosomes can produce hydrogen peroxide (H 2 O 2 ) to combat pathogens. [104][105][106] Therefore, it's crucial to create a reliable method for measuring H 2 O 2 in inammatory tissues to assess the physiological and pathological link between lysosomal H 2 O 2 and inammation.
Inspired by the excellent optical properties of naphthalimide derivatives (donor-p-acceptor structured), Zhou et al. constructed probe 22 based on naphthalimide, benzylboric acid (H 2 O 2 responsive group), and pyridine group (lysosometargeting group) for monitoring H 2 O 2 in living tissue and in inamed tissue (Fig. 12, Table 1). 37 In the presence of H 2 O 2 , probe 22 displayed ratiometric uorescence change, assigned to H 2 O 2 mediated removal of the boric acid group from probe 22 and ICT effect. The uorescence color of the solution changed from bright blue to light yellow. Furthermore, tissue imaging experiments using a confocal microscope demonstrated a potential application for H 2 O 2 detection in inamed tissues.
TBET-based probes offer several advantages, such as high energy transfer efficiencies, improved imaging resolution, and a large Stokes-shi. 107,108 With this in mind, Shen et al. synthesized probe 23 based on the imidazo[1,5-a]pyridine moiety (donor) and rhodamine moiety (acceptor) (Fig. 12, Table 1). 15 The free probe 23 exhibited emission at 462 nm attributed to the emission of the imidazo[1,5-a]pyridine uorophore. Upon the gradual addition of aliquots of HOCl, the emission intensity at 462 nm almost remained constant, while a new emission maximum at 589 nm emerged and increased, assigned to the rhodamine moiety. The sensing mechanism was the change of the rhodamine spiro form to the ring-open state in the presence of HOCl and the TBET process between the rhodamine unit and imidazo[1,5-a]pyridine. Applied probe 23 successfully monitored the HOCl changes in the lysosomes.
Using a similar mechanism to the one described above, which converts the rhodamine spiro-form into the ring-open form when HOCl is present, Yuan et al. provided the coumarin and rhodamine based FRET platform 24 for HOCl detection in living cells (Fig. 12, Table 1). 14 Upon excitation at 410 nm, probe 24 displayed emission at 480 nm (which belonged to the coumarin moiety). However, in the presence of HOCl, the emission spectrum of probe 24 showed a decrease in intensity at 480 nm and an increase in new maxima at 580 nm (which belonged to rhodamine), assigned to a ring-opening and FRET (FRET efficiency = 93.75%) based detecting mechanism.
Liu et al. constructed probe 25 from phenothiazine coumarin and a morpholine unit for hypochlorite detection (Fig. 12, Table  1). 109 The uorescence titration of probe 25 with ClO − showed a blue shi in emission from 610 to 535 nm. The sensing mechanism was inhibition of the ICT process due to the oxidation of the phenothiazine moiety. Furthermore, the uorescence imaging experiment demonstrated the application of probe 25 for detecting ClO − in living cells (RAW264.7 cells) and zebrash (Fig. 13).

