Fluorescence Imaging Of ROS And Polarity In Organelles

Cell,as the fundamental unit of life,is comprised of numerous organelles,including mitochondria,endoplasmic reticulum,lysosme,Golgi apparatus,ribosome and so on.The cell exerts the biological function by interplay and regulation of all these organelles.The various bioactive molecules such as reactive oxygen species?ROS?,metal ions as well as microenvironment including polarity,viscosity,p H values and temperature play an essential role in sustaining the normal function of the cell and organelles.ROS are a series of active molecules or free redicals derived from oxygen and play important role in maintaining redox balance,regulating cell proliferation,differentiation and apoptosis.The overprouduction of ROS will lead to oxidative stress by oxidation reaction,which will result in cell death and generate multiple diseases through damaging lipids,proteins and DNA.It is widely accepted that mitochondria are the main source and regulation core of ROS,however,other organelles,such as endoplasmic reticulum will also produce ROS during the synthesis of proteins and lipids.The ROS in endoplasmic reticulum are closely associated with metabolic diseases.Cell polarity as one of the important parameters in cellular microenvironment plays a crucial part in chemical and biological processes.Many cellular events,such as adipogenic differentiation,membrane fusion and proteins accumulation will lead to polarity variation in cell.In live cells,especially in organelles,the changes of polarity will alter the activity of enzymes or proteins,further affecting many physiological and pathological processes and inducing diseases,such as liver cirrhosis and diabetes.Therefore,accurate detection of ROS in organelles is beneficial to elucidating the production,conversion and correlation of ROS,which will provide new ideas for studying the pathogenesis of ROS-associated diseases.Meanwhile,it's vital to real-time detection of polarity in organelles for the early diagnosis and preventive therapy of diseases caused by polarity variation.More impoertantly,study of interaction of ROS and polarity has more comprehensive and profound meaning for illuminating regulation of cell function as well as occurrence and development of disease.Fluorescence imaging is a powerful tool to track various bioactive molecules in live cell and in vivo because of its remarkable advantages,such as high temporal-spatial resolution,excellent biocompatibility and high sensitivity.In recent years,many fluorescent probes for detection of ROS and polarity have been developed,however,these probes have many shortcomings.For example,although many ROS-responsive probes have been reported,it can't achieve the simultaneous fluorescence imaging of ROS in two even more organelles.It's hard for the existing probes to monitor polarity within specific organelle.Meanwhile,due to the poor sensitivity and short excitation and emission wavelengths,it can't realize the polarity detection in deeper tissue and in vivo.Moreover,current method can't accomplish the simultaneous fluorescence imaging of ROS and polarity in specific organelle.For all the above reasons,by targeting mitochondria and endoplasmic reticulum,we develop many approaches to achieve simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during different apoptotic stimuli,near-infrared fluorescence ratiometric as well as fluorescent/photoacoustic dual mode imaging of polarity in mitochondria and endoplasmic reticulum,simultaneous fluorescence imaging of the synergetic changes of superoxide anion and polarity in endoplasmic reticulum during stress.The main results of this dissertation are shown as follows:1.We have designed and synthesized two organelle-targetable fluorescent probes termed MI-H₂O₂ and ER-H₂O₂ for imaging H₂O₂ in mitochondria and endoplasmic reticulum of live cells during apoptosis.ER-H₂O₂ and MI-H₂O₂ can selectively and sensitively respond to H₂O₂,displaying distinct excitation and emission spectra,which favors dual-color fluorescence imaging in live cells.These two probes have excellent organelle targeting capabilities,and thus are used to successfully image exogenous or endogenous hydrogen peroxide in mitochondria and endoplasmic reticulum.In addition,during diverse apoptotic stimuli,dual-color fluorescence imaging results reveal that the changes of H₂O₂ levels in mitochondria and endoplasmic reticulum are obviously different.2.We have designed and synthesized a new near-infrared ratiometric fluorescent probe,MCY-BF2,for ultrasensitive sensing mitochondria polarity.The long conjugated system and asymmetry structure of the probe account for the near-infrared excitation/emission spectra and large Stokes shift,respectively.By combining with cardiolipin with its n-hexadecyl,MCY-BF2 preferentially accumulates in mitochondria.MCY-BF2 is ultrasensitive to the polarity due to the excited state intramolecular charge transfer?ICT?,exhibiting weak fluorescence and longer emission wavelength with increase of polarity.Therefore,a plot of the fluorescence intensity ratios at two different wavelengths versus dielectric constant may be achieved,thereby can be utilized to estimate the polarity of certain media and live cells.MCY-BF2 can locate in mitochondria exclusively in various cells and discriminate polarity differences between normal and cancer cells.Also,the intrinsic polarity variance at different developmental stages in Caenorhabditis elegans was reported for the first time here.In addition,it was successfully applied for monitoring the polarity distinction in normal and tumor tissue in live animals,suggesting that tumor tissue of mice display obviously less polarity than that in normal tissue.3.We have designed and synthesized a new fluorescent and photoacoustic dual-mode probe,ER-P,for detection of endoplasmic reticulum polarity.ER-P displayed near-infrared excitation/emission spectra.The methyl sulphonamide moiety is introduced into ER-P to assist the probe to accumulate into endoplasmic reticulum by binding to the sulphonamide receptor.Due to the intramolecular charge transfer?ICT?,the maximum absorption wavelength of ER-P obviously red-shifts with the increase of environmental polarity,resulting in the changes of PA signal intensities at two selected wavelengths-700 nm?PA700?and 800 nm?PA800?.The PA signal intensity ratios between these two wavelengths?PA700/PA800?can quantify the certain polarity of the media.In addition,the fluorescence intensity of ER-P at 800 nm decreases dramatically along with increasing polarity of the media,which can accomplish the fluorescence detection of polarity upon 633 nm excitation.ER-P can exclusively accumulate into endoplasmic reticulum and indicate the change of the polarity during endoplasmic reticulum stress.Also,ER-P can distinguish the ER polarity of normal and cancer cells by imaging flow cytometry.More importantly,due to the excellent photoacoustic signal,the polarity difference of liver tissue in normal and diabetic mice were visualized by photoacoustic ratiometric detection,indicating the polarity in liver tissue of diabetic mice is larger than that in liver tissue of normal mice.4.We have designed and synthesized a new reversible fluorescent probe,ER-NAPC,for detection of superoxide anion in endoplasmic reticulum.The probe can selectively and sensitively respond to superoxide anion and has outstanding endoplasmic reticulum targetable ability.The probe can real-timely visualize the increase of superoxide anion level during endoplasmic reticulum stress.More importantly,ER-NAPC and polarity probe ER-P display distinct excitation and emission spectra,which favors dual-color fluorescence imaging in live cells.With these two probes,we successfully image the synergetic changes of superoxide anion and polarity in endoplasmic reticulum during stress.The results demonstrated that endoplasmic reticulum stress will result in rise of superoxide anion and polarity.