Two Photon Microscopy and Second Harmonic Generation Leif Gibb and David Matthews Introduction and Background Nonlinear microscopy has become an important tool for imaging biological samples such as neurons In two photon fluorescence a fluorophore absorbs two photons at a low energy state one of its electrons briefly converts to a higher energy state and it emits a single excited photon at an energy level higher than that of the absorbed photons In second harmonic generation SHG laser light is focused on a sample to generate frequency doubled light two photons of one wavelength are annihilated and a single photon of half the wavelength is generated As neurobiology increasingly addresses the cellular and circuit level underpinnings of behavioral and systems level phenomena pushing nonlinear microscope technology to diffraction limited spatial and unlimited temporal extents is necessary The development of novel nonlinear techniques has revealed much in the last decade and a half of neuroscience and promises the possibility of a much richer functional understanding of the brain In light of the importance of this technique we were interested in using a prefabricated laser to build a two photon and SHG microscope Thus in the present work we had several goals 1 to generate 2 photon fluorescence from a cuvette of Rhodamine dye 2 to create labeled lipid membranes on planar glass supports for use in SHG 3 to achieve 2 photon fluorescence and SHG from labeled lipid membranes 4 to demonstrate SHG from a nonlinear crystal Two Photon Microscopy and Fluorescence Two photon laser microscopy TPLM is a technique in which photons of a pulsed laser collide in a femtoliter order volume of sample It is especially useful to biology because of its selective excitation the theory of which came in 1931 from Maria G ppert Mayer She recognized that a fluorophore could simultaneously absorb two photons at a low energy state and emit one photon at a higher energy state The technology was gradually developed in 1960 Ted Maiman built the first working laser by 1982 two photon microscopy was used for spectroscopic examination of molecular excitation states Friedrich 1982 and within a decade Winfried Denk and Watt Webb had developed the two photon laser for application to neurobiology Denk et al 1990 A laser is a coherent beam of intense light generated by energizing one or a set of ions from a ground to a metastable molecular state Traditional laser methods achieve this state change by a population inversion in which intense radiation at room temperature moves the majority of ions from the ground to the excited state Their inevitable relaxation at thermal equilibrium from this excited state back to ground state releases photons in a beam by a process called stimulated emission Hecht 2002 When photons coherently leave the laser encasement they have sufficient energy to excite certain fluorophores in a sample In traditional fluorescence microscopy if the emission wavelength of the laser and the absorption spectrum of the fluorophores align excitation occurs In two photon fluorescence the simultaneous absorption of two photons of half the frequency of the absorption spectrum of the fluorophore is required for fluorescence The likelihood of simultaneous absorption of two photons in natural light is small a back of the envelope calculation suggests that sunlight might cause 1 event 106 years Webb 1997 Therefore fluorescence using TPLM involves a modelocked laser in which photons are emitted at a frequency of order femtoseconds 1 or greater That is the intensity of light emitted is high for short bursts of time Because the likelihood of simultaneous absorption events increases supralinearly with the population of photons emitted just as a Hill coefficient of n 2 in physical chemistry this greater intensity leads to many more absorption events For example mode locking in a Ti Sapphire laser increases the probability of absorption by 105 relative to a continuous wave with the same average power Webb 1997 Mode locking and two photon absorption render TPLM advantageous over its predecessors for many applications In particular with such a small focal volume in which fluorophores are excited there is little wasted fluorescence the fluorescence emission of all excited molecules is captured and imaged at once This is in stark contrast to confocal microscopy in which a pinhole blocks all but a small section of the fluorescence from a section of a sample while intense laser light excites fluorophores throughout the entire sample Many problems that come with confocal microscopy fast fluorescence decay with time increased light scattering wasted fluorescence excitation increased photodamage and increasingly poor resolution with depth in the sample are therefore avoided with the mode locked slower wavelength laser light of TPLM Nonlinear Optics and Second Harmonic Generation Although a complete review of nonlinear optics is beyond the purview of this paper what follows is a brief discussion as a primer for our experiments with second harmonic generation Nonlinear optics is concerned with phenomena resulting from light induced changes to the optical properties of a system Such phenomena are nonlinear in that they depend quadratically on the strength of the optical field Second harmonic generation is an appropriate example of such nonlinearity the intensity of the light resulting from second harmonic generation scales with the square of the applied laser light The following is an informal mathematical description of second harmonic generation starting with an intuitive description of nonlinear optics The polarization P t the dipole moment per volume of a system depends on the strength t of the optical field In conventional optics this relationship is linear P t 1 t where the constant 1 is called the linear susceptibility However in nonlinear optics the generalization of this equation attains in which the polarization goes with the power series of the field strength P t 1 E t 2 E2 t n En t P t P 1 t P 2 t P n t where n is the nth order nonlinear optical susceptibility and P n t is called the nthorder polarization When a laser with an electric field strength given by t Ee i t complex conjugates impinges on a system with a nonzero second order susceptibility 2 the polarization created is P 2 t 2 2 EE 2 E2 e 2i t complex conjugates Clearly this second order polarization has a zero frequency component in which 0 and a frequency 2