Study On Femtosecond Laser Two-photon Fluorescence Microscopy

Two-photon fluorescence microscopy is one of the most important inventions inthe field of biological imaging. This technology enables noninvasive study of biologicalspecimens in three dimensions with submicrometer resolution. Two-photon excitationof fluorophores results from the simultaneous absorption of two incident photons.Thanks to this nonlinear nature, two-photon fluorescence microscopy has a number ofunique advantages, such as enhanced resolution and contrast and limiting specimenphotobleaching to the focal point. Furthermore, NIR femtosecond laser excitation pulsescan not only reduce photodamage of biological specimens, but also increase imagedepth to about1000μm. And this is also the most important advantage over confocalmicroscopy. In this dissertation, a femtosecond Ti:sapphire laser (Tsunami,Spectra-Physics) is utilized as the excitation light source, a two-photon fluorescencemicroscopy system is built under current condition, and some researches on two-photonmicrocsopy has been conducted based on this home-built system.In theory, the process of fluorescence generation by two-photon excitation isinvestigated, and the advantage of using a mode-locked laser is proved based on theinvestigation. Calculations show that compared with a CW laser, two-photon excitationprobablity increases by105with a mode-locked laser when excitated with the sameaverage power. Saturation power and image depth of two-photon fluorescencemicroscopy are discussed, concrete expression of saturation power is deduced and wealso find that decrease the pulse width and repetition rate of the laser can effectivelyinprove the penetration depth. Based on the Fresnel diffraction theory and thediffraction theory of high-NA imaging given by Richards and Wolf, the resolution oftwo-photon fluorescence microscopy is calculated and found to be submicrometer.In experiment, we first develop a high voltage power supply used as power unit ofphotomultiplier tube, the photoelectric detector used in our two-photon microscopysystem. And the dark noise of R3896type PMT is measured based on the power supply.The result shows that the dark noise increases significantly when the supply voltageexceeds500V. We also manufacture a laser power diagnosis device with a PINphotodiode to real-time monitor the jitter of the incident laser beam. Software to controlthe scanning is written in C#languange. Based on the software and current hardware, atwo-photon fluorescence microscopy system is constructed. Because sometimes thefemtosecond Ti:sapphire laser will be out of mode-lock and the output power will beinstable when affected by the surrounding environment. In the system, a fibrespectrometer (HR400, Ocean Optics) and the home-made laser power diagnosis deviceare emplyed to real-time monitor the output of laser. Then one-and two-photon fluorescence spectra of Rhodamine B solution with different concentrations aremeasured. The consequences show that as the concentration increases, the centrewavelength of two-photon fluorescence spectrum of Rhodamine B solution approachesthe maximum fluorescence emission wavelength,610nm, and the FWHM increasesgradually. Moreover, the shapes of one-and two-photon fluorescence spectrum ofRhodamine B solution are almost the same when the concentration is quite low. At last,we apply the two-photon fluorescence microscopy system into the microscopy andphotobleaching study of Rhodamine B specimen. The image ability and resolutioncapability of the system are proved by the results. We also find that the photobleachingrate of Rhodamine B specimen increases with high-order power (>3) of excitationpower.