Correction methods for fluorescence quantitation in thick specimens by confocal microscopy

Ingjerd Lien; Toril A. Nagelhus; Stig M. Knutsen; Leif M. Sande; Tore Lindmo

doi:10.1117/12.239504

10 May 1996 Correction methods for fluorescence quantitation in thick specimens by confocal microscopy

Ingjerd Lien, Toril A. Nagelhus, Stig M. Knutsen, Leif M. Sande, Tore Lindmo

Proceedings Volume 2678, Optical Diagnostics of Living Cells and Biofluids; (1996) https://doi.org/10.1117/12.239504
Event: Photonics West '96, 1996, San Jose, CA, United States

Abstract

The problems of fluorescence bleaching and fluorescence attenuation in thick specimens need to be solved for quantitative fluorescence imaging of thick specimens by confocal microscopy. Methods for fluorescence bleaching correction based on data collected from the same specimen as the one used for quantitative investigation, have been devised (Nagelhus et al. Cytometry, In press). The present work describes model systems for the estimation of degradation effects such as attenuation caused by scattering and absorption and decreasing resolution with increasing depth in thick specimens. Monodisperse, fluorescently stained particles with fluorescence spectrally separate from the tissue fluorescence to be investigated, were placed on top of and underneath the tissue specimen to be studied. From these particles imaging parameters such as the lateral and axial point spread function and attenuation coefficients for excitation and emission light, were estimated. By recording the particle fluorescence and specific tissue fluorescence on separate detectors, correction parameters may be estimated for localized areas throughout the specimen. If, based on detailed investigation, the change in imaging parameters with increasing imaging depth in the tissue can be modeled, correction factors for each depth and lateral location can be estimated from recordings of particle fluorescence from the top and bottom of the biological specimen. We have investigated such problems by means of 0.2 μm and 6 μm fluorescent particles imaged through frozen sections of various thicknesses of tumor tissue grown in athymic nude mice, using a Bio-Rad MRC-600 confocal microscope. Together with correction factors for bleaching of the specific tissue fluorescence, we hope to use the estimated imaging parameters to correct for image deterioration with imaging depth, and thus obtain quantitative 3D data on fluorescence intensity throughout thick specimens.

Citation Download Citation

Ingjerd Lien, Toril A. Nagelhus, Stig M. Knutsen, Leif M. Sande, and Tore Lindmo "Correction methods for fluorescence quantitation in thick specimens by confocal microscopy", Proc. SPIE 2678, Optical Diagnostics of Living Cells and Biofluids, (10 May 1996); https://doi.org/10.1117/12.239504

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