Building Of Multiphoton Imaging System At The 1700-nm Window And Its Application To Multiphoton Fluorescence Microscopy Of Labeled Blood Vessels And Cells

Multiphoton microscopy?MPM?is capable of subcellular spatial resolution?on the order of submicron?and large imaging depth in biological tissue?typically millimeter?,due to the use of long wavelength excitation and the underlying nonlinear excitation principle.MPM has found widespread applications in imaging biological samples,especially in brain research in vivo,including mapping neural network and investigating neuronal function.Currently,the development trend of MPM is to get higher spatial resolution,larger field of view,faster imaging speed and larger imaging depth.Although the imaging depth of 3-photon microscopy?1350?m?is deeper than 2-photon microscopy?1000?m?,there are still problems hampering the development of its applications as follows:1.The imaging depth hasn't reached the theoretical limit yet?calculated to be 3.75 mm?because of the signal depletion deep in the tissue,which is due to the depletion of the excitation energy and relatively inadequate fluorescence signal intensity.2.1700-nm 3-photon microscopy is mostly confined to imaging blood vessel of brain,which lacks the ability of imaging versatile structures in the brain.In this paper,we demonstrate development of excitation laser sources,including a 500-nm tunable range soliton source and a high-energy?>100 nJ?soliton source in a photonic crystal?PC?rod.Using these excitation sources,we are able to perform both deep imaging,in versatile structures such as blood vessels and cells.Through the works demonstrated in this thesis,we will provide advanced imaging solution for brain research.We have performed the following research in this thesis:1.We build a broadband large-mode-area fiber source and a high-energy PC rod source.Soliton self-frequency shift was used to generate tunable optical solitons with amultiphoton fluorescence microscopy of labeled blood vessels and cells500-nm tunable range in LMA fiber and generate high-energy?>100 nJ?excitation pulses at the 1700-nm window in PC rod,pumped by a 1550-nm fiber laser.We further optimize the transmittance of the multiphoton microscope and improved transmittance to 46%from 20%.2.In order to prevent water vapor absorption by D₂O and the resultant multiphoton signal decrease,we demonstrate an easy to implement,yet very effective technique to seal D₂O from water vapor,using paraffin liquid.Ex vivo imaging results and in vivo deep-tissue THG imaging of the whiter matter in the mouse brain justify the effectiveness of this technique for long-span in vivo imaging.3.In order to prepare a high-quality cranial window for deep-brain imaging,we designe a titanium alloy circlets and improve the craniotomy details step by step.We compare different intravenous injection techniques for labeling blood vessels,concluding that retro-orbital injection is much easier to implement.We also demonstrate a procedure for preparing acute brain slice at room temperature.4.Based on the optimized laser and microscope systems,we demonstrate several applications of MPM at the 1700-nm window.?1?Equipped with this energetic laser source delivering pulses at the optimal excitation wavelength,we performe in vivo brain imaging.Blood vessels labeled by SR101,dextran-conjugated Texas red and quantum dot can still be clearly imaged1340?m,1600?m and 1800?m below the surface of the brain,which are so far the deepest imaging depth,respectively,with organic dye molecule and quantum dots labeling.?2?Utilizing the deep imaging ability of the source and the microscopy,we performe 3-photon microscopy of blood vessels and astrocytes labeled by SR101 in the deep mouse brain in vivo.We also demonstrate a multiphoton imaging guided measurement system,which allows us to accurately measure the most efficient excitation wavelength for a certain fluorophore at the 1700-nm window.Our results clearly indicate that the optimal excitation wavelength for tdtomato is 1620 nm,which provide guidelines for excitation wavelength selection when transgenic mousemultiphoton fluorescence microscopy of labeled blood vessels and cellsexpressing tdtomato RFP in their neurons are to be imaged.The main innovation in this thesis can be summarized as follows:1.A 500-nm tunable range fiber source in LMA fiber and a 1620-nm high-energy excitation pulses?>100 nJ?fiber source in PC rod are built.The PC rod fiber source enables deep-brain MPM at the 1700 nm window.The broadband tunable soliton source enables quantitative measurement of the signal generation efficiency from the fluorophores at the 1700 nm window.2.An effective technique for isolating D₂O and multiphoton signal maintenance at the 1700 nm window enables long imaging span of over 5 h,quite suitable for brain imaging.3.Using the optimized laser and microscope system,blood vessels labeled by dextran-conjugated Texas red and quantum dot,can clearly imaged 1650?m and 1800?m below the surface of the brain,which is so far the largest MPM imaging depth in mouse brain in vivo.4.3-photon microscopy of astrocytes labeled by SR101 in the deep mouse brain in vivo is demonstrated,which enables us to visualize astrocytes 910?m below the surface of the mouse brain in vivo,30%deeper than that could be achieved with 2PM.A multiphoton imaging guided measurement system was built for wavelength selection of the red fluorescent protein-labeled neurons in mouse brain.