Characterization of a reflective objective with multiphoton microscopy

Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending ~1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of ~25% for TPF images of sub-diffraction fluorescent beads and ~10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of ~4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.

[1]  Nien-Hui Ge,et al.  Rapid vibrational imaging with sum frequency generation microscopy. , 2011, Optics letters.

[2]  Vincent Couderc,et al.  Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation). , 2012, Optics express.

[3]  K. Kieu,et al.  All-reflective multiphoton microscope. , 2017, Optics express.

[4]  R. Barer,et al.  Infra-Red Spectroscopy with the Reflecting Microscope in Physics, Chemistry and Biology , 1949, Nature.

[5]  C. Burch,et al.  Reflecting Microscopes , 1943, Nature.

[6]  N. Durr,et al.  Annular Beam Shaping in Multiphoton Microscopy to Reduce Out-of-Focus Background , 2017 .

[7]  D. S. Grey,et al.  A new series of microscope objectives; catadioptric Newtonian systems. , 1949, Journal of the Optical Society of America.

[8]  Thomas M Bartol,et al.  Hybrid reflecting objectives for functional multiphoton microscopy in turbid media. , 2006, Optics letters.

[9]  L. Bonacina,et al.  Multi-Order Investigation of the Nonlinear Susceptibility Tensors of Individual Nanoparticles , 2016, Scientific Reports.

[10]  S. Hell,et al.  Annular aperture two-photon excitation microscopy , 1995 .

[11]  S. Fujiyoshi,et al.  Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures , 2015, Scientific Reports.

[12]  Karl Kilborn,et al.  Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy , 2014, Journal of microscopy.

[13]  Isaac Freund,et al.  Second harmonic generation in collagen , 1979 .

[14]  K. Fujita.  [Two-photon laser scanning fluorescence microscopy]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.