DEVELOPMENT OF HIGH-SPEED FOCAL MODULATION MICROSCOPY FOR VISUALIZATION OF THICK BIOLOGICAL SPECIMENS

Abstract

The study of morphology and functional processes of biological tissues at submicron spatial resolution using optical techniques is always limited by 1) shallow penetration depth accompanied by poor imaging contrast and 2) the relatively slow recording speed of fluorescence imaging tools such as confocal microscopy. To overcome these limitations and allow the in vivo study of biological tissues, we have developed Focal Modulation Microscopy (FMM) that permits large imaging depth with better image contrast; and at high frame rates in the line scan mode.

FMM utilizes the coherence property of the light source, through spatio-temporal modulation scheme to differentially phase modulate segments of the excitation beam. These segments of the beam, when being focused by the objective lens, generate an intensity modulation exclusively at the focal region. This modulation excitation focus therefore results in a modulated fluorescence signal from the focal volume. In principle, out-of-focus excited fluorescence should not be modulated. Demodulation of the collected fluorescence signal at the designated modulation frequency could allow us to discriminate the in-focus fluorescence from the multiple-scattered background, thus greatly enhance the signal-to-background ratio (SBR) in the image. More importantly, the penetration depth of FMM can be significantly improved as the degradation of the image contrast is considerably much slower, and thus could potentially revolutionize the clinical and biomedical applications of FMM for in vivo high-resolution visualization of thick biological specimens. Up to date, imaging using FMM with penetration depth up to 600 microns has been demonstrated with biological specimens, without the necessity of longer wavelength excitation or non-linear light mechanisms.

In this thesis, theoretical studies have been performed to understand the efficiency of FMM in rejecting out-of-focus fluorescence background, as well as the effect of aperture configurations in modulation depth of FMM. Three implementations of FMM which varies in term of phase modulation schemes - double reflecting mirrors, tilting plate as well as FMM based on acousto-optical modulators that are significantly progressive in term of stability and image performance are being demonstrated. Additionally, a line scan mode of galvanometer-based FMM is described which provide much higher effective frame rate for real-time imaging of biological processes.

In total, this research has progressed FMM for clinical applications in in vivo imaging of biological tissues.