Reactive sulfur species (RSS) detection in lysosome
The endogenous oxidation of hydrogen sulde or sulfurcontaining amino acids produces bisulte, which remains in equilibrium with sulfur dioxide and sulte in aqueous media. [110][111][112][113] As a result, the discovery of bisulte in lysosomes is of great interest.
Tamima et al. pioneered probe 26 by introducing a morpholine moiety (targeting group) to the benzopyronin dye ( Fig. 12, Table 1). 114 Aer adding bisulte, the absorption and uorescence spectrum of probe 26 displayed complete peak separation from 613 nm to 426 nm (spectral shi = 187 nm) and from 704 nm to 512 nm (spectral shi = 192 nm), respectively. The sensing mechanism was the 1,6-conjugate addition reaction. Probe 26 specically targeted lysosomes and displayed application for monitoring intracellular bisulte levels.
Cysteine, homocysteine, hydrogen sulde, and glutathione are examples of cellular thiols frequently studied employing lysosome-targeting probes. 115 These biothiols are signicant indications of lysosomal function because they are produced by lysosomal proteolysis. 116 Tamima et al. devoted benzo[b]xanthene-derived probe 27 for the two-photon ratiometric uorescence-based detection of cysteine (Fig. 12, Table 1). 117 The detecting mechanism of cysteine was a 1,6-conjugate addition reaction to the benzoxanthene core, resulting in the formation of a cysteine adduct that emitted red. However, in the presence of hydrogen peroxide, the 1-cysteine adduct was reverted to 27. Furthermore, probe 27 demonstrated practical application to quantify cysteine levels in biological samples (human blood plasma).
Zhang et al. applied a cleavable FRET-based strategy for designing a lysosome-targeting probe 28, based on a coumarin-NBD (nitrobenzofurazan) cassette (Fig. 12, Table 1). 118 Free probe 28 showed two emission maxima at 415 nm and 560 nm, belonging to the coumarin and NBD groups. Upon treatment with H 2 S, probe 28 underwent ratiometric uorescence change, the solution's emission color changed from yellow to blue, and the intensity ratio increased from 0.22 to 67.7 (300-fold approx.). Furthermore, probe 28 preferentially targeted lysosomes and displayed potential application for detecting H 2 S in lysosome.

Cu 2+ detection in the lysosome
Many essential physiological processes involve the role of copper. Copper plays important roles in a variety of fundamental physiological processes. 119 At the organelle level, improper copper homeostasis can cause several severe illnesses. [120][121][122] As a result, it is still essential to monitor the concentration of copper levels in cells, particularly in lysosomes.
Liu et al. applied FRET-based strategies for constructing ratiometric probe 29 (Fig. 12, Table 1). 123 The probe 29 displayed high selectivity, sensitivity (detection limit = 1.45 nM), and ratiometric uorescence response towards Cu 2+ . From uorescence imaging experiments, live cells (L929 cells) incubated with probe 29 showed blue and weak green uorescence. However, cells pre-treated with Cu 2+ and then incubated with probe 29 showed solid green and diminished blue uorescence. In addition, probe 29 was applied for uorescence imaging of Cu 2+ in the lysosome of living cells.
Inspired by Czarnik's report on Cu 2+ -induced ring opening of rhodamine, 124 Wu et al. synthesized probe 30 for Cu 2+ detection (Fig. 12, Table 1). 125 In CH 3 CN-H 2 O (8 : 2) solution, probe 30 behaved as a selective and sensitive probe for Cu 2+ over other tested ions. The sensing mechanism was Cu 2+ -induced ring opening and the FRET process from pyrrole to rhodamine.

Hg 2+ detection in the lysosome
Mercury is a hazardous and pervasive heavy metal that can result in numerous serious health issues, including renal failure, damage to the central nervous system, etc. 126 Therefore, it is crucial to have an effective method for detecting mercury ions.
Zhang et al. developed an NBD-based probe 31 by introducing a morpholine moiety to the NBD uorophore for Hg 2+ detection (Fig. 12, Table 1). 127 In the presence of Hg 2+ , the absorption spectrum of probe 31 showed a red shi of the peak from 470 nm to 494 nm, and the color detected by the naked eye changed from light yellow to red. Under similar conditions, the uorescence spectrum also revealed a red shi of emission maxima from 535 nm to 595 nm. Herein, the morpholine unit played dual roles as a ligand for Hg 2+ and lysosome-targeting group. The sensing mechanism was assigned to the inhibition of the PET process from the nitrogen atom of morpholine to the NBD uorophore upon coordination with Hg 2+ .

