This Small Business Innovation Research Program (SBIR) Phase I project aims to develop and demonstrate a superpenetration multiphoton microscope (S-MPM) that will more than double the imaging depth achievable in highly scattering biological tissue. MPM technology has revolutionized the field of subsurface biological imaging, but its depth of penetration is limited. The severe scattering introduced by biological tissue - especially neural tissue - prevents most commercial MPM instruments imaging beyond a few scattering mean free paths. With this limitation, research on cells and cell networks at the frontier of neuroscience is constrained. A recent breakthrough in coherent light propagation and control through highly scattering media demonstrated the possibility of enhancing focal intensity by factors of several hundred on the far side of a medium, despite any amount of scattering, by using a spatial light modulator to modify the phase of the coherent light on the near side of the medium. This project will combine MPM and BMC's fast microelectromechanical spatial light modulators (MEMS SLMs) to offer a compelling and affordable way to exploit this breakthrough in optical science that will make a substantial impact on biomedical and neurobiological research.
The broader impact/commercial potential of this project is to substantially improve multiphoton microscopy techniques that have grown exponentially in importance over the past two decades. If successful, the research outcomes will have broad impact on the field of neurobiology by allowing researchers to routinely probe molecular-scale structures and functions at depths of up to 1mm in brain tissue. This enhanced capability will effectively double the achievable penetration depth, with only modest additional instrument cost. The MEMS SLM work that comprises the bulk of the Phase I work plan will generate an important new commercial component, a fast, low cost, MEMS SLM subsystem. Since the proposed instrument will only require use of less than half of the MEMS SLM dynamic range, the drive electronics can be reduced in size and simplified allowing easy integration and significantly reducing cost by using off-the-shelf components and at the same time increase the temporal bandwidth of the system.. The result will be a fivefold reduction in cost and a fivefold increase in speed, which will also have a significant impact on other commercial applications such as free-space laser communication, femtosecond laser.
The overall objective of this SBIR program was to demonstrate the feasibility of a superpenetration multiphoton microscope (S-MPM) capable of more than doubling the imaging depth achievable in highly scattering biological tissue. The instrument’s unprecedented performance is enabled by a new low-cost high-speed microelectromechanical spatial light modulator (MEMS SLM) subsystem and will be capable of real-time imaging deep within living tissue. The objectives of the Phase I work were to design and fabricate a low-cost, high-speed MEMS spatial light modulator (SLM) driver, integrate the MEMS SLM and the driver subsystem into a microscopy test bed, and demonstrate focus intensity enhancement through scattering media. MEMS SLM drive electronics A low-cost prototype electronics driver for the MEMS SLM was developed as an alternative to the existing electronics driver, which is prohibitively expensive for this application and had an insufficient frame update rate for in vivo image enhancement in S-MPM. In addition to reducing the cost of the driver by more than a factor of two, the achievable frame update rate was increased by a factor of five, to 20KHz. S-MPM Testbed Results An S-MPM testbed was constructed (see Figure 1) in which we achieved focus optimization and subsequent imaging in using various test samples including biological scattering media (e.g. ex vivo mouse skull). An example of optimization and imaging through a real and quite challenging biological scattering sample is depicted in Figure 2. The scattering sample was a portion of a mouse skull, measuring approximately 200µm thick. The skull, which had significant curvature, was immersed in water, and pressed against an underlying glass slide measuring 150µm thick. On the back side of the glass slide were clusters of 1µm diameter fluorescent polystyrene beads. For this experiment, the beads were imaged first without the skull in place, to provide a reference for what would be expected when imaging without the interference induced by scattering media. Then the skull was inserted in the imaging path, and a second image of the fluorescent bead layer was recorded. Next, the two-photon signal was optimized using feedback to the SLM, and then a final image of the fluorescent bead layer was recorded. Conventional MPM yields no discernable image of the bead clusters. S-MPM yields image quality in the central 20µm of the image that compares well with the image obtained without the mouse skull The results demonstrate clearly that the proposed S-MPM technique will far surpass the performance of conventional MPM in imaging applications characterized by strong scattering. The original image, without the mouse skull in the imaging path, shows clearly resolved bead clusters across the entire field of view of the microscope. With the skull in place, however, all capacity to resolve the bead clusters is lost. Optimization using the SLM restores near-perfect imaging, albeit over a reduced field of view.
