Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. To understand the performance limits of SIDH, we calculate the Cram´er-Rao lower bound (CRLB) of the localization precision for SIDH. We further compare the calculated precision bounds to the 3D single molecule localization precision from different Point Spread Functions. SIDH results in almost constant localization precision in all three dimensions for a 20 µm axial range. For high signal-to-background ratio (SBR), SIDH on average achieves better localization precision. For lower SBR values, the large size of the hologram on the detector results in more overall noise, and PSF models perform better.
KEYWORDS: Super resolution microscopy, Holograms, Photons, Signal to noise ratio, 3D image reconstruction, Digital holography, Michelson interferometers, Microscopes
Self-interference digital holography (SIDH) is a promising approach for three-dimensional imaging as it offers the ability to view a complete three-dimensional volume from a single image. SIDH has so far, largely been limited to image samples that emit a large number of photons. We report the use of a Michelson interferometer based SIDH setup which provides higher light throughput compared to previous systems that employed spatial light modulators (SLM). SIDH microscopy incorporating a Michelson interferometer in the proposed optical configuration and high-numerical-aperture oil immersion objectives can be used to perform super-resolution single-molecule localization microscopy (SMLM) and single-particle tracking (SPT) over large axial ranges. We demonstrate this by localizing a single 0.1 µm diameter fluorescent nanosphere using a custom built wide-field microscope. With 49,000 photons detected, the proposed system achieves a localization precision of 4.5 nm in x, 5 nm in y and 39.8 nm in z over a 20 µm axial range. Further, we also discuss the SNR requirements to image photon-limited light sources such as a single-molecules using SIDH.
Super-resolution imaging of volumes as large as whole cells in three-dimensions (3D) is required to reveal unknown features of cellular organization which cannot be resolved by conventional fluorescence microscopy. We propose a new 3D high resolution imaging technique based on the principles of single-molecule localization microscopy (SMLM) and fluorescence incoherent correlation holography (FINCH). FINCH enables hologram acquisition and three-dimensional (3D) imaging of large objects emitting incoherent light. This technique combines FINCH and SMLM to enable single-molecule volumetric imaging over large axial ranges without scanning the sample using a simple and robust setup, hence making it a viable solution for whole cell super-resolution imaging of biological samples. Here, we present the underlying theory and simulations demonstrating the extended depth of field. We image a single 0.2-μm fluorescent microsphere using this approach and discuss the signal-to-noise ratio (SNR) requirements for an experimental implementation.
Superresolution microscopy is rapidly becoming an essential tool in the biological sciences allowing imaging biological structure at length scales below 250 nm. Currently, superresolution microscopy has been applied successfully on single cells achieving resolutions of 100nm down to 20nm over a few microns of depth. When superresolution microscopy is applied in thicker samples the resolution rapidly degrades. Optical aberrations and scattering distort and reduce the point spread function causing different superresolution techniques to fail in different ways. I will discuss our work on combining structured illumination microscopy and stochastic optical reconstruction microscopy with adaptive optics to achieve sub-diffraction resolution in thick tissue.
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