Challenges
Lysosome-based research has advanced signicantly in recent years. Nevertheless, there are still several challenges for these probes. The synthetic probe cannot differentiate between autolysosomes, autophagosomes, endosomes, and other acidic compartments. These probes are harmful to live cells and inappropriate for long-term detection because they make lysosome alkaline. As a result, lysosome-specic probes devoid of the alkalization effect are necessary. Most of the developed probes have emissions in the visible region, thus, cannot be used for deep-tissue imaging due to the poor penetration power.

Endoplasmic reticulum-targeting ratiometric fluorescent probes
The most prominent organelle in a cell, the endoplasmic reticulum (ER), is crucial for protein synthesis, folding, distribution, and calcium ion storage. Literature reports revealed that ER stress, which is linked to signicant diseases, can cause autophagy and even cell death. Therefore, studying ER is an exciting eld of research. 128 In the past, the diaminomalenonitrile-based Schiff base has been used by several researchers for developing ClO − specic sensors. [132][133][134] On the other hand, the sulfonamide group has been known to target ER. 135 Taken together, Hou et al. constructed biosensor 33 for ClO − detection in ER (Fig. 14, Table  1). 39 Upon the addition of ClO − , the uorescence spectrum of biosensor 33 displayed a blue shi in emission wavelength from 554 nm to 480 nm, attributed to the reaction-based sensing mechanism. The plot of emission intensity ratio (I 480 /I 554 ) vs. ClO − concentration was linear between 0 and 120 mM concentrations. The detection limit was 0.59 mM for ClO − . The applied biosensor 33 examined exo/endogenous ClO − in the ER of living cells.
Inspired by the excellent optical properties and ICT process of 4-aminonaphthalimide moiety, 136 Ma et al. constructed biosensor 34 using a 4-aminonaphthalimide moiety (uorescent group), (2-aminoethyl) thiourea unit (HClO recognition site), and p-toluenesulfonamide unit (ER-targeting group) (Fig. 14, Table 1). 41 In ethanol-H 2 O (1 : 1, v/v) solution, free sensor 34 showed emission at 533 nm (green emission), attributed to the ICT process. Aer adding HOCl, biosensor 34 revealed ratiometric uorescence change with blue-shi (49 nm) of the emission maxima from 533 nm to 484 nm, which was assigned to inhibition of the ICT process. Furthermore, biosensor 34 demonstrated an application for HOCl detection in the ER of PC-12 cells.
In the past, aryl boronic acid has been widely utilized in designing uorescent sensors for biological species, 137   over other ROS (Fig. 14, Table 1). 18 Sensor 35 followed an electrophilic oxidation mechanism that involved B-H bond cleavage. Sensor 35 successfully targeted ER and demonstrated an application for monitoring HOCl in living cells (Raw 264.7 cells) and rat hippocampal slices by a two-photon uorescence microscopy experiment.

Reactive sulfur species (RSS) detection in endoplasmic reticulum
Taking into account the essential role of H 2 S during ER stress and ER functions, 142,143 Shu et al. pioneered ratiometric NIR-uorescent biosensor 36 based on dicyano-isophorone (NIR emission, large Stokes-shi) and an O-carboxybenzaldehyde unit (H 2 S recognition site) for H 2 S detection in ER (Fig. 14, Table 1). 144 Sensor 36 showed uorescence emission at 560 nm due to the ICT effect. Upon the gradual addition of H 2 S, a new emission peak at 650 nm appeared and increased with the simultaneous decrease in l em at 560 nm. The sensing mechanism was a nucleophilic addition reaction. Furthermore, sensor 36 demonstrated an application for monitoring H 2 S in living cells (HeLa cells) and zebrash. Interestingly, sensor 36 successfully detected endogenous H 2 S produced during ER stress incited by Tunicamycin.
Nowadays, physiological functions of SO 2 and its derivatives are gaining increasing attention. According to literature reports, SO 2 is vital in cardiovascular processes 145 and can regulate hippocampal neuron apoptosis. 146 Thus, SO 2 detection is essential. Li et al. developed a FRET-based platform 37, constructed from benzoindole-based hemicyanine (acceptor) and naphthalimide derivatives (donor), for the detection of SO 2 derivatives in ER (Fig. 14, Table 1). 147 Sensor 37 exhibited emission maxima in buffer solution at 610 nm (l ex = 440 nm), attributed to the FRET process. The sensing mechanism was a nucleophilic addition reaction and inhibition of the FRET process. Furthermore, sensor 37 successfully targeted ER and demonstrated an application for imaging exogenous and endogenous SO 2 derivatives in living cells.

