The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo

Che-Hang Yu; Yiyi Yu; Liam M Adsit; Jeremy T Chang; Jad Barchini; Andrew H Moberly; Hadas Benisty; Jinkyung Kim; Brent K Young; Kathleen Heng; Deano M Farinella; Austin Leikvoll; Rishaab Pavan; Rachel Vistein; Brandon R Nanfito; David G C Hildebrand; Santiago Otero-Coronel; Alipasha Vaziri; Jeffrey L Goldberg; Anthony J Ricci; David Fitzpatrick; Jessica A Cardin; Michael J Higley; Gordon B Smith; Prakash Kara; Kristina J Nielsen; Ikuko T Smith; Spencer LaVere Smith

doi:10.1038/s41592-023-02098-1

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo

Nat Methods. 2024 Jan;21(1):132-141. doi: 10.1038/s41592-023-02098-1. Epub 2023 Dec 21.

Authors

Che-Hang Yu¹, Yiyi Yu², Liam M Adsit³, Jeremy T Chang⁴, Jad Barchini⁴, Andrew H Moberly⁵, Hadas Benisty⁵, Jinkyung Kim⁶, Brent K Young⁷, Kathleen Heng^{7

8}, Deano M Farinella⁹, Austin Leikvoll⁹, Rishaab Pavan¹⁰, Rachel Vistein¹¹, Brandon R Nanfito¹², David G C Hildebrand¹³, Santiago Otero-Coronel^{13

14

15}, Alipasha Vaziri^{14

15}, Jeffrey L Goldberg⁷, Anthony J Ricci^{16

17}, David Fitzpatrick⁴, Jessica A Cardin⁵, Michael J Higley⁵, Gordon B Smith⁹, Prakash Kara⁹, Kristina J Nielsen¹², Ikuko T Smith^{3

18

19}, Spencer LaVere Smith^{20

21}

Affiliations

¹ Department of Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara, CA, USA. chehangyu@ucsb.edu.
² Department of Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara, CA, USA.
³ Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA, USA.
⁴ Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA.
⁵ Department of Neuroscience, Yale University, New Haven, CT, USA.
⁶ Department of Otolaryngology, Washington University School of Medicine, St. Louis, MO, USA.
⁷ Spencer Center for Vision Research, Byers Eye Institute, School of Medicine, Stanford University, Palo Alto, CA, USA.
⁸ Neurosciences Interdepartmental Program, Stanford University, Stanford, CA, USA.
⁹ Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA.
¹⁰ Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.
¹¹ Department of Molecular and Comparative Pathobiology, and Zanvyl Krieger Mind/Brain Institute, Johns Hopkins University, Baltimore, MD, USA.
¹² Solomon H. Snyder Department of Neuroscience, and Zanvyl Krieger Mind/Brain Institute, Johns Hopkins University, Baltimore, MD, USA.
¹³ Laboratory of Neural Systems, The Rockefeller University, New York, NY, USA.
¹⁴ Laboratory of Neurotechnology and Biophysics, The Rockefeller University, New York, NY, USA.
¹⁵ Kavli Neural Systems Institute, The Rockefeller University, New York, NY, USA.
¹⁶ Department of Otolaryngology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA.
¹⁷ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA.
¹⁸ Department of Psychology and Brain Sciences, University of California Santa Barbara, Santa Barbara, CA, USA.
¹⁹ Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA, USA.
²⁰ Department of Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara, CA, USA. sls@ucsb.edu.
²¹ Department of Psychology and Brain Sciences, University of California Santa Barbara, Santa Barbara, CA, USA. sls@ucsb.edu.

Abstract

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm² field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo

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