Biophysics / Internet Invited Lecture
SECOND-HARMONIC GENERATION MICROSCOPY WITH HIGH NUMERICAL APERTURE OBJECTIVES
Elijah Y S Yew, Graduate Programme in BioEngineering, National University of Singapore, Singapore
Colin J R Sheppard, Division of BioEgineering, National University of Singapore, Singapore
ABSTRACT
Second-harmonic generation (SHG) microscopy is a novel imaging modality that is fast gaining popularity. Much of the work done with SHG microscopy is with objectives of low numerical aperture (NA < 1.0) whereby the paraxial approach may be used to describe the electric field at the focus as having only one ‘dominant field’. On the other hand, high NA objectives allow for sub-micron resolution of the SHG signals and is useful for determining the nonlinear tensor coefficients through polarimetry. We have previously shown through theory that for high NA focusing the axial component of the electric field at the focus can contribute significantly to the SHG process. We now present experimental results validating the above theory. For the case of axially sectioned rat-tail tendons a paraxial approach to describing the electric field at the focus will result in little or no SHG a tightly focused. Accounting for the axial component on tightly focused beams, an x polarized beam (60x water, NA1.2) resulted in a cos2 type dependency of the SHG intensity as a function of the analyzer angle. On the other hand, a tightly focused, y polarized beam resulted in a sin2 dependency on the analyzer angle. The results indicate that it is possible to determine the various components of the tensor coefficients through varying the sample orientation or polarization control when sample orientation is not feasible. As SHG is coherent and its output polarization depends on the input polarization and sample tensor, it is thus possible to use nonlinear processes as a ‘mode-converter’ to generate various output polarizations given a single input polarization.
Representing Author:
Elijah Yew, National University of Singapore, (Singapore, Singapore)
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