Cu 2+ detection in endoplasmic reticulum
Mitochondria and lysosomes implicate the cellular homeostasis of copper. 148 Furthermore, lysosomes in damaged tissue contained high concentrations of copper ions. 149 However, it is unclear whether or not the copper ions in these organelles are the cause of the ER's harmful actions. Thus, to understand copper-related diseases, a reliable technique for imaging copper at the level of organelles is crucial.
Park et al. presented a naphthalimide and hydrazide based biosensor 38 for copper ion detection in living cells (Fig. 14,  Table 1). 150 Upon the addition of copper ion to the solution of sensor 38 in HEPES buffer, the emission maxima were red-shied (380 nm to 440 nm), and solution color changed from blue to yellowish-green. The detecting mechanism was assigned to the copper-mediated hydrolytic reaction of sensor 38, forming aminonaphthalimide. Additionally, the biocompatible sensor 38 specically targeted the ER of living cells and displayed potential for qualitative and quantitative detection of Cu + /Cu 2+ under physiological conditions.

Monitoring pH in endoplasmic reticulum
The physiological functions of the ER, such as targeting during secretion, protein sorting, and retrieving resident chaperones, are regulated by the pH of the ER, which serves as a crucial parameter. 151 The ER pH is the same as that of the cytoplasm under normal physiological conditions. 152 Recent research has shown that ER stress potently stimulates autophagy, strongly linked to many diseases (such as cancer, infectious disorders, and neurodegeneration), causing the ER pH to drop. 153,154 Therefore, it is crucial to quantitatively evaluate the pH change in ER to explain the biological functions of ER fully.
Fluorescent probes with dual responsive sites have shown improved sensitivity to pH and enhanced emission wavelength. 155 With this in mind, Dong et al. pioneered probe 39 based on the naphthalimide-coumarin platform and employed hydroxyl and morpholine groups as the pH-responsive sites (Fig. 14, Table 1). 40 On excitation at 405 nm, the uorescence spectrum of probe 39 showed ratiometric change with a single emission band at 527 nm (acidic pH, 4.09-5.08) and two emission maxima at 446 nm and 527 nm between pH 5.08 and 7.73, and further single emission maxima at 446 nm at pH 7.73 (basic pH). The sensing mechanism was assigned to the FRET-PET-ICT process (Fig. 15). Furthermore, due to the presence of the ptoluenesulfonamide group (ER-targeting group), probe 39 successfully targeted ER. It demonstrated an application for quantitatively detecting the pH changes in the dexamethanosetreated cell and during ER stress.

Challenges
Over the past few years, scientists engineered several ER-targeting uorescent probes for the selective detection of various substances like NO, H 2 S, H 2 O 2 , and HOCl. However, the mechanism behind ER selectivity still needs to be claried. Many biological species present in ER still need to be detected due to a lack of efficient probes. In addition, most of the probes discussed above emit visible light. Thus, their in vivo applicability is restricted.

Golgi apparatus-targeting ratiometric fluorescent probes
The Golgi apparatus is an organelle with a phospholipid membrane composed of cisterna. It transforms proteins from the rough ER, then separates them into vesicles for transport to other cell regions. According to recent ndings, CO is crucial for the Golgi apparatus. [159][160][161] Thus, to study in depth various functions of the Golgi apparatus, it is essential to develop efficient Golgi apparatus-targeting probes.

