Adding Dimensions to Multiplex Molecular Imaging

Imaging of living specimens (intravital imaging) offers a means to draw upon the growing body of high-throughput molecular data to better understand the underlying cellular and molecular mechanisms of complex events ranging from embryonic development to disease processes. However, imaging approaches are challenged by major tradeoffs between spatial resolution, temporal resolution, field of view and the limited photon budget.

We are attempting to advance this tradeoff by constructing faster and more efficient light sheet microscopes that maintain subcellular resolution. Our two-photon light-sheet microscope combines the deep penetration of two-photon microscopy and the speed of light sheet microscopy to generate images with more than ten-fold improved imaging speed and sensitivity. As with other light sheet technologies, the collection of an entire 2-D optical section in parallel dramatically speeds acquisition rates. By adopting two-photon excitation the light sheet illumination is far less subject to light scattering, permitting subcellular resolution to be maintained far better than conventional light sheet microscopes. This combination of attributes permits 4D cell and molecular imaging with sufficient speed and resolution to generate unambiguous tracing of cells and signals in intact systems.

To increase the 5th Dimension, the number of simultaneous labels, we are refining new multispectral image analysis tools that exceed the performance of our previous work on Linear Unmixing by orders of magnitude in speed, error propagation and accuracy. These new analysis tools permit rapid and unambiguous analyses of multiplex-labeled specimens.

In parallel, we have refined label-free approaches so that imaging and sensing can be more extended to patient-derived tissues and even human subjects. The low concentrations and low sensitivity of the techniques can make single cell approaches challenging. We have refined fluorescence lifetime approaches (FLIM), combining it with multispectral tools to optimize intravital imaging in these challenging settings.

Combined, these imaging and analysis tools offer the multi-dimensional imaging required to follow key events in intact systems as they take place, and to allow us to use noise and variance as experimental tools rather than experimental limitations.