Design And Synthesis Of Molecular Rotors And Their Applications In Imaging Mitochondrial Viscosity

Mitochondrion is an organelle coated by double membranes that presents in most eukaryotic cells.Mitochondria are energy-producing structures in cells and the primary site for aerobic respiration.When the human body suffers from some diseases(such as Parkinson’s disease)it will cause changes in the mitochondrial membrane viscosity.Mitochondrial viscosity is highly relative to respiration and tricarboxylic cycles.The molecules produced in respiratory process could mechanically or osmotically induce the viscosity changes in mitochondria,and the changes of mitochondrial viscosity could in return regulate metabolite diffusion during respiratory.In addition,mitochondrial function could be restrained by decreasing its membrane fluidity,which resulting in reducing electron transport chain activating,increasing ROS production,and facilitating cytochrome c release in the mitochondrial matrix viscosity.Therefore,in situ and real-time monitoring mitochondria viscosity,such a key parameter,is greatly crucial for biological and pathological researches.Fluorescent probes have been broadly applied as powerful tools due to the noninvasive character to biosamples,facile operation,and capability of in situ and real-time visualization of intracellular entity and events.Generally,fluorescent probes can be divided into turn-on ones upon single channel intensity change and ratiometric ones by means of dual channel intensity ratio.As known well,the fluorescent signals of turn-on probes are greatly susceptible by variable factors such as dye concentration,the excitation intensity,and environmental parameters(pH,polarity,temperature,and so on).Comparatively,Ratiometric probes can reduce background interference and provide more precise methods to normalize variation in path length,dye concentration,and photobleaching,and make quantitative detection possible.Thus,ratiometric probes have gained more intense interest because they can address the troubles posed by single-intensity measurement.Compared to imaging intracellular entity such as ions and lysosomes et al,the advantages of ratiometric probes is more obvious in imaging cellular physical parameters,such as intracellular pH,temperature,viscosity.By means of a ratiometric probe,fluorescent images with a scaleplate to targeting parameter can be obtained via calculation of the fluorescent intensity ratio in the two channels by pixels.Recently,various ratiometric probes for pH values and viscosity of cytoplasma or organelles have been reported intensively and extensively.Furthermore,the fluorescent images of these intracellular parameters have been acquired in confocal and two-photon microscopes.However,a ratiometric probe to mitochondrial viscosity suitable for a confocal microscope is still challenged.We constructed a fluorescent probe(probe PP-1)by incorporating an unsaturated pyridinium to a pyrene fluorophore.We think that probe PP-1 should be a good ratiometric probe to mitochondrial viscosity.From our previous work,a fluorophore with pyridinium can exclusively accumulate in the mitochondria.Similarly,to a mitochondrial probe presented by us,probe PP-1 possessed internal rotation bond.Thus,in low-viscosity environment only moiety of pyrene can emit,comparatively in high-viscosity condition whole conjugation assembly emit.Furthermore,according to scientific report of Liu et al,various ions and molecules such as F-,Cl-,Br-and Cys do not change the fluorescence of probe PP-1 except sulfite.Generally,unless biosamples have been polluted,the content of sulfite in normal cells is too low to affect fluorescence of probe PP-1.When excited by 405 nm as a routine source in a confocal microscope,probe PP-1 showed dual emission in 470 nm or 550 nm,and the log(I550 nm/I470 nm)of PP-1 has a linear relationship with the logarithm of the solution viscosity(η)in the viscosity range of mitochondria.As a result,the probe has been successfully applied for the monitoring of mitochondrial viscosity under confocal microscopes.Molecular rotors are sensitive to viscosity,so we can use them to monitor the intracellular viscosity changes.Meanwhile,these fluorescent dyes are good at imaging the high-viscosity targets with high signal to noise ratio(SNR).In this work,we synthesized three molecular rotors with D-π-A structures,TPA-1,TPA-2 and PP-2.These probes can respond to viscosity and exhibit weak fluorescence in low-viscosity solvent,such as water.The fluorescence can be greatly enhanced with the increasing amount of high-viscosity solvent,such as glycerol.Amongst,all of the three probes can target mitochondria in living cells.These results indicate that TPA-1,TPA-2 and PP-2 can be used in monitoring the viscosity change in mitochondria.The red-emitting fluorescent probe has advantages for imaging in living cells and tissues.Comparing probes that emit fluorescence in the blue to yellow range,the red probe has better spectral separation with cellular absorption and autofluorescence,so It is of great significance to design a dye with a large two-photon absorption cross section,long wavelength emission,and strong fluorescence properties.Two-photon fluorescence has a very large advantage,such as strong penetration of active biological samples,and less damage to the sample.In addition,two-photon fluorescence can only be excited by a strong laser near the focal point,which makes photobleaching,photodamage effects decrease.Two-photon fluorescence has its unique advantages in bioimaging applications and has therefore been a hot area of research.We designed three probes which emitted red fluoresce.They can image mitochondria clearly and have the potential of imaging organization.In summary,we synthesized a series of mitochondrial probes.Cross-energy transitions were used to design a proportional mitochondrial probe;a single peak viscosity mitochondrial probe was designed using a D-π-A structure.Using the enlarged resonance area of the molecule,a red fluorescence probe was designed,and several probes also had good two-photon performance.These probes can make some contributions to the further study of the structure and viscosity of mitochondria.