Reactive oxygen species (ROS) detection in Golgi apparatus
Golgi oxidative stress is closely linked to the occurrence and progression of hypertension, and the concentration of hydrogen peroxide (H 2 O 2 ) plays a critical role in this process. To address this issue, Wang et al. developed a two-photon uorescent probe 41, which targets the Golgi apparatus with the aid of a phenylsulfonamide group. The response mechanism was a reaction-based process (Fig. 17). 162 Upon reacting with H 2 O 2 , the boric acid ester is transformed into a hydroxyl group that donates electrons, thereby promoting the push-pull electron effect of the naphthalimide-conjugated system. This leads to the production of strong uorescence emission. The probe 41 enables in situ H 2 O 2 ratiometric imaging in living systems and provides a highly effective means to monitor Golgi oxidative stress. The probe 41 was able to identify the generation of H 2 O 2 during Golgi oxidative stress and demonstrated increased levels of Golgi H 2 O 2 in the kidneys of hypertensive mice.

Reactive nitrogen species (RNS) detection in Golgi apparatus
The detection of peroxynitrite (ONOO − ) is essential for the study and treatment of drug-induced liver injury (DILI) associated with oxidative stress. Targeting the Golgi apparatus has emerged as a new approach for DILI research and treatment. Feng et al. developed a new probe, 42, by conjugating a sulfanilamide moiety to a coumarin-hemi-cyanine conjugated system (Fig. 18). 163 The probe displayed high sensitivity, selectivity, and low cytotoxicity, and showed a rapid ratiometric uorescence response to ONOO − . Unexpectedly, probe 42 also displays unique targeting properties in living cells, with the ability to label the cell membrane rst and then the Golgi. Imaging experiments with probe 42 showed it to be effective in monitoring ONOO − under Golgi oxidative stress and in DILI using mice models.

CO monitoring in Golgi apparatus
Inspired by the recent reports on the uorescent probe for CO detection based on the Tsuji-Trost reaction and to understand in depth the role of CO in subcellular organelles, Zheng et al. constructed probe 43 (Fig. 19, Table 1). On excitation at 360 nm, probe 43 exhibited emission at 425 nm. 164 Upon the addition of CORM-3 (CO donor), probe 43 showed a signicant 95 nm red shi with a decrease and increased emission maxima at 425 nm and 560 nm, respectively. The detecting mechanism was attributed to the cleavage of the allycarbamate group of probe   43 to form 43a mediated by a Pd 0 Tsuji-Trost reaction. Furthermore, probe 43 found application for CO imaging in cells and zebrash and to visualize CO levels during cellular oxidative stress stimulated by lipopolysaccharide (Fig. 20).

Glutathione (GSH) detection in Golgi apparatus
The high expression of antioxidants like glutathione in cancer cells helps them withstand oxidative stress within the Golgi apparatus. Thus, monitoring changes in glutathione concentration within the Golgi could serve as an effective way to track the occurrence and progression of tumor cells. Rong et al. created a Golgi-targeting probe 44 that could detect GSH with high accuracy. 165 The uorescence titration experiment of probe 44 with GSH revealed a decrease in intensity at 425 nm and emergence of a new peak at 510 nm. The detection limit was 0.49 mM for GSH. The response mechanism was a reactionbased process (Fig. 21). Furthermore, the Golgi stress response experiment revealed the ability of probe 44 for in situ endogenous GSH detection in the Golgi apparatus during oxidative stress.

Challenges
The Golgi apparatus-targeting ratiometric uorescent probes are rare in the literature. Furthermore, the targeting mechanism is not clear.

