There is a fundamental limit to the imaging speed of a point-scanning confocal fluorescence microscope due to the limited amount of fluorescence signal that is emitted from a biological sample. To improve the speed of scanning, several techniques have been developed in the past, such as Nipkow spinning disk confocal microscope and line-scanning confocal microscope. However, to scan a large volume, their speed is still limited by the field of view (FOV) of the microscope objective. Additionally, they ae difficult to be miniaturized into portable handheld devices. We propose to develop an array confocal fluorescence microscope (ACFM) that can image large 3D volumes at a speed one order of magnitude faster than conventional confocal microscope over a large FOV. The proposed ACFM consists of an array of miniature high-NA confocal fluorescence objectives, each of which scans a small sub-FOV. Multiple point scanning and detection will not only increase the overall scanning speed dramatically, but also reduce photobleaching or phototoxicity significantly in live cells because it requires a lower level of liht intensity per unit area. The FOV of the proposed ACFM will not be limited by the FOV of individual objective;it will only be limited by the scan range of the scanning mechanism. Most importantly, instead of scanning stage or objective for depth imaging, the proposed ACFM will develop a novel tunable liquid plate located in the image space of the objective to perform high speed depth scan in the object space. An array of fibers, one for each channel in the ACFM, delivers the excitation illumination and collects the emitted fluorescence signal. The fibers will also act as the confocal pinholes to eliminate out-of-focus light. With this unique configuration the confocal head of the proposed ACFM can be very compact and scalable, particularly suitable for handheld clinical applications. In this proposed three-year effort, we will design, build, and test a compact high NA (NA=0.7) and large FOV ACFM with 5x5 confocal objectives. The objectives will be designed with optical plastics and fabricated using diamond turning techniques. We will calibrate the system, measure the lateral and axial resolution, and demonstrate system capabilities through imaging mouse tissue samples. This effort will require interdisciplinary collaboration of various areas in biomedical engineering optical engineering and fabrication, and system engineering and electronics. We will apply experience learned from developing bright-field array microscope to the proposed development of ACFM. If successful, the proposed ACFM will have significant impacts on biomedical imaging, especially in in-vivo clinical applications over large FOVs and whole slide imaging. The proposed ACFM can be used as a scalable platform for other imaging modalities, such as confocal Raman microscope, multiphoton microscope, and hyperspectral microscope. It will also be a platform for more advanced imaging applications, such as parallel depth imaging and multiple-band fluorescence imaging. The research will provide an excellent opportunity to train the next generation of interdisciplinary scientists and engineers, at both the undergraduate and graduate levels.
This proposed project will design, build, and test an array confocal fluorescence microscope (ACFM) that can image large 3D volumes at a speed one order of magnitude faster than conventional point scanning confocal fluorescence microscopes. Development of this technology will have significant impacts on biomedical imaging, especially in in-vivo clinical applications over large FOVs and handheld clinical applications.
|Pacheco, Shaun; Wang, Chengliang; Chawla, Monica K et al. (2017) High resolution, high speed, long working distance, large field of view confocal fluorescence microscope. Sci Rep 7:13349|
|Pacheco, Shaun; Milster, Tom; Liang, Rongguang (2015) Analysis of grating doublets for achromatic beam-splitting. Opt Express 23:22939-52|
|Pacheco, Shaun; Brand, Jonathan F; Zaverton, Melissa et al. (2015) Sensitivity analysis and optimization method for the fabrication of one-dimensional beam-splitting phase gratings. Opt Express 23:11771-82|
|Pacheco, Shaun; Salahieh, Basel; Milster, Tom et al. (2015) Transfer function analysis in epi-illumination Fourier ptychography. Opt Lett 40:5343-6|