Bleed-through elimination method in a dual-channel fluorescence microscopy system

Reddikumar Maddipatla; Patrice Tankam

doi:10.1117/12.2544412

14 February 2020 Bleed-through elimination method in a dual-channel fluorescence microscopy system

Reddikumar Maddipatla, Patrice Tankam

Proceedings Volume 11244, Multiphoton Microscopy in the Biomedical Sciences XX; 1124420 (2020) https://doi.org/10.1117/12.2544412
Event: SPIE BiOS, 2020, San Francisco, California, United States

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.

Citation Download Citation

Reddikumar Maddipatla and Patrice Tankam "Bleed-through elimination method in a dual-channel fluorescence microscopy system", Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 1124420 (14 February 2020); https://doi.org/10.1117/12.2544412

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available