Research On Cellular Function Modulation Based On Femtosecond Laser Stimulation

Biophotonics is a frontier interdiscipline combines photonics and biology.The development of femtosecond laser provides a series of new technologies,methods,opinions and theories for biophotonics remarkably.Nowadays biophotonics based on femtosecond laser has been investigated and applied in multiple research fields such as multiphoton microscopy and micromanipulation widely and deeply.However,former research about effection on cells and injury mechanism of femtosecond laser stimulation was relatively rough.What's more,mechanism of cell's biological reaction after femtosecond laser stimulation and interaction between laser and cell is not clearly demonstrated.Based on such background,this thesis investigated the cellular molecular transduction induced by femtosecond laser exposure and promoted a new method of modulating cellular molecules.Besides,ultrafast microscopy is discussed and researched experimentally.This thesis firstly reviews the development history of biophotonics based on femtosecond laser,current research focuses and present research status.Afterwards,calcium,which is a basic ion in biological analysis in cell,is introduced in detail,including the storage of calcium in cells,multiple functions of calcium in cells and other molecular signaling related to calcium.Furthermore,micromanipulation system consists of femtosecond laser,confocal microscope and CCD is introduced.Meanwhile,femtosecond laser induced calcium ion density change is discussed experimentally and theoretically.Based on this,we found that femtosecond laser could further modulate other molecular action.By precisely adjusting optical exposure stimulation of femtosecond laser,cellular calcium ion release and high level calcium maitainance could be achieved,after which calcineurin is activated.Then transcription factor NFAT will be dephosphorylated and go into nuclear and initiate gene expression,which means the NFAT signaling passway is activated.In experiment,femtosecond activated NFAT translocation from cytoplasm to nuclear was realized,which could further induced gene expression,this was tested by luciferase reporter gene.Base on this theory,gene expression in cells such as TNF-? could be influenced by femtosecond laser treatment.Except for normal somatic cell,molecular action in stem cell could also be modulated by femtosecond laser.Then we applied the optical stimulation method to mesenchymal stem cells(MSCs),calcium ion density could also be modulated by femtosecond laser in MSCs.Furthermore,transcription factors such as Runx2 and Osterix could be modulated accordingly to some extent.Owing to the fact that these transcription factors are critical in bone marrow stem cell differentiation,the optical method might be of great potential value in related research fields such as inducing stem cell differentiation.However,other effect such as injury is not ignorable when cell is stimulated by light.We found that both stimulation of femtosecond laser exposure for a short time and long time scanning by continuous laser could induce autophagy in cell.We systematically observed the influence of different optical stimulation on cellular autophagy and analysed the mechanism of two methods of stimulation,then demonstrated the sensitiveness of autophagy to laser excitatioin.We deemed that although the optical stimulation is relatively safe and would not effect cellular activity very much,it could still cause special cellular stress responses such as autophagy.In the mechanism of autophagy induced by femtosecond laser exposure,mitochondrial membrane might be one origination of autophagosome.Because the process of femtosecond laser exposure in cells belongs to ultrafast course,it is necessary to observe it to get further understanding.Thus at last,ultrafast imaging technologies such as STEAM,STAMP and FIRE are introduced.Features,state of the art,application fields based on these technologies are discussed.Moreover,new fluorescence microscopy system based on FIRE is discussed in detail experimentally,and image based on new FIRE system was gotten.Besides,subordinate problems in system building were investigated and concluded.Most of the work in this thesis investigated the stimulation mechanism of femtosecond laser in cell,and provided relatively new theories and technological basis of cellular modulation by optical method.