Three Photon Excited Image Scanning Microscopy for in-Depth Super-Resolution Studies of Biological Samples

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 <a:math xmlns:a="http://www.w3.org/1998/Math/MathML"><a:mrow><a:msup><a:mn>4</a:mn><a:mo>′</a:mo></a:msup><a:mo>,</a:mo><a:mn>6</a:mn></a:mrow></a:math>-diamidino-2-phenylindole. Deconvolution improves the resolving power further and allows for imaging with better than <b:math xmlns:b="http://www.w3.org/1998/Math/MathML"><b:mrow><b:mi>λ</b:mi><b:mo>/</b:mo><b:mn>8</b:mn></b:mrow></b:math> resolution with respect to the 3PE wavelength <c:math xmlns:c="http://www.w3.org/1998/Math/MathML"><c:mi>λ</c:mi></c:math>. With the ISM pixel reassignment procedure, we demonstrate a resolution enhancement of <d:math xmlns:d="http://www.w3.org/1998/Math/MathML"><d:mrow><d:mo>∼</d:mo><d:mn>1.6</d:mn></d:mrow></d:math> laterally, compared to the resolution attained using a photomultiplier tube in a non-descanned detection arrangement, and a factor of <e:math xmlns:e="http://www.w3.org/1998/Math/MathML"><e:mrow><e:mo>∼</e:mo><e:mn>1.8</e:mn></e:mrow></e:math> enhancement in axial resolution. The experimentally measured three-dimensional point spread function volume is shrunk <f:math xmlns:f="http://www.w3.org/1998/Math/MathML"><f:mrow><f:mo>∼</f:mo><f:mn>4.4</f:mn></f:mrow></f:math>-fold, which is close to the theoretically expected enhancement. Published by the American Physical Society 2024