A deeper understanding of the biological mechanisms that govern stem cells requires detailed, real-time image analysis of living cells. Currently, conventional live microscopy techniques are ineffective at imaging features like the nucleus in the center of a cell, principally due to aberrations caused by imaging through cytoplasm, organelles and other molecules inside the cell. Similar image distortions occur when looking at an object at the bottom of a pool, where motion of the water causes the image to “shimmer.” We therefore propose to develop an improved wide-field microscope with dynamically adjustable optics for live imaging deep within tissues containing stem cells.
Application of technical innovations recently developed by astronomers that provide clearer images of stars is central to our approach. 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 imaging of stem cell samples. 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 near a feature of interest in cells to serve as beacons to acquire necessary measurements to correct for most distortions.
In year one, we propose to build a device to measure the magnitude of image distortions as a function of depth in living tissue. During this time, we will also design a microscope with sufficient correction to adjust for the measured distortions. In year two, we will build the microscope and characterize its performance. We will test the new imaging system in early fruit fly embryos. With amazing similarity to human cell and developmental biology, these insects are emerging as a key system to safely address many outstanding issues in stem cell biology. Once the imaging system is fine tuned, we will use the technology to examine mechanisms of stem cell self-renewal in the fruit flies.
Once the value of this technology is demonstrated, it will be possible to use it in a wide range of stem cell research, as well as for other applications in cell and developmental biology.
Successful development of an adaptive optics 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 if we are understand how the mechanisms of polarization, asymmetric, self renewal and differentiation. Further, if stem cell biology is to has a therapeutic future this mechanisms must be well understood because we need to control them both in tissue culture as well as in vivo.
The adaptive optics scope we are developing should enable us to perform live fluorescent imaging at depths well below the current 30um to 50um limit. This will greatly accelerate research and discovery in fundamental mechanisms of stem cell biology. For example in the studies proposed in this grant, we will be able us to the first time to image live over extended periods, the divisions of germline stem cells of the Drosophila ovary. In addition, we will be able to simultaneously follow the differentiation and of the daughter cells. Once this is achieved this new technology can be readily applied to mammalian stem cell systems.
Twenty years ago confocal microscopes were rare. However as they have proven their worthiness and the price has gone down, they are now universal and essential tools in stem cell labs. We envision our adaptive optics microscope as having the same fate. Significantly we are designing the scope so that it can be added on to existing fluorescent scopes thus significantly reducing cost. We hope that within the decade AO microscopes will be a standard tool for stem cell biologists.
On a final note, this project will have direct benefit to the California economy as the manufacturing, distribution and selling of the AO scopes would take place in California. We have already had interest from local optics companies in developing the AO scope for market once a suitable prototype is developed.
This application focuses on the development of an adaptive optic, wide-field microscope that can be used to image cells deep within living tissues in real time. To achieve these ends, the applicants propose to use techniques conventionally employed by astronomers to quantify and correct optical aberrations caused by imaging through tissues. First, a number of fluorescent beads will be embedded at defined locations within a tissue and serve as reference beacons for quantifying the magnitude of aberration as a function of depth. Next, a microscope will be designed with sufficient correction to adjust and compensate for these measured aberrations. Finally, after building and testing its performance, the applicants propose to use this new microscope to examine mechanisms of stem cell self-renewal in a Dropsophila model system.
The reviewers were enthusiastic about the proposed technology and its potential to advance stem cell science. The proposal was straightforward, well written and based on a sound and innovative premise. While technical concerns were noted, the potential utility and applicability of the described technology convinced the reviewers of its overall merit.
The reviewers were uniformly enthusiastic about the potential of the proposed technology to advance the field of stem cell biology. Due to limitations of current instrumentation, it has not been possible to faithfully visualize cellular and subcellular processes that occur deep within living tissues. If successful, the proposed technology would enable stem cells and other objects of interest to be imaged in real time in their native environment, thus enabling a variety of studies to be performed that would not have been practical otherwise. Such a tool would be widely utilized and could greatly benefit all aspects of cell biology, including studies of stem cell behavior as well as in vivo engraftment for therapy.
In terms of feasibility, the reviewers were confident in the qualifications of the research team. The proposal was well written, the research plan was straightforward, and the underlying premise was both innovative and sound. Although not definitive, the preliminary data were encouraging and spoke to the overall feasibility of the method. Despite their enthusiasm, however, the reviewers discussed several technical weaknesses that could diminish the overall impact of this effort. For example, it was unclear as to whether the bead quantity and types that were proposed would be sufficient for achieving the desired resolution. Furthermore, due to the observed increase in aberration at 25 microns, the reviewers questioned whether it would be realistic to image objects at a depth of several hundred microns, the level that would likely be necessary for this technology to be useful in the study of mouse tissues.
In general, the reviewers felt that the qualifications of the research team were excellent. Although the track record of the principal investigator was judged to be appropriate but not very strong, the co-investigators were considered to be oustanding. Due to the complementary strengths of the collaborators, the team as a whole was considered an asset.
Overall, this was an innovative and largely feasible proposal from a talented team of investigators with the potential to make significant contributions to the field of stem cell biology.
A motion was made to move this proposal from Tier 2, Recommended for Funding if Funds Available, to Tier 1, Recommended for Funding. Despite some technical uncertainties, the reviewers argued that this technology is needed, could be widely employed, and could be of practical utility both before and during translational research. The motion carried.