The challenge

Imagine gaining admittance inside the cells of living organisms and seeing up-close the inner workings of the molecular machinery of life. What new insights might be achieved? What subtle models for bio-inspired engineering? What inspirations for new therapies?

While advanced microscopy and imaging techniques have begun to provide us with reconstructed images of the basic building blocks of matter (atoms), as well as intricate details of the basic building blocks of life (cells), we have yet to be able to witness and record molecular life processes at work inside living cells.

Until recently, optical microscopy has been limited to the resolution of the smallest wavelengths of visible light, which are still too large to resolve the intricate nanoscale molecular machinery inside cells. Electron microscopy can reveal much of this subcellular detail, however, it cannot be used to image cells in living organisms. Scientists who want to study the processes by which cells live and grow and perform their different functions typically have no choice but to try to isolate, kill, and section cells, and then to try to infer from these static images the interplay of structure and functionality. Such post-mortem images provide intimate awareness of molecular-scale structure inside the cell, but afford much less insight into the molecular-scale processes that make up life itself.

Molecular and cellular biologists long to see the actual millisecond-to-millisecond interplay of molecular-scale elements inside the specialized cells of muscle, bone, and brain tissues, as together they carry out the intricately coordinated activities of life.

The approach

The QSTORM team has come together to pursue one particular pathway to tackle this challenge. Their approach requires expert knowledge and experience from several different scientific and engineering disciplines. The full title of their research project is “Collaborative Research: QSTORM: Switchable Quantum Dots and Adaptive Optics for Super-Resolution Imaging.” The acronym QSTORM stands for Quantum dots with STtochastic Optical Reconstruction Microscopy. Stochastic Optical Reconstruction Microscopy (STORM) is one of several new techniques for enhancing the resolution of optical microscopy beyond the limit set by the wavelength of visible light. As the Howard Hughes Medical Institute describes it, “To create an image with STORM, researchers label the molecules they want to study with fluorescent probes, and then use a burst of light to activate the fluorescence in a small percentage of labeled molecules. The microscope captures an image of the fluorescing probes. The technique is designed to activate a sufficiently low percentage of the probes to allow the image of each fluorescing molecule to be seen separately. This allows the molecules to be localized individually. The process is repeated many times, capturing a different subset of molecules with each iteration. A final compilation of the images shows each molecule in its precise location in the cell with nanometer accuracy.” The STORM process also can be used to produce reconstructed images in three dimensions.

Another technique, known as Adaptive Optics (AO), allows researchers to use complex computer algorithms to correct for the “wavefront distortions” that occur when the waves of closely located point light sources interfere with each other. These two techniques have been used together to obtain super resolution images of cellular structures, but not in living cells. Furthermore, the flourescent dyes that are currently used to tag particular structures within cells have significant drawbacks. Their florescence is limited, and it fades rather quickly.

The QSTORM team plans to substitute quantum dot probes for the florescent dyes typically used in biological imaging. Quantum dots are very bright, and last indefinitely. The challenge here is to develop a technique for “switching on and off” the quantum dots from outside the organism. The QDs need to be switchable in order the control how many are glowing at any one time. This will help reduce the wavefront interference or “airy disk” problem. The switchable QDs will then be microinjected into the cells of living organisms. So QSTORM plans to combine three elements — user-controlled quantum dot imaging probes, STORM imaging algorithms, and adaptive optics (AO) — to produce the world’s first super-resolution in vivo imaging technology. If the team is successful, this tool will allow biologists to observe biological structures and processes in action at a resolution below the limit of light microscopy (~ 200 nanometers).

To achieve success, the team must:
(1) Design, synthesize, and characterize specialized fluorescent quantum dots (QDs) that can be tagged to specific molecular structures inside specialized cells and can be reliably switched on and off from outside the model organism.
(2) Test the QSTORM integrated approach in non-living (ex vivo cells).
(3) Test the approach in living (in vivo) cells, by:
(A) Micro-inject the specialized QDs into the targeted cells of model organisms.
(B) Demonstrate single QD imaging in vivo using wide-field microscopy with adaptive optics.
(C) Evaluate effectiveness of QSTORM technique in model organisms.
(4) Share the process and results of QSTORM research broadly.

The two model organisms and systems selected are:
(i) Zebrafish, to study myofilament behavior in muscle cells.
(ii) Drosophila, to study vesicle transport in neural cells

These systems were selected because (a) they consist of components below the resolution limit for traditional optical microscopy; (b) they are thin specimens in which microinjection has been previously performed; and (c) they pose biological problems that cannot be adequately addressed with existing microscopy techniques.

An important aspect of the proposed approach is that several of the individual research components have the potential to advance the field whether or not all the aims are achieved. For example, several of the proposed experiments do not require switchable QDs or can be performed with alternative fluorescent molecules. Significant progress in biological imaging can still be achieved through these experiments, including possible first-of-a-kind demonstrations of single QD detection in vivo, the use of non-switchable QDs and adaptive optics, and high resolution ultrastructural analyses in vivo (using fluorescent dyes or traditional QDs and adaptive optics).