Introduction Multi photon microscopy MPM is a noninvasive method of fluorescence microscopy used in examining tissue sections and living animals It has been used to study calcium dynamics in the brain1 2 neuronal plasticity3 cancer angiogenesis4 5 and lymphocyte trafficking6 It has also been used to measure cerebral blood flow CBF 7 and as a tool for cell cutting and ablation8 Three dimensionally localized excitation depends on two photons interacting with a molecule nearly simultaneously on the order of 10 16 s This results in the occurrence probability of two photon excitation TPE having a quadratic dependence on light intensity9 This is in contrast to conventional fluorescence which has a linear dependence on light intensity Due to this quadratic dependence TPE can be highly localized to a focal plane of choice as away from the focal plane the TPE probability drops off rapidly Fig 1 Figure 19 However to make two photon microscopy practical the beam must be concentrated in both space and time This will increase the probability of TPE and number of fluorescence photons generated for measurement To concentrate the light in time a pulse laser is used This increases laser intensity while keeping the average power relatively low9 In our system this is established with mode locking and is apparent when the measured frequency of the beam becomes less well defined as the uncertainty principle dictates will occur with ultra short pulses in time Two photon scanning microscopy has advantages over conventional microscopy which has limited spatial signal localization and major scattering of light in living tissues Conventional microscopy is thus limited in its applications to cells in culture sectioned tissues and select small organisms such as nematodes TPE on the other hand has excellent spatial localization as noted above and it is also associated with less light scattering since it uses photons of longer wavelength and lower frequency This allows TPE to penetrate into deeper tissue with greater accuracy This translates into the ability to make noninvasive measurements as deep at 500 mm into living tissue whereas conventional fluorescence is limited to at least half of this10 In addition TPE does not generate out of focus fluorescence since its photon absorption is confined to a narrow region at the plane focus Thus while conventional microscopy requires a pinhole to eliminate photons from out of focus areas TPE does not require such a pinhole This results in increased fluorescence collection efficiency over conventional microscopy because scattered light emitted from an excited fluorophore can still be collected11 Finally TPE also minimizes the occurrence of photobleaching and photodamage due to its ability to achieve high spatial localization12 For this project we explored our ability to build a two photon microscopy system Once the system was assembled we used it to simulate measurements of blood flow using a capillary phantom In addition we tested its ability to perform cell ablation Methods System Overview Our 2 photon laser system consisted of a Millennia V CW Visible Laser a Tsunami mode locked Ti sapphire Laser spectrophotometer power meter gold mirrors scanning mirrors lens a 220 focal length objective a condenser filter photodiode AC amplifier and analog to digital converter ADC Laser Alignment The first consideration in building the two photon laser was ensuring proper alignment of the Millennia and Tsunami lasers To start the quarter they were aligned but became misaligned due to user error After reading through the manual we determined that adjusting the beam steering mirrors P1 and P2 would fix the problem Fig 2 Figure 2 Tsunami User s Manual Spectra Physics June 2002 We performed fine tuning of P1 and P2 by ensuring the beam projection onto mirror M4 did not have astigmatism or different foci in its two perpendicular planes We also adjusted the tuning slit to improve beam intensity while maintaining the ability to modelock the laser Beam Characteristics Mode locking is a critical ability of the two photon system It produces ultra short laser pulses that concentrate beam intensity in a very brief amount of time This increases the probability that two photons will interact to produce fluorescence To achieve modelocking we adjusted the prism dispersion compensation control and less often we adjusted the output coupler M10 and the high reflector M1 When mode locking is achieved the uncertainty principle dictates that the ultra short pulses will result in decreased ability to accurately determine the frequency Fig 3 Figure 3 We determined the beam intensity to be 0 49W using a power meter and the wavelength to be about 780 nm using a spectrophotometer Using gold mirrors we routed the beam to an optical track with scanning mirrors lens and a filter and photodiode for detection Details of the scanning mirror are below We used a lens with focal length of 76 2 mm to focus the beam on a 210 mm objective We mounted the blood flow phantom on a condenser f 50 mm in order to maximize the number of emitted photons collected We placed an additional 50 mm lens to move the focal point closer and placed a filter with a transmission greatest in the 340 600 nm range To maximize the power of the laser while keep the laser tightly focused on the sample we must correctly fill the back aperture of the objective The beam width of the laser is necessary to calculate the expanding factor and the parameters necessary in order to achieve this Usually the profile of a laser beam is a 2 D Gaussian function Assuming the beam is circularly symmetric either the full width at half maximum HMFW or two standard deviations 2s of the Gaussian function could be used as its effective beam width Here we use the definition of 2s To accurately measure the beam width we followed these steps Mounted a sharp edged razor on a base whose Figure position can be accurately controlled Fig 4 4 Put this mounted razor in between the laser beam and a power meter Slowly moved the razor from not blocking the laser at all to completely blocking the laser Recorded the position of the razor and the power of the laser on the power meter Fig 5A Took the first order difference of the measured curve to get the beam profile Fitted the beam profile to Gaussian function Fig 5B using 2s as its beam width Measured and fitted data is shown Fig 5 Effective diameter is 2 1mm Figure 5 A B Fig 5A Measured laser power at different positions 5B