This Small Business Innovation Research (SBIR) Phase I project is aimed at developing an integrated system to stabilize a conventional high-end, inverted optical microscope to the precision required to routinely achieve the imaging power of emerging super-resolution (SR) microscopy techniques. These recent methods, enabled by particular photo-physical properties of certain fluorescent probes, have circumvented the diffraction limit normally associated with light microscopy and pushed it into realms thus far only achievable with electron microscopy. These methods put very high demands on the stability of the microscopy system. Conventional microscopes fail to meet these demands, and sample drift occurs relative to the image detector that can destroy the SR capabilities of the imaging system if this drift is not compensated. This project will integrate a 3-axis piezo-driven nanopostioning microscope stage with a closed-loop feedback system using data generated from an EMCCD camera, which will also serve as the image detector for the system. Fluorescent fiduciary references sparsely distributed within the sample will serve as anchor points from which to assess sample drift, and produce compensatory movement using the nanopositioning stage to stabilize the sample relative to the image detector in all 3 dimensions, in real-time.
The broader impact/commercial potential of this project lies in making localization-based SR imaging methods routinely useful to working biologists. This means making them technically straightforward to implement, and economically accessible. Presently, there is no single source for the components required to stabilize a conventional microscope and make it routinely useful for SR microscopy. Scientists hoping to implement these very broadly applicable techniques are left with the daunting task of assembling a working system from its many individual components, and then getting these components to work together seamlessly as required. Our goal is to develop and then commercialize a fully-integrated microscope stabilization and imaging system based on a nanopositioning stage, an EMCCD camera, and software enabling real-time image-based feedback control of the sample?s position. It will be developed with localization-based SR microscopy in mind, but will be useful for any imaging experiment that requires long-term sample stability and image acquisition, such as extended live-cell imaging. This system will greatly simplify the technical challenges faced by biologists wanting to utilize SR microscopy as an experimental tool, enabling them to convert and extend their current inverted microscopes into potentially a large number of SR-capable imaging systems.
Research microscopy in the biological sciences is in the midst of a period of fundamental change. Recent advances on several fronts have enabled the development of several so-called "super-resolution" (SR) methods, each of which surpasses the diffraction-limit on resolving power once thought to be insurmountable. Fundamentally based on fluorescence microscopy, these SR methods allow biologists to image cellular structures with the resolution previously seen only with Electron Microscopy (EM), while being far less invasive and far more versatile. Although SR techniques are "game changing" research tools, they are also technically challenging and expensive to implement, and place physical demands on the microscope platform it was not designed to meet. A phenomenon commonly referred to as "drift" poses serious practical problems for biological research microscopy. Drift is the result of small movements in components throughout an entire microscopy system over the course of an experiment, and these cumulative instabilities ultimately manifest in the sample moving relative to the detector being used to collect its image. Over the long time periods and at the small distance scales becoming more commonly relevant in advanced microscopy methods, this instability is inevitable. In even the best cases, this system drift complicates experiments and limits their power, and at worst it can interrupt an experiment in progress or render it impossible. Simply put, if you can’t hold something still enough, you can’t really measure where it is. Mad City Labs, Inc. has developed and will now commercialize a microscope-stabilization and imaging system targeted to the basic biological and biophysical research market. Our product is most succinctly described as a "Steady-Cam" system for research microscopy. It uses the imaging pathway from the sample to the detector as the reference frame for the stability of the imaging system, and actively adjusts for any measured movement in this pathway by producing compensatory movement in the stage on which the sample rests. While using a single core technical innovation, our microscope-stabilization and imaging system addresses the needs of two separate but related microscopy markets: (1) the "super-resolution" imaging market; and (2) the "live-cell" imaging market. We have separated our product into two versions to meet the specific needs of each of these markets.
