One-step calibration of specimen induced focal shifts and spherical aberration for quantitative 3D microscopy: Approach and first results

D. Thomann, E. Stüssi, G. Danuser

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Scopus citations

Abstract

With the advent of laser scanning confocal and multiphoton microscopy, 3D life tissue characterization has been rendered possible. This involves the restoration of thick section images (in the depth range of 100 microns) of biological samples. In contrast to thin samples new effects become important when imaging thick samples: Because of changes of the refractive index across the specimen or the embedding medium, strong (> 1 micron) focal shifts and spherical aberration occur and scattering effects get more prominent. For tissue mapping it is essential to correct for such aberrations and distortion effects. In this paper, we propose a calibration framework, which allows us to determine thick section focal shifts and spherical aberrations in a one-step-procedure. Gradients in the focal shift induce a scaling in the Z-direction of the observed sample. A second effect arises with depth-dependent spherical aberration. We model our microscope as a linear-shift-NON-invariant system (LSNI) where multiple depth classes are assigned distinct point spread functions (PSFs). We measure these two effects in a 3D sample of randomly distributed fluorescent focal check beads. The beads are embedded in gelatine, a medium with high resemblance to real biological tissue. The PSF is approximated by a mathematical parametric model. While estimating the parameters of the PSF with object-constrained deconvolution, we track depth dependent changes of the observed bead diameter. This allows us to determine the gradient of the focal shift across a thick section. By numerical integration along the optical axis we obtain the focal shift values as a function of sample depth. In the end, these values will be applied as a correction to compensate depth distortions in the tissue images.

Original languageEnglish (US)
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsJ. Conchello, C.J. Cogswell, T. Wilson
Pages69-80
Number of pages12
Volume4261
DOIs
StatePublished - 2001
EventThree-Dimensional and Multidimensional Microscopy: Image Asquisition and Processin VIII - San Jose, CA, United States
Duration: Jan 21 2001Jan 22 2001

Other

OtherThree-Dimensional and Multidimensional Microscopy: Image Asquisition and Processin VIII
CountryUnited States
CitySan Jose, CA
Period1/21/011/22/01

Keywords

  • Calibration
  • Confocal Microscopy
  • Deconvolution
  • Focal Shifts
  • Spherical Aberration

ASJC Scopus subject areas

  • Electrical and Electronic Engineering
  • Condensed Matter Physics

Fingerprint Dive into the research topics of 'One-step calibration of specimen induced focal shifts and spherical aberration for quantitative 3D microscopy: Approach and first results'. Together they form a unique fingerprint.

  • Cite this

    Thomann, D., Stüssi, E., & Danuser, G. (2001). One-step calibration of specimen induced focal shifts and spherical aberration for quantitative 3D microscopy: Approach and first results. In J. Conchello, C. J. Cogswell, & T. Wilson (Eds.), Proceedings of SPIE - The International Society for Optical Engineering (Vol. 4261, pp. 69-80) https://doi.org/10.1117/12.424521