ABSTRACT
The locations and volumes of the contents of a single HepG2 cell were visualized under three-dimensional (3D) holographic and tomographic (HT) laser microscopy, colored by refractive index, not staining. After trapping the specific area of a target cell in a nanospray tip, quantification was performed by live single-cell mass spectrometry. Comparison of the HepG2 cells' before and after 3D-HT images allowed the inference of the precise volume and original location of the trapped cell contents. The total amount of a trapped molecule was estimated. The images also revealed morphological changes in the cell structure caused by the manipulation.
Subject(s)
Holography/methods , Imaging, Three-Dimensional/methods , Mass Spectrometry/methods , Microscopy, Confocal/methods , Single-Cell Analysis/methods , Tomography/methods , Cytosol/metabolism , Hep G2 Cells , Humans , Image Processing, Computer-Assisted , RefractometryABSTRACT
We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-of-principle measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited.
Subject(s)
Microscopy, Interference/methods , Holography/methods , Holography/statistics & numerical data , Microscopy, Electron, Scanning/methods , Microscopy, Electron, Scanning/statistics & numerical data , Microscopy, Interference/statistics & numerical data , Microscopy, Phase-Contrast/methods , Microscopy, Phase-Contrast/statistics & numerical data , Models, Theoretical , Nanostructures/ultrastructure , Optical Phenomena , Scattering, Radiation , Signal-To-Noise RatioABSTRACT
We present a novel technique for three-dimensional (3D) image processing of complex fields. It consists in inverting the coherent image formation by filtering the complex spectrum with a realistic 3D coherent transfer function (CTF) of a high-NA digital holographic microscope. By combining scattering theory and signal processing, the method is demonstrated to yield the reconstruction of a scattering object field. Experimental reconstructions in phase and amplitude are presented under non-design imaging conditions. The suggested technique is best suited for an implementation in high-resolution diffraction tomography based on sample or illumination rotation.
ABSTRACT
Based on truncated inverse filtering, a theory for deconvolution of complex fields is studied. The validity of the theory is verified by comparing with experimental data from digital holographic microscopy (DHM) using a high-NA system (NA=0.95). Comparison with standard intensity deconvolution reveals that only complex deconvolution deals correctly with coherent cross-talk. With improved image resolution, complex deconvolution is demonstrated to exceed the Rayleigh limit. Gain in resolution arises by accessing the objects complex field - containing the information encoded in the phase - and deconvolving it with the reconstructed complex transfer function (CTF). Synthetic (based on Debye theory modeled with experimental parameters of MO) and experimental amplitude point spread functions (APSF) are used for the CTF reconstruction and compared. Thus, the optical system used for microscopy is characterized quantitatively by its APSF. The role of noise is discussed in the context of complex field deconvolution. As further results, we demonstrate that complex deconvolution does not require any additional optics in the DHM setup while extending the limit of resolution with coherent illumination by a factor of at least 1.64.
Subject(s)
Algorithms , Holography/methods , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Microscopy/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We report the experimental observation of systematically occurring phase singularities in coherent imaging of sub-Rayleigh distanced objects. A theory that relates the observation to the sub-Rayleigh distance is presented and compared with experimental measurements. As a consequence, the limit of resolution with coherent illumination is extended by a factor of 1.64x.