Nucleus-targeting ratiometric fluorescent probes
For both cancer treatment and genetic engineering, the cell nucleus has been a primary target since it stores the genetic material that is protected by the nuclear envelope, which is made up of two lipid bilayer membranes. 166

Reactive sulphur species (RSS) detection in the nucleus
Organisms' excessive generation of formaldehyde (FA) and sulfur dioxide (SO 2 ) is directly linked to several ailments, such as genotoxicity, respiratory disease, and neurological abnormalities. However, the protective barrier of the cell nucleus membrane makes it challenging for uorescent probes to investigate the correlation of FA and SO 2 in the nucleolus regions. Ma et al. took this challenge and constructed probe 45 based on the benzopyryllium-dansyl FRET platform (Fig. 23, Table 1). 52 The probe 45 was used for SO 2 reversible sensing and recovered by FA. The sensing mechanism of SO 2 was the interruption of the FRET process between dansyl (donor) and benzopyryllium (acceptor). The FA addition restored the FRET process because of the reversible Michael addition reaction. Probe 45 found application for quantitatively monitoring endogenous SO 2 /FA in the nucleus region of live cells and living animals.

Reactive nitrogen species (RNS) detection in the nucleus
Nitric oxide (NO) is a signicant signal molecule involved in a variety of physiological and pathological processes. Thus, to understand these processes, real-time detection of short-living NO in the biological medium is crucial. Based on their previous reports on NO detection, 167 Li et al. developed probe 46 for NO detection in living cells. 168 The uorescence spectrum of probe 46 showed a ratiometric change in uorescence with NO, and the plot of intensity ratio I 530 /I 424 vs. NO concentration was linear between 0 and 40 mM. The sensing mechanism was a reaction-based process in which 46m reacted with NO to generate product 46p (Fig. 22, Table 1). In living RAW 264.7 cells, 46m can detect both exogenous and endogenous NO. It's interesting to note that 46m and its sensing product 46p both show localization to the nucleus and the mitochondria,   Chemical Science respectively. In the presence of ctDNA, 46m showed high sensitivity to NO (LOD = 2.8 nM). However, due to nucleus localization, 46p could be an excellent green-uorescent probe for the nucleus.

Nucleus DNA detection
The nucleus viscosity and G-quadruplex have several critical roles in biological processes, such as controlling gene expression, preventing tumorigenesis, etc. Thus, to better understand their molecular process and function, an efficient tool that can target the nucleus is in demand. Sun et al. developed D-p-A type probe 47 based on triphenylamine (donor) and N-methyl benzothiazole (acceptor and nucleus-targeting group) (Fig. 23, Table 1). 169 With a considerable ratiometric increase in uorescence, probe 47 revealed strong selectivity to G-quadruplex DNA and viscosity inside the nucleus. However, the notable limitation of probe 47 is that it cannot distinguish between Gquadruplex DNA and viscosity change. Ferroptosis controls cell death by accumulating lipid peroxide-associated reactive oxygen species, which is predicted to change the shape and polarity of lipid droplets (LDs). However, there needs to be concrete proof of this. Wang et al. reported dual-organelle targeting (LD and nucleus) uorescent probe 48 for monitoring cellular microenvironment polarity change (Fig. 23, Table 1). 170 The uorescence titration of probe 48 with ds26DNA revealed ∼11-fold increase in emission intensity at 670 nm, while emission maxima at 470 nm remained constant. From molecular modeling calculations, the selectivity of probe 48 to DNA could be due to its binding to the minor grooves of DNA through hydrogen bonding and electrostatic interactions.
Yang et al. developed a ratiometric uorescent probe 49 based on a naphthalimide dye and a Hoechst (nucleus-targeting unit) (Fig. 23, Table 1). 171 Upon gradually increasing the concentration of ctDNA, the emission maxima of probe 49 at 450 nm and 505 nm increased signicantly, ascribed to the FRET process from the Hoescht to the naphthalimide unit. Probe 49's remarkable benet is its ability to provide precise and wash-free nuclear DNA staining in living cells, including normal cells COS-7 and L02, as well as cancer cells MCF-7, SMMC-7721, and HeLa. Furthermore, probe 49 found a potential application for monitoring nucleus DNA damage induced by the anticancer drug etoposide and hydroxyl radicals.

