Non-axial-scanning multifocal confocal microscopy with volume holography

Wide-field fluorescence microscopy is a commonly used imaging technique by researchers and clinicians. A standard wide-field microscope has no optical sectioning capabilities and this limits its use in imaging thick biological samples. Although standard wide-field fluorescence microscopy with deconvolution techniques can improve image quality [1], it does not provide true optical sectioning, due to the missing cone in system’s transfer function. The most commonly used optical sectioning imaging method with good background rejection in biomedicine is based on the confocal approach [2-4]. However, the price to pay for improved image quality in 3D confocal microscopy is a point-by-point scan time that is proportional to the number of desired voxels (i.e. the 3D space-bandwidth product). Here, we demonstrate the first experimental realization of a non-axial-scanning multi-focal confocal microscope for 3D imaging where contrast and speed are achieved from a combination of confocal imaging pinholes and multiplexed holographic Bragg illumination filters. Figure 1: Schematic drawing of the proposed microscope in epi-illumination format. Figure 2: Experimental measurement of point-spread function at different planes. References: [1] McNally, J.G., Karpova T., Cooper, J., Conchello, J.A., Three-dimensional imaging by deconvolution microscopy, Methods 19, 373-385, 1999. [2] C.J.R. Sheppard, A. Choudhury, “Image Formation in the Scanning Microscope”, Optica Acta, Vol. 24, No.10, pages 1051-1073, 1977. [3] M.Minsky, “Memoir on inventing the confocal scanning microscope”, Scanning, Vol. 10, Issue 4, pages 128–138, 1988 [4] Yuan Luo, Vijay Raj Singh, Dipanjan Bhattacharya, Elijah Y. S. Yew, Jui-Chang Tsai, Sung-Liang Yu, Hsi-Hsun Chen, Jau-Min Wong8, Paul Matsudaira, Peter T. C. So and George Barbastathis, "Talbot Holographic Illumination Non-scanning (THIN) Fluorescence Microscopy," Laser & Photonics Reviews ,Volume 8, Issue 5, pages L71–L75, September 2014.