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Proceedings Paper

Bleed-through elimination method in a dual-channel fluorescence microscopy system
Author(s): Reddikumar Maddipatla; Patrice Tankam
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Paper Abstract

Bleed-through is a common problem in multi-channel fluorescence microscopy when simultaneously imaging multiple organelles tagged with different fluorophores. This is majorly due to the large emission spectra of fluorophores. The correction of bleed-through in a multi-channel fluorescence microscopy system is essential to accurately identify or track the location of multiple fluorophores simultaneously. This paper presents a method for eliminating the bleed-through in a dual-channel fluorescence microscopy system using highly sensitive photomultiplier tubes. Fluorescein and Alexa fluor 594 dyes that were diluted in phosphate buffer solution at different dilution ratios were used to establish the relationship between the intensities of both channels. The bleed-through intensity in the red channel was quantified using a nonlinear polynomial model. Bleed-through correction was performed using the derived polynomial coefficients and the detected intensity in the green channel. The approach was experimentally validated on a mixed solution of fluorescein and Alexa fluor 594 using the scanning mode dual-channel fluorescence microscopy system.

Paper Details

Date Published: 14 February 2020
PDF: 5 pages
Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 1124420 (14 February 2020); doi: 10.1117/12.2544412
Show Author Affiliations
Reddikumar Maddipatla, Indiana Univ. (United States)
Patrice Tankam, Indiana Univ. (United States)

Published in SPIE Proceedings Vol. 11244:
Multiphoton Microscopy in the Biomedical Sciences XX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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