Development Of High-Speed Holographic Fluorescence Microscopy System With Submicron Resolution

The group has realized a scanless 3D imaging system and an algorithm for high-speed measurement

The National Institute of Information and Communications Technology (NICT), Tohoku University, Toin University of Yokohama, and Japan Science and Technology Agency (JST) have succeeded in developing a scanless high-speed holographic fluorescence microscopy system with submicron resolution for a 3D space. The system is based on digital holography. The developed microscopy system has an algorithm to acquire 3D information of fluorescent objects toward scanless 3D measurement in less than 1 millisecond. Scanless 3D sensing with submicron resolution and color-multiplexed holographic fluorescence imaging have been demonstrated using the algorithm. The microscopy system will be further developed to achieve holographic 3D motion-picture sensing of specimens with incoherent light.

This achievement was published in Optics Letters as an open-access paper on January 29, 2021.

The scanless high-speed holographic fluorescence microscopy system shown in was constructed. The system is based on digital holography and is applicable to the sensing of incoherent light such as fluorescence light and natural light. The developed algorithm enables the adoption of a phase modulator to generate two phase values, which is expected to increase the measurement speed. Submicron resolution for a 3D space was successfully demonstrated using fluorescent objects with a diameter of 0.2 μm. The experimental results shown in indicate that the developed microscopy system achieves 3D sensing of nanoparticles and has submicron resolution quantitatively for a 3D space. Scanless 3D measurement in less than 1 millisecond is achievable by using the algorithm with either a ferroelectric liquid crystal on silicon (FLCOS) or an electro-optic (EO) device. Color-multiplexed holographic fluorescence imaging with the algorithm and only four exposures has also been demonstrated by combining the proposed algorithm and computational coherent superposition (CCS). The number of exposures is reduced by the algorithm, and the number of photons per hologram is increased even for ultimately weak light.

• High-speed holographic motion-picture imaging for 3D dynamics and multiple moving objects in a 3D space.
• Improvements of the system such as recording of a quantitative phase, sensing of ultimately weak light, and construction of a compact optical setup.

This work was supported by the Cooperative Research Program of "Network Joint Research Center for Materials and Devices" (No. 20201164); Precursory Research for Embryonic Science and Technology (PRESTO) (JPMJPR15P8, JPMJPR16P8, JPMJPR17P2); Japan Society for the Promotion of Science (JSPS) (18H01456).

Digital holography
Digital holography is a technique used to record 3D information of a specimen as digital holograms with an image sensor and to reconstruct a 3D image of the specimen numerically using a computer. shows the system constructed for recording digital holograms. Incoherent light such as fluorescence light and natural light can be recorded as holograms using the specially designed optical system shown in. An image sensor records fluorescence light that passes through the optical system as digital holograms. A computer numerically reconstructs a 3D image from the recorded holograms. Object-wave extraction from the recorded hologram(s) is numerically calculated using an algorithm such as the proposed algorithm and CCS. Numerical wave propagation is calculated and then images focused on arbitrary depths are reconstructed.

Computational coherent superposition (CCS)
CCS is an in-line holographic multiplexing scheme based on phase-shifting interferometry. schematically illustrates the system for recording digital holograms and the basic concept for object-wave extraction from the recorded holograms. In CCS, a wavelength-multiplexed interferometer in the real world that records wavelength-multiplexed phase-shifted holograms is constructed to conduct color-multiplexed holographic imaging. In the numerical calculation process used to extract object waves at multiple wavelengths from the wavelength-multiplexed holograms, an interferometer in a computer is assumed to reconstruct a color holographic image from the recorded holograms. On the basis of phase-shifting interferometry, the numerical interference is calculated in the computer and only object-wave information at the desired wavelength is extracted.