scholarly journals Two-photon PSF-engineered image scanning microscopy

2019 ◽  
Vol 44 (4) ◽  
pp. 895 ◽  
Author(s):  
Omer Tzang ◽  
Dan Feldkhun ◽  
Anurag Agrawal ◽  
Alexander Jesacher ◽  
Rafael Piestun
Keyword(s):  
Scanning Microscopy ◽  
Image Scanning ◽  
Two Photon

scholarly journals EASY TWO-PHOTON IMAGE-SCANNING MICROSCOPY WITH SPAD ARRAY AND BLIND IMAGE RECONSTRUCTION

2019 ◽  
Author(s):  
S. V. Koho ◽  
E. Slenders ◽  
G. Tortarolo ◽  
M. Castello ◽  
M. Buttafava ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Single Photon ◽  
Imaging Modality ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Reconstruction Method ◽  
Image Scanning ◽  
Two Photon ◽  
Blind Image

ABSTRACTTwo-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo. However, the resolution in two-photon microscopy is poor, below confocal microscopy, and the lack of an optical pinhole becomes apparent in complex samples as reduced quality of optical sectioning. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on a new single-photon avalanche diode array detector – coupled with a novel blind image reconstruction method, is shown to improve the optical resolution, as well as the overall image quality in various test samples. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user – it works like any old and familiar two-photon system, but simply produces higher resolution images (in real-time). Making the complexity disappear, in our view, is the biggest novelty here, and the key for making 2PE-ISM mainstream.

Image formation in image scanning microscopy, including the case of two-photon excitation

2017 ◽  
Vol 34 (8) ◽  
pp. 1339 ◽  
Author(s):  
Colin J. R. Sheppard ◽  
Marco Castello ◽  
Giorgio Tortarolo ◽  
Giuseppe Vicidomini ◽  
Alberto Diaspro
Keyword(s):  
Image Formation ◽  
Scanning Microscopy ◽  
Image Scanning ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals Two-photon image-scanning microscopy with SPAD array and blind image reconstruction

2020 ◽  
Vol 11 (6) ◽  
pp. 2905 ◽  
Author(s):  
Sami V. Koho ◽  
Eli Slenders ◽  
Giorgio Tortarolo ◽  
Marco Castello ◽  
Mauro Buttafava ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Scanning Microscopy ◽  
Image Scanning ◽  
Two Photon ◽  
Blind Image

scholarly journals Two-photon laser scanning microscopy with electrowetting-based prism scanning

2017 ◽  
Vol 8 (12) ◽  
pp. 5412 ◽  
Author(s):  
Omkar D. Supekar ◽  
Baris N. Ozbay ◽  
Mo Zohrabi ◽  
Philip D. Nystrom ◽  
Gregory L. Futia ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Two Photon

scholarly journals Assessment of Fluorochromes for Two-Photon Laser Scanning Microscopy of Biofilms

2002 ◽  
Vol 68 (2) ◽  
pp. 901-909 ◽  
Author(s):  
Thomas R. Neu ◽  
Ute Kuhlicke ◽  
John R. Lawrence
Keyword(s):  
Nucleic Acid ◽  
Laser Scanning ◽  
Single Photon ◽  
Selective Excitation ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Excitation Wavelength ◽  
Laser Microscopy ◽  
Two Photon ◽  
Photon Excitation

ABSTRACT A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F420-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

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