Real-time focal modulation microscopy

Abstract

Focal modulation microscopy is a novel microscopy method that can achieve a large penetration depth with single photon excited fluorescence. In this method, excitation intensity within the focal volume is modulated by the use of a time dependent spatial phase modulator. The resultant fluorescence emission is filtered by the spatial gating provided by the pinhole and demodulated with a lock-in amplifier. Previous implementation of the spatial modulator is realised through a mechanical tilt plate phase modulator. This mechanical implementation affords stable modulation and provides satisfactory image quality. However, the slow modulation rate(in kHz range) depreciates the prospect of its employment in real time imaging. A new modulation scheme is proposed in which an acoustic optical modulator(AOM) is utilised. This new arrangement boost the modulation speed and makes real time imaging possible. We report on the development of such a modulation system together with the accompanying electronics. Images obtained with this new modulation system will also be presented. © 2010 Copyright SPIE - The International Society for Optical Engineering.