Developments And Biological Applications Of Mitochondrial Targetable Fluorescent Dyes

Mitochondria,which feature dynamic such as fission,fusion,transport,and mitophagy,are important organelles for maintaining stability.Real-time and accurate monitoring of the dynamic of the mitochondrial microenvironment and morphology,which is the pivotal topics of cell biology,put forward high requirements for the fluorescent dyes.Most traditional mitochondrial targeting dyes are limited in precision imaging of biomedical applications,due to their non-specific labeling,and inaccurate measurement,which used mitochondrial inner membrane potential to label mitochondria.Herein,a series of mitochondrial stably targeting fluorescent dyes have been designed and synthesized,which aim to enhance the detection accuracy and biocompatibility of fluorescent sensing.It is suitable for precision fluorescence imaging under various physiological and pathological.Thereby,the accurate information,that the fluorescence signals reflected change of mitochondrial microenvironment and micromorphological,can become a reliable indicator for medical diagnosis and biological research.Firstly,a change of mitochondrial temperature can be an important indicator of mitochondrial metabolism,in which mitochondria generate considerable heat with ATP production..For this reason,the development of fluorescent probes to detect mitochondrial temperature has become an attractive topic.Herein,Mito-TEM is designed and synthesized,which is a thermosensitive probe with stable mitochondrial labeling,and overcome the drawback of off-target the most mitochondrial fluorescent probes.Mito-TEM is based on a positively charged rhodamine B fluorophore that tends to be attracted to mitochondria,which have a negative potential,and the p-formyl phenyl group of Mito-TEM can be used as an anchor group to condense with an amino group in proteins to realize stable labeling.By using Mito-TEM,the change of mitochondrial temperature and morphology can be monitored under the drug stimulation with grayscale imaging and super-resolution imaging in live cells.Secondly,single fluorescence intensity as signal for detection is difficult to guarantee quantitative accuracy,because such detection might be interfered by some uncertain factors such as local probe concentration and excitation intensity.To overcome this limitation,a ratiometric thermometer Mito-TEM 2.0 is developed for the accurate quantification of mitochondrial temperature.Mito-TEM 2.0 exhibits different fluorescence ratios of the rhodamine B-BODIPY dyad when varying the temperatures,and can anchor in mitochondria via covalent bonds of the formyl aryl group reacts with the amino group of protein.With fluorescence ratiometric imaging,Mito-TEM 2.0 can quantitatively monitor the temperature changes of dysfunctional mitochondria and visualized indicate the inflammation of cells and zebrafish in different models.Furthermore,abnormal mitochondrial morphology is related to metabolic dysfunction and many diseases.To detect the changes of ultra-structures of mitochondria,a novel dye,RhBTPP-A is synthesized for the application of super-resolution imaging.The switching between fluorescence "on-off" states of RhB-TPP-A is finely regulated by the proton gradient in the mitochondria,which results in a self-blinking property to meet the requirement of singlemolecule localization imaging.RhB-TPP-A exhibits high switching frequency(14.7 per 100 frames)and high positioning accuracy(22 nm)after low light intensity excitation.By using RhB-TPP-A,the mitochondrial network fragmentation has been observed under light or drug stimulation with super-resolution imaging.Inspired the applicability of RhB-TPP-A in single-molecule localization imaging,a series of "self-blinking" long-wavelength analogue dyes are developed on silicon-rhodamine scaffold(HMSiR)through a modular approach.HMSiR is synthesized through metalized aryl silyl ether and phthalide,exhibiting excellent optical properties and biocompatibility.A series of sub-organelle-targeted dyes,named Lyso-HMSiR,Mito-HMSiR,ER-HMSiR,Halo-HMSiR,are constructed by using the "Click" reaction from the alkynyl group functioned platform dye HMSiR-Alk.These derivatives can label the corresponding subcellular organelles or fusion protein tag,and produce the high-quality images of ultra-structures with the super-resolution imaging technology.