Development of coherent Raman depletion microscopy for probing bacteriorhodopsin’s proton pumping activity as a function of structural organization

Abstract

The resolution of optical microscopy techniques is inherently limited by the wavelength of light used. This limitation, known as the diffraction limit, prevents optical microscopy from having a resolution below 200 nm while using visible light. However, biology exists on the nanoscale and to properly observe complex biological systems with optical microscopy, we need to be able to surpass the diffraction limit. Fluorescence-based super-resolution techniques are able to observe features below the diffraction limit, but require fluorescent tags that suffer from photobleaching, difficult multiplexing, and may perturb their environment. It would be useful to have a super-resolution method that doesn’t require labels, but instead is able to use the difference in the chemical structure of the sample as contrast. My thesis has focused on developing coherent Raman depletion microscopy (CRDM) to make efficient label-free super-resolution imaging a reality. Beyond just imaging below the diffraction limit, I have also worked to develop a method to measure the activity of a well-studied ion channel, bacteriorhodopsin (bR), that can be combined with CRDM to measure activity below the diffraction limit. By comparing Raman spectra taken during and after excitation of bR with a weak green excitation beam, my assay is able to directly measure the activity of bR. Using the assay, I have been able to monitor the activity of bR both in vitro and in vivo. My results show that the activity of bR responds differently to changes in external pH in vivo than in vitro. These results indicate that the local environment of bR affects how it responds to changes in its bulk environment. Future measurements that combine the Raman-based assay with CRDM will be able to measure the activity of bR below the diffraction limit to determine if the activity of bR has a spatial dependence on its location in the membrane. To better understand the suppression mechanism of CRDM, I investigated how the femtosecond depletion beam is able to drive other four-wave mixing pathways. I was able to show that for the stimulated Raman version of CRDM, stimulated Raman depletion microscopy (SRDM), the depletion of signal appears to be driven by competing four-wave-mixing pathways that are nonlinearly dependent on the power of the femtosecond depletion beam. In this study, I also showed that the same depletion scheme could be used to deplete inverse Raman signal, paving the way for inverse Raman depletion microscopy (IRDM). This investigation indicated that optimizing the interaction between the depletion and Raman probe beams is required to maximize signal depletion. In an effort to build a system more accommodating to biological samples, I constructed a CRDM system using an 80 MHz pump laser and a photonic crystal fiber (PCF) as the Raman probe source. With the system, I was able to deplete both SRS and IRS signal at 80 MHz, proving that the depletion necessary for CRDM is possible at lower peak powers. The low output power and low spectral density of the PCF prevented the system from being able to measure biological samples. However, with a different Raman probe source, the system will be able to image biological systems, such as bR in a cell membrane, below the diffraction limit without the need for fluorescent probes.