2D simultaneous spatial and temporal focusing multiphoton microscopy for fast volume imaging with improved sectioning ability

Qiyuan Song; Keisuke Isobe; Kenichi Hirosawa; Katsumi Midorikawa; Fumihiko Kannari

doi:10.1117/12.2078093

5 March 2015 2D simultaneous spatial and temporal focusing multiphoton microscopy for fast volume imaging with improved sectioning ability

Qiyuan Song, Keisuke Isobe, Kenichi Hirosawa, Katsumi Midorikawa, Fumihiko Kannari

Author Affiliations +

Proceedings Volume 9329, Multiphoton Microscopy in the Biomedical Sciences XV; 93292N (2015) https://doi.org/10.1117/12.2078093
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

Simultaneous spatial and temporal focusing (SSTF) multiphoton microscopy offers us widefield imaging with sectioning ability. As extending the idea to 2D SSTF, people can utilize a 2D spectral disperser. In this study, we use a 2D spectral disperser via a virtually-imaged phased-array (VIPA) and a diffraction grating to fulfill the back aperture of objective lens with a spectrum matrix. This offers us an axial resolution enhanced by a factor of ~1.7 compared with conventional SSTF microscopy. Furthermore, the small free spectral range (FSR) of VIPA will reduce the temporal self-imaging effect around out-of-focus region and thus will reduce the out-of-focus multiphoton excited fluorescence (MPEF) signal of 2D SSTF microscopy. We experimentally show that inside a sample with dense MPEF, the contrast of the sectioning image is increased in our 2D SSTF microscope compared with SSTF microscope. In our microscope, we use a 1 kHz chirped amplification laser, a piezo stage and a sCMOS camera integrated with 2D SSTF to realize high speed volume imaging at a speed of 50 volumes per second as well as improved sectioning ability. Volume imaging of Brownian motions of fluorescent beads as small as 1μm has been demonstrated. Not only the lateral motion but also the axial motion could be traced.

Citation Download Citation

Qiyuan Song, Keisuke Isobe, Kenichi Hirosawa, Katsumi Midorikawa, and Fumihiko Kannari "2D simultaneous spatial and temporal focusing multiphoton microscopy for fast volume imaging with improved sectioning ability", Proc. SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV, 93292N (5 March 2015); https://doi.org/10.1117/12.2078093

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available