Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging

Abstract

Fresnel Incoherent Correlation Holography (FINCH) enables holograms to be recorded from incoherent light with just a digital camera and spatial light modulator. We previously described its application to general three dimensional incoherent imaging and specifically to fluorescence microscopy, wherein one complex hologram contains the three dimensional information in the field of view, obviating the need for scanning or serial sectioning. We have now further analyzed FINCH in view of linear system theory and in comparison to conventional coherent and incoherent two dimensional imaging systems. We demonstrate, theoretically and experimentally, improved resolution by FINCH, when compared to conventional imaging.

Incoherent digital holography with phase-only spatial light modulators

“…The resolving power of FINCH, as previously shown, 21 can exceed the resolving power of a regular incoherent imaging system of a similar NA. In Fig.…”

“…3, showing a FINCH system with an extra glass lens L c . 21,24 An object point, positioned at a working distance from the objective lens L o , emits a spherical beam which is introduced into the system. An input polarizer, P 1 , oriented at an angle of 45 deg to the SLM active axis, allows the operation of two different imaging systems in the same physical single-channel setup.…”

“…Finally, reducing the optical path difference (OPD) in FINCH, for processing wide-bandwidth light sources, was done by inefficiently increasing the length of the systems. These difficulties were fruitfully solved in the later versions of FINCH; 19,21,24 some of them are reviewed in Sec. 2.2.…”

“…52 However, note that the original confocal FINCH 44 is motionless and accomplishes optical sectioning of the observed specimen without losing the inherent transverse super-resolution capabilities of the original FINCH. 21 Therefore, confocal FINCH can image different planes of interest at various depths while blocking light from other planes. By that, the axial imaging resolution of the imager is enhanced and small details that otherwise would be lost are discovered.…”

“…Similar to FINCH, in order to achieve optimal resolution, the system is configured to fulfill the requirement of a perfect overlap between the two interfering waves on the camera plane. 21 The advantage of working with two spherical waves, rather than with one spherical wave and one plane wave, is manifested by the system (either FINCH or SAFE) capability of handling light of a wider bandwidth, while keeping the visibility of the recorded interference patterns sufficient. This is because the maximal OPD between two interfering waves becomes shorter as the two image points, generated by focusing the two spherical waves, get closer to each other.…”