Challenges
For chemical biology purposes, nucleus-targeting systems have been thoroughly investigated. However, commercially available nucleus-targeting uorescent probes are limited to staining DNAs. 172 Nowadays, newly developed nucleus-targeting probes are more specic to a particular analyte but are still rare. Furthermore, each developed method needs a clear explanation of organelle-specic/targeting mechanism, cellular toxicity, targeting ability in the altered environment, probe stability, response time, etc.
9 Membrane-targeting ratiometric fluorescent probes Cytomembrane is an essential target for the study of dynamics and morphology due to the recent discovery of membrane microdomains (ras) in cancer 173 and viral infection. 174,175 The cytomembrane also contributes to amyloid formation in neurodegenerative diseases. 176,177 9.1 Reactive sulphur species (RSS) detection in the membrane Zhang et al. developed probes 50 and 51 based on coumarin derivatives (Fig. 24, Table 1). 178 Notably, the uorescence spectrum of probe 50 and 51 exhibited ratiometric response with SO 2 and visible color change of the solution from dark purple to colorless. The nucleophilic addition reaction was the sensing   mechanism of probes 50 and 51 for SO 2 . From cell imaging experiments, probes 50 and 51 exhibited good cytomembranetargeting and mitochondrion-targeting ability and can detect SO 2 in mice. Due to the negatively charged cell inner membrane, the cationic property of probe 50 allowed it to target the cell membrane through electrostatic interactions. However, the probe 51 can selectively accumulate in mitochondria because of its positively charged nature, long alkyl chain, and appropriate hydrophobic characteristic (Fig. 25).

Monitoring pH changes in the membrane
The cell's various biomolecules, pathogens, and uids are regulated by intracellular vesicles that the plasma membrane produces. 179 Intravesicular pH changes during trafficking depend on endosomal maturation and signaling. 180 The cell's various biomolecules, pathogens, and uids are regulated by intracellular vesicles that the plasma membrane produces. Inspired by the work of Liu et al. on red-shied chromenoquinoline based probes, 181 Michelis et al. designed probe 52 based on chromenoquinoline for imaging the distribution and acidi-cation of intracellular vesicles and to measure the pH of individual vesicles (Fig. 24, Table 1). 182 From the uorescence experiment, probe 52 underwent an increase in emission intensity upon binding with the plasma membrane (10-fold). Furthermore, probe 52 monitored the acidication of the vesicles throughout the endocytic pathway. The main limitation associated with probe 52 is that we cannot use it for long-term tracking due to the instability of the basic form.

Challenges
Despite several efforts in designing membrane-targeting ratiometric uorescent probes, literature reports are rare. To date, no commercially available membrane-targeting uorescent probes are available. In the future, researchers must consider several parameters for designing efficient membrane-targeting uorescent probes, such as probe stability, selectivity, sensitivity, response time, orientation/location in the membrane, and detailed mechanism of interaction with the membrane.
10 Multi organelle-targeting ratiometric fluorescent probes Developing a powerful molecular tool that can target other organelles simultaneously is necessary to study the relationship between different organelles.

Reactive sulphur species (RSS) detection in lysosome and mitochondria
From a recent study, SO 2 is a critical gas messenger that plays a vital role in many cellular processes, including apoptosis in lysosomes and mitochondria. Therefore, to know the relationship between lysosome and mitochondria in regulating SO 2related cellular activities, Kong et al. pioneered probe 53 (Fig. 26, Table 1). 183 Free probe 53 showed emission at 600 nm (red uorescence) owing to the FRET process between the naphthalimide unit and the semi-cyanine unit. Upon the addition of SO 2 , the emission intensity at 600 nm decreased. In comparison, a new emission maximum at 530 nm appeared and increased, assigned to Michael's addition reaction of SO 2 in the semi-cyanine unit and inhibition of the FRET process. In living cells, probe 53 found an application for simultaneously detecting endogenous SO 2 in lysosome and mitochondria by one and two-photon modes.

