• Current Optics and Photonics
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• v.5 no.4
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• pp.421-430
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• 2021

The multifocal multiphoton microscopy (MMM) enables high-speed imaging by the concurrent scanning and detection of multiple foci generated by lenslet array or diffractive optical element. The MMM system mainly suffers from crosstalk generated by scattered emission photons that form ghost images among adjacent channels. The ghost image which is a duplicate of the image acquired in sub-images significantly degrades overall image quality. To eliminate the ghost image, the photon reassignment method was established using maximum likelihood estimation. However, this post-processing method generally takes a longer time than image acquisition. In this regard, we propose a novel strategy for rapid noise reduction in the MMM system based upon Monte-Carlo (MC) simulation. Ballistic signal, scattering signal, and scattering noise of each channel are quantified in terms of photon distribution launched in tissue model based on MC simulation. From the analysis of photon distribution, we successfully eliminated the ghost images in the MMM sub-images. If the priori MC simulation under a certain optical condition is established at once, our simple, but robust post-processing technique will continuously provide the noise-reduced images, while significantly reducing the computational cost.

• Molecules and Cells
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• v.26 no.2
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• pp.113-120
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• 2008

Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon microscopy, has enabled the quantification of spatiotemporal patterns of $[Ca^{2+}]_i$ and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocytosis" for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.