INVESTIGATING THE LIMIT OF THREE-PHOTON MICROSCOPY IN BRAIN IMAGING

One of the main challenges to understand how brain works is to map out all of the neurons as well as their activities. The requirement of breadth, depth as well as precision triggers a search for new tools. Two-photon microscopy (2PM) has been widely used in brain studies since its invention in 1990. However, the fundamental limit of signal-to-background-ratio (SBR) stops 2PM from imaging deep into biological sample. Three-photon microscopy (3PM) combines longer-wavelength excitation and higher order of nonlienarity to achieve deeper tissue penetration. Light attenuation in thick biological tissues, caused by a combination of absorption andscattering, limits the imaging depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the right wavelength with minimum attenuation for deep imaging. Tissue scattering and absorption impact the excitation and emission light in different ways for multiphoton imaging. In this thesis, I will describe the key points in selecting the optimum excitation wavelengths and emission wavelengths theoretically and experimentally. We show that the excitation wavelength has more impact on imaging depth than emission wavelength and the advantage of long wavelength dyes for multiphoton deep imaging is almost entirely due to the long excitation wavelengths. Then we applied three-photon microscopy to image the subventricular zone (SVZ), which is a heterogeneous neurogenic stem cell niche deep in the brain. We show 3PM visualisation of typical neural stem cells (NSC), intermediate progenitors and neuroblasts in both postnatal and adult SVZ. 3PM provides the first non-damaging opportunity to image the SVZ, distinguish cell morphologies in live animals and has the potential of dynamically imaging stem cell lineage pro- gression in situ at various ages. Lastly, we did chronic neural activity imaging in Drosophila and captured neural structure and activity through the intact fly cuticle. We performed chronic functional imaging of odour-evoked neural activity in the mushroom body Kenyon cells and found odour responses changed over time; sharp odour evoked responses gradually switched to persistent neural activity. This demonstrates that three-photon microscopy extends the time limits of the current in vivo imaging methods used in flies for anatomical and functional imaging, and opens up new ways to chronically capture neural activity from the fly brain