Monitoring pH uctuation in mitochondria and lipid droplets (LDs)
Understanding the fundamental connection between uctuating mitochondrial pH and lipid droplet (LD) generation is crucial for understanding cell physiology. Bai et al. developed probe 54 based on hemicyanine and rhodamine dyes for selectively monitoring mitochondria and lipid droplets under different pH values through a dual-emission channel ( Fig. 27 and 28, Table 1). 19 The pH (from 2.52 to 10.50) sensing behavior of probe 54 revealed a notable decrease in the emission band at 580 nm, accompanied by the increase in a new emission peak at 450 nm, attributed to two different structural forms under acidic and basic medium. Under acidic conditions, the ringopen form of probe 54 targeted mitochondria and displayed solid red emission. In contrast, the ring-closed form of probe 54 targeted LDs and gave blue emission. Furthermore, applied probe 54 monitored the pH uctuation in living cells in the presence of different exotic chemicals.

Challenges
Dual organelle-targeting probes hold great promise for elucidating the relationship between organelles and deepening our  understanding of the biological processes behind the biological species. However, building molecular probes with two or more sensitive moieties is challenging. Furthermore, dual organelletargeting probes are rare in the literature.

Conclusion and future outlook
This review article highlighted recent advances in organelletargeting ratiometric uorescent probes reported since 2015. In particular, we discussed synthetic uorescent probes with potential applications in biological systems such as biological species detection, uorescence imaging, studying physiological and pathological processes, etc. Additionally, we highlighted the methods utilized to construct these probes and sensing mechanisms for their response to particular species.
Nowadays, signicant research has been put into developing organelle-targeting uorescent probes to create uorescent tools with improved resolution and sensitivity and a better understanding of the molecular mechanisms behind various biological processes. In conclusion, high selectivity, high reactivity, fast response time, low detection limit, good solubility, and organelle-targeting ability are the multiple advantages of the uorescent probe. Currently, ratiometric probes that meet all the above properties are rare in the literature.
Researchers will always be very interested in the advancement of ratiometric probes and their commercial applications. Despite the abundance of current outcomes, the following areas require additional work in the future. First, compared to traditional onephoton uorescence probes, two-photon uorescent probes provide several advantages, such as deep visualization, reduced photo-toxicity, minimal light scattering, and highly bright and contrast images. 184 Thus, the development of ratiometric probes with two-photon properties is greatly needed. Second, for in vivo application, near-infra-red (NIR) probes provide several advantages, such as deep tissue penetration, reduced photon scattering, and reduced photodamage to the living organism. 185 Thus, ratiometric probes with emissions in the NIR region are in demand. Third, Reversible probes hold great promise for revealing the dynamic states of relevant analytes in various processes. 52 Thus, developing ratiometric probes for biological species detection based on reversible reactions is essential. Fourth, probes targeting organelles such as the nucleus, membrane, lipid droplet (LD), melanosome, and Golgi apparatus are rare in the literature (Table 1). Fih, there are several species in biological systems. However, only a limited number of species, such as ROS, RNS, RSS, metal ions, and pH, have been the subject of interest (Table 1). Sixth, most of the discussed ratiometric probes have found applications for the bio-imaging of biological species in living cells and mice. To date, no commercially available ratiometric probes are available. By taking the factors mentioned above into account in the probe design, uorescent probes can be utilized as signicant materials in the coming days, and their commercial availability will be anticipated in the future.

Data availability
All the data were collected freely from websites such as https:// scholar.google.com/ and https://www.sci-hub.se/. Microso Office Word 2007 was used for writing the article, while Microso Office PowerPoint 2007 was used for graphical presentation. ChemBioDraw was used for chemical structure drawing.

Author contributions
Dr Manoj Kumar Goshisht and Dr Neetu Tripathi contributed equally to writing the original dra and to reviewing and editing the manuscript. Dr Goutam Kumar Patra and Dr Manohar Chaskar contributed to reviewing and editing the manuscript.