Transmittance Characterization Of The Optical Components And Immersion Media For The Multiphoton Microscope At The 1700-nm Window And Its Application

1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy?MPM?.A system with high transmittance of excitation wavelength and signal wavelength is the key for MPM,which can improve the imaging depth and speed directly.However,the transmittances of optics and immersion media used for objective lens at 1700nm window are largely unknown,which prevents the optimization of the transmittance of the MPM system.In order to improve MPM system performance,we characterize the transmittance of a wide variety of optics?long pass filter?LPF?,dichroic beamsplitters?DC?objective lens?and the absorption spectra of several immersion media?water,deuterium oxide?D₂O?,and commonly used immersion oils?,and demonstrate applications to signal enhancement and even new imaging modalities in MPM at the 1700-nm window.Our research works include:?1?We characterized some LPFs and DCs'transmittance precisely,chose the optics based on our measured results,the MPM system excitation power improve 50%at1700nm.?2?We develop a method for measuring the broadband transmittance of the high numerical aperture?NA?objective lenses.Using this method,we perform measurement of two commonly used objective lenses for MPM,one with regular coating and the other with customized coating for high transmittance at the 1700-nm window.Based on our measured results,we demonstrate fourth harmonic generation imaging of biological tissue and 5-photon fluorescence of carbon quantum dots.Through comparing two objective lens in deep-brain imaging,we achieve a 3-photon fluorescence imaging depth of>1600?m in mouse brain in vivo.These results will provide guidelines for objective lens selection for MPM at the 1700-nm window.?3?As for the immersion,we accurately measured the absorption spectrum of D₂O and demonstrated its application at 1700nm window.As a result,compared with water immersion,D₂O immersion enhances signal levels in second-harmonic generation imaging,3-photon fluorescence imaging,and third-harmonic generation imaging by8.1,24.8,and 24.7 times with 1662-nm excitation,in good agreement with theoretical calculation based on our absorption measurement.This suggests D₂O a promising immersion medium for deep-tissue imaging at the 1700-nm window.?4?In order to enhance signal levels in MPM for deep-skin at the 1700-nm window with oil immersion,we measure the absorption spectra of several commonly immersion oils.We further demonstrate new immersion medium with lower absorption and tunable refractive index.Based on absorption spectra characterization of immersion oils,we propose optimal selection of excitation wavelength within this window.Second and third harmonic generation imaging of mouse tissue corroborate our selection:1600-nm excitation leads to notable orders-of-magnitude increase in MPM signal,compared with 1700-nm excitation,enabling 200-?m imaging depth of mouse skin while 1700-nm excitation could resolve virtually no structure.Finally,we summarize our experiment results and provide some perspectives on the development of MPM based on our results.