We propose to build and use custom designed microfluidics chips that, when combined with associated optics, will greatly facilitate several modalities of fluorescence super- resolution imaging. The essential concept is the presence of a reflective, planar surface that makes an angle of near 45 degrees with respect to the cover slip normal. This reflective surface will allow a single imaging objective to also provide planar illumination at an image focal plane placed anywhere inside a mammalian cell. Since only the imaged focal plane is illuminated, the fluorescence background is greatly reduced and fluorophore longevity is improved. The dimensions of the cell accessible volumes of the chip will be designed such that cells will always locate to regions amenable for imaging by the reflected light. We will focus on three imaging modalities: 3D Single-Molecule Super-Resolution (SM-SR), extended time series, 3D imaging, and 3D Structured Illumination Microscopy (SIM). The project will focus around design of the microfluidics device and optics, and assessing the imaging performance of each of the three above mentioned imaging modalities. For each, we will compare results when using the device to conventional methods in terms of resolution, both spatial and temporal, and probe photo-stability. The developed devices and techniques will be immediately used for studying FceR1 associated signal transduction in RBL-2H3 cells, which is the central focus of the 'The New Mexico Center for Spatiotemporal Modeling of Cellular Signaling'(STMC). The STMC is an NIH funded center for systems biology, of which both senior personnel are members.

Public Health Relevance

We will develop a microfluidics chip device that will be used for fluorescence imaging of fluorescently tagged proteins within single cells. The device will allow imaging at faster speed and at better resolutions than conventional microscopy approaches by making use of techniques that would otherwise have to be implemented at much greater cost or sophistication. This device could be used by many researchers as an addition to existing equipment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21GM104691-03
Application #
8707498
Study Section
Special Emphasis Panel (ZRR1)
Program Officer
Friedman, Fred K
Project Start
2012-08-15
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of New Mexico
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
City
Albuquerque
State
NM
Country
United States
Zip Code
87106
Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S et al. (2016) Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution. Biomed Opt Express 7:2219-36
Liu, Sheng; Lidke, Keith A (2014) A multiemitter localization comparison of 3D superresolution imaging modalities. Chemphyschem 15:696-704
Liu, Sheng; Kromann, Emil B; Krueger, Wesley D et al. (2013) Three dimensional single molecule localization using a phase retrieved pupil function. Opt Express 21:29462-87