2026-11-09 テキサスA&M大学
＜関連情報＞
- https://stories.tamu.edu/news/2026/11/09/new-microscopy-technique-brings-cellular-detail-into-deeper-focus/
- https://www.spiedigitallibrary.org/journals/journal-of-biomedical-optics/volume-31/issue-08/086501/Breaking-the-100-nm-resolution-barrier-with-multiphoton-microscopy-using/10.1117/1.JBO.31.8.086501.full
- https://journals.aps.org/prxlife/abstract/10.1103/PRXLife.2.033010
画像走査顕微鏡と光変動イメージングを用いた多光子顕微鏡による100nm解像度の壁突破 Breaking the 100-nm resolution barrier with multiphoton microscopy using image scanning microscopy and optical fluctuation imaging
Anton Classen, Alma Fernández, Ajithamithra Dharmasiri, Cristobal Rodriguez, Rahul Srinivasan, Alexei M. Zheltikov, Girish S. Agarwal, Aart J. Verhoef
Journal of Biomedical Optics Published:2 Aug 2026
DOI:https://doi.org/10.1117/1.JBO.31.8.086501
Abstract
Significance
Multiphoton fluorescence microscopy is the technique of choice for investigations of thick, highly scattering samples, but is outperformed by single-photon super-resolution techniques in spatial resolving power.
Aim
We combine two-photon microscopy with two super-resolution microscopy methods, namely, image scanning microscopy and super-resolution optical fluctuation imaging to overcome the reduction in resolution of laser scanning multiphoton microscopy compared with confocal microscopy. Making use of higher-order cumulants and image deconvolution a resolution better than 100 nm can be achieved.
Approach
Two-photon image scanning optical fluctuation imaging is achieved by detecting the descanned signal of fluorescence on a 23-element single photon avalanche detector and analyzing (higher order) cumulants of the temporal evolution of the signals. We test the performance of our method with samples of dispersed quantum dots. We show the applicability of two-photon image scanning optical fluctuation imaging to biological samples with fixed mouse ventral midbrain neurons with quantum dot labeled tubulin.
Results
Combining two photon laser scanning microscopy with image scanning microscopy allows to overcome the reduction in resolution caused by the longer wavelength excitation inherent to multiphoton excitation. Analyzing the temporal fluctuations of the signals by calculating cumulants allows to surpass the resolution achieved with conventional confocal imaging of the same fluorophores, and the use of higher-order cumulants and deconvolution allows to achieve a lateral resolution of 75 nm when imaging quantum dots emitting at 625 nm.
Conclusions
Combining two-photon microscopy with image scanning microscopy and optical fluctuation imaging allows to achieve a 5-fold improvement in resolution over standard two-photon microscopy, and a 3.5-fold improvement over conventional widefield imaging of the same fluorophores. This represents the first time, to our knowledge, that sub-100 nm imaging is achieved using multiphoton laser scanning microscopy.
生物試料の超高解像度詳細研究のための3光子励起画像走査顕微鏡 Three Photon Excited Image Scanning Microscopy for in-Depth Super-Resolution Studies of Biological Samples
Anton Classen, I. Michel Antolovic, Stanislav Vitha1, Mia Pacheco, Dylan McCreedy, Alexei V. Sokolov, Girish S. Agarwal, Aart J. Verhoef, and Alma Fernández
PRX Life Published: 5 September, 2024
DOI:https://doi.org/10.1103/PRXLife.2.033010
Abstract
Multiphoton laser scanning microscopy is a powerful tool for deep imaging of thick biological samples. Image scanning microscopy (ISM) has demonstrated significant improvements in the signal-to-noise ratio in confocal laser scanning microscopy, while at the same time improving upon the effectively attainable resolution. Two-photon excitation (2PE), combined with ISM, has been shown to allow for deep tissue imaging with enhanced resolution compared to 2PE microscopy. Three-photon excitation (3PE) has enabled record imaging depth and contrast for multiphoton imaging, due to the superior suppression of out-of-focus signal generation. In this paper, we demonstrate super-resolution 3PE ISM. This is achieved using a single-photon avalanche detector array, and 1040-nm pulses for 3PE of blue fluorescence. This method enables subdiffraction limited resolution imaging of biological samples stained with blue fluorescent markers, such as mouse myocardial and spinal cord tissues stained with 4′,6-diamidino-2-phenylindole. Deconvolution improves the resolving power further and allows for imaging with better than /8 resolution with respect to the 3PE wavelength . With the ISM pixel reassignment procedure, we demonstrate a resolution enhancement of ∼1.6 laterally, compared to the resolution attained using a photomultiplier tube in a non-descanned detection arrangement, and a factor of ∼1.8 enhancement in axial resolution. The experimentally measured three-dimensional point spread function volume is shrunk ∼4.4-fold, which is close to the theoretically expected enhancement.
