Development and application of multi-photon microscopy in neuroscience and nanoscience

Abstract

Neuroscience is at crossroads. Several interdisciplinary optical developments centred around nonlinear optical processes helps in better understanding of brain function at sub-micron and millisecond precision. Traditional neurophysiological approaches measure the activities from one or group of neurons at a time. Multi-Photon microscopy paves the way towards network level understanding of brain circuit in slices and also in behaving animals. The technological advancement in the field of nonlinear microscopy and a rich toolkit provided by developments in nanoscience and nanotechnology (Rubinov 2015) now enable us to record activity of thousands of neurons. Given the importance of two-photon microscopy and repertoire of nanofabricated devices due to advancement in nanoscience and engineering, we investigate the application of custom-built multi-photon microscopy which can be used to understand synaptic and network level physiology and characterization of nanofabricated devices with future application in the field of neuroscience. The work presented in this thesis was divided into two main parts. In my first part of study we begin with the development of two photon microscope which can perform submicron structure (spine) imaging in a neural tissue. In addition, we combined the two photon imaging set-up with the whole cell patch clamp recording to perform simultaneous patch clamp recording and two photon calcium imaging. The realized electro-optic setup can help in understanding of single or interaction of group of neurons in a network under a physiological condition like in slices or in live mice brain. Two-photon uncaging combined with calcium imaging is a critical experimental tool to investigate synaptic plasticity rules in a spatiotemporal scheme. To perform simultaneous uncaging and imaging, typically ultrafast laser pulses from two separate laser systems are used in conjunction with two independent sets of galvanometric mirrors. However, such a setup is instrument intensive and requires synchronized operation of two laser systems making it relatively tedious to operate and maintain. Here, in this section we report a single ultrafast laser system based optical setup that uses a single set of galvanometric mirrors based regular scanning assembly to perform simultaneous two photon uncaging and calcium imaging in a hippocampal neuron. A fast operating optical shutter operating along with our delay line optics and galvanometric mirror is used to generate patterned uncaging excitation. Spatial control of uncaging is measured and shown to be close to the optical resolution. The accuracy and synchrony are shown to be within few microseconds. We put to use the good control of uncaging location in these experiments to investigate the cooperative and associative plasticity. However, multi-photon microscopy despites its extensive application in the field of neuroscience also enable the researchers to perform the characterization of nanofabricated devices. My second part of study catered on characterization of nanofabricated devices using the above custom built two-photon optical set-up. One such application was the photo response characterization of in-house developed light detectors using state-of-the-art nanofabrication techniques to each mode locked pulses from ultrafast laser using phase locked techniques. We used the modified two photon optical set-up to measure the frequency response of the photo detector using high-speed sampling scope. The 3dB cut off frequency is estimated from the 9 relation, f3dB = 0.45/FWHM, where FWHM is full width at half maximum of time response of photo detector. The highest bandwidth of 15.5 GHz corresponding to FWHM of 29 ps is obtained at 10V reverse bias. At lower reverse bias voltages broadening of the pulse response (FWHM) indicates the photodetector bandwidth is transit time limited. At 20 V reverse bias pulse response broadens slightly due to impact ionization build up time in the photo detector. In short, we have successfully demonstrated an integrated lateral silicon pin photo detector on SiN-SOI platform using the two-photon imaging set-up. We obtained the highest responsivity of 0.44 A/W at 25 V bias and estimated the best 3dB cut off frequency of 15.5 GHz at 10 V reverse bias. To the best of our knowledge, this is the highest ever reported bandwidth of a waveguide integrated Silicon photo detector at 850 nm wavelength band. Another application was the investigation of the circular differential two photon luminescence response from a three-dimensional chiral metamaterial, comprising a system of achiral (spherical) metal nanoparticles arranged on a chiral (helical) dielectric template. The enhanced dipolar response of the individual particles arising from their strong electromagnetic coupling resulted in strong photoluminescence under peak illumination intensities as low as 2 ×103 W/cm2. The strong chiro-optical effect observed in these experiments may be relevant to technologies related to nonlinear plasmonics, in particular imaging applications where control over the polarization state of the imaged photons may be desirable.