Acquisition geometry analysis with point‐spread functions

AbstractSub-diffraction limited localization of fluorescent emitters is a key goal of microscopy imaging. Here, we report that single upconversion nanoparticles, containing multiple emission centres with random orientations, can generate a series of unique, bright and position-sensitive patterns in the spatial domain when placed on top of a mirror. Supported by our numerical simulation, we attribute this effect to the sum of each single emitter’s interference with its own mirror image. As a result, this configuration generates a series of sophisticated far-field point spread functions (PSFs), e.g. in Gaussian, doughnut and archery target shapes, strongly dependent on the phase difference between the emitter and its image. In this way, the axial locations of nanoparticles are transferred into far-field patterns. We demonstrate a real-time distance sensing technology with a localization accuracy of 2.8 nm, according to the atomic force microscope (AFM) characterization values, smaller than 1/350 of the excitation wavelength.

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Resolution-Improvement of Confocal Fluorescence Microscopy via Two Different Point Spread Functions

Lecture Notes of the Institute for Computer Sciences, Social Informatics and Telecommunications Engineering - Industrial Networks and Intelligent Systems ◽

10.1007/978-3-030-63083-6_6 ◽

2020 ◽

pp. 77-84

Author(s):

Xuanhoi Hoang ◽

Vannhu Le ◽

MinhNghia Pham

Keyword(s):

Fluorescence Microscopy ◽

Confocal Fluorescence Microscopy ◽

Point Spread Functions ◽

Point Spread ◽

Confocal Fluorescence ◽

Resolution Improvement

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Two simple criteria to estimate an objective’s performance when imaging in non design tissue clearing solutions

10.1101/799460 ◽

2019 ◽

Author(s):

Sabrina Asteriti ◽

Valeria Ricci ◽

Lorenzo Cangiano

Keyword(s):

Image Quality ◽

High Refractive Index ◽

Optimal Conditions ◽

Point Spread Functions ◽

Tissue Clearing ◽

Fluorescent Microscope ◽

Immersion Medium ◽

Quality Degradation ◽

Point Spread ◽

The Impact

ABSTRACTTissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescence deep in biological samples without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions, which require expensive optimized objectives to avoid aberrations. One may thus need to assess whether an available objective is suitable for a specific clearing solution, or the impact on imaging of small mismatches between cleared sample and objective design RIs. We derived closed form approximations for image quality degradation versus RI mismatch and other parameters available to the microscopist. We validated them with computed (and experimentally confirmed) aberrated point spread functions, and by imaging fluorescent neurons in high RI solutions. Crucially, we propose two simple numerical criteria to establish: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several, achieves the highest resolution in a given immersion medium. These criteria apply directly to the widefield fluorescent microscope but are also closely relevant to more advanced microscopes.

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