Funding opportunities

RT2-01962 Adaptive Optics Two-Photon Microscope for Single Cell Imaging of Stem Cells Embedded in Deep Tissue

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01962
Funds requested: 
$1 230 462
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
To speed the delivery of stem cell therapies from research in the laboratory to practice in the clinic, our collaborative research team composed of biologists, physicists, astronomers and engineers proposes to develop a new two-photon microscope with Adaptive Optics (AO) that can see more clearly when imaging deeply into the brain. Adaptive optics are used by astronomers to provide clearer images of stars. In astronomy, lasers are used to create reference beacons which can be used to adjust the optics in the telescope to correct for image distortions caused by changes in the atmosphere, such as winds and dynamic temperature changes that cause the stars to “twinkle.” Such distortions can then be corrected by using a dynamically deformable mirror, similar to the curved mirrors in fun houses, which make you look short or tall. In principle, reference beacons and deformable mirrors can also be used to improve image depth and resolution of stem cell samples when looking through thick tissue. Here the reference beacons control the mirrors that remove warping of the image caused by looking through the materials inside the cell. Our preliminary results indicate small fluorescent beads can be implanted in tissue to serve as beacons to acquire the necessary measurements to correct for most distortions. The new microscope that will be developed uses two photons to excite fluorescence, which enables it to image more deeply into tissue. The new two-photon AO microscope builds upon our prior success using adaptive optical techniques for a single-photon AO microscope which has been shown to be capable of improving the image resolution by ten times the resolution of conventional microscopes. Many promising stem cell-based therapies will require injecting in-vitro grown stem cells into a specific afflicted tissue. However, previous attempts at reviving or repairing damaged tissues by injecting stem cells have had limited success. The current lack of understanding of the cellular basis of this failure has severely impeded advancement of such therapies. A major technical barrier has been the limitation for visualizing individual living stem cells in deep tissue, thus preventing investigators from answering many critical questions. For example, do such injected cells continue to undergo normal stages of self renewal and differentiation? Once the stem cells migrate to particular niches, what is the fate of stem cells that subsequently differentiate? We will use this new microscope for tracking transplanted stem cell derivatives within a living organism to study how stem cells can help repair brain damage after a stroke. By using adaptive optics we will be able to increase the imaging depth and resolution to near diffraction limited imaging.
Statement of Benefit to California: 
Successful development of an adaptive optics two-photon microscope will have an immediate benefit to stem cell research, particularly that research funded by the California Institute for Regenerative Medicine (CIRM). Stem cells usually reside deep within dense cell layers and thus have proven extremely difficult for live fluorescent imaging. Live fluorescent imaging has become an essential tool for revealing the molecular and cell biology of dynamic biological events. With respect to stem cell biology, live cellular imaging will be essential to address the translational bottleneck for the development of sensitive imaging and molecular techniques for tracking transplanted stem cell derivatives in vivo. Many promising stem cell-based therapies will require injecting in vitro grown stem cells into a specific afflicted tissue. However, previous attempts at reviving or repairing damaged tissues by injecting stem cells have had limited success. The current lack of understanding of the cellular basis of this failure has severely impeded advancement of such therapies. A major technical barrier has been the limitation for visualizing individual living stem cells in deep tissue, thus preventing investigators from answering many critical questions. The adaptive optics two-photon microscope we are developing should enable us to perform live fluorescent imaging at depths more than double the current limit of 250 microns. We hope that within the decade AO two-photon microscopes will be a standard tool for stem cell biologists. We will use this new microscope for tracking transplanted stem cell derivatives within a living organism to study how stem cells can help repair brain damage after a stroke. In addition, it will have general applicability for treatment of many other neurological diseases. Although two-photon microscopy is best applied to tracking transplanted stem cells in small animal models, the results will be important for understanding disease in humans. We believe this project will also have direct benefit to the California economy as the manufacturing, distribution and selling of the microscope would take place in California. We have already had interest from local optics companies in developing the AO microscope for market once a suitable prototype is developed.
Review Summary: 
This project seeks to improve an existing adaptive optics (AO) microscope by adding more powerful optics and a laser for 2-photon microscopy, in order to image deeper into tissue than is currently possible. Required algorithms and software to operate and process the system will also be developed. If successful, the microscope will offer an increased capability to image dynamic stem cell events deeper within tissue than was previously possible in animal model systems. The applicant proposes to use a mouse stroke model system to evaluate the capability of the microscope for imaging individual stem cells injected deep into the central nervous system. They propose two specific aims. Aim 1 will build on the existing AO confocal microscope to enable two-photon microscopy for deep tissue imaging and then evaluate the performance of this system. The goal of Aim 2 is to use the new AO 2-photon microscope system to perform live in vivo imaging of the adult mouse cortex following induction of focal cortical stroke. The investigators will image the migration of adult neural stem cells (NSCs) to the site of injury in this model and examine the spine dynamics of newly generated neurons in the deep layers of the cortex. Reviewers agreed that this is an innovative proposal that combines adaptive optics with 2-photon microscopy together with a reference beacon and mathematical analysis, to better focus excitation light and better recover and refocus emitted light. The microscope that will be developed is unique, innovative, and should result in a cutting edge deep tissue imaging system that should have multiple uses. Reviewers further emphasized that since there are still major limitations to visualize dynamic cellular events greater than 300um into the tissue, improving depth penetration and allowing for visualization of single stem cell behavior would be advantageous. Reviewers noted that while the value of this technology is for experimental purposes, it might eventually be possible to adapt this kind of imaging for clinical diagnostic purposes. Although the reviewers liked the overall approach, they expressed concern that the proposal provided few details on what the increase in the depth of tissue imaging will become. In addition, their enthusiasm for the proposed project was lessened by the fact that there are two emerging technologies for deep tissue imaging that are already commercially available, and they were unable to judge how much deeper the proposed tool would be able to image compared to these technologies. They also cited the lack of preliminary data that would indicate that the investigator can perform in vivo imaging through the mouse skull opening and they expressed doubts regarding how significant the increase in optical resolution would be. Other concerns included the lack of details provided about the stroke model, and the lack of information regarding the temporal and spatial patterns of neural stem cell migration towards a stroke site, as well as the origin of these cells. The principal investigator was judged to be excellent and an expert in adaptive optics. The team was deemed appropriate, complementary and synergistic in their capabilities since it also includes biologists with experience in mouse models for stroke. Overall, the reviewers expressed support for the development and improvement of current imaging technology for deep tissue imaging. However, lack of experimental details about the stroke model and proposed use of neural stem cells in addition to the fact that this proposal is not translatable to humans dampened the reviewers’ enthusiasm and they therefore did not recommend this application for funding.
Programmatic review: 
  • A motion was made to move this application into Tier 1, Recommended for Funding, given that this was one of the few applications using a light based method for deep tissue imaging. Reviewers reiterated that it was unclear how much additional depth and resolution would be achievable with the proposed technology improvements over current technologies and that the technology, while potentially useful for small animal models, was not likely to be translatable to humans. The motion failed.
Conflicts: 

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