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

Che-Hang YuYiyi YuLiam M AdsitJeremy T ChangJad BarchiniAndrew H MoberlyHadas BenistyJinkyung KimBrent K YoungKathleen HengDeano M FarinellaAustin LeikvollRishaab PavanRachel VisteinBrandon R NanfitoDavid G C HildebrandSantiago Otero-CoronelAlipasha VaziriJeffrey Louis GoldbergAnthony J RicciDavid FitzpatrickJessica A CardinMichael J HigleyGordon B SmithPrakash KaraKristina J NielsenIkuko T SmithSpencer LaVere Smith
Published in: Nature methods (2023)
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 mm 2 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.
Keyphrases
  • high resolution
  • mass spectrometry
  • living cells
  • single molecule
  • fluorescence imaging
  • minimally invasive
  • photodynamic therapy
  • fluorescent probe