Fluorescent Bleed-Through

The first FRET-based biosensors employing fluorescent proteins were developed over 10 years ago. These protease sensors consisted of a BFP donor fused to a GFP acceptor by a protease-sensitive linker [44, 119]. BFP and GFP have well separated emission spectra, resulting in little fluorescence bleed-through (Figs. 5.5A and 5.6A). This facilitates data analysis for FRET ratio imaging... 

FRET applications employing CFP and YFP are complicated due to considerable bleed-through between CFP and YFP fluorescence (Figs. 5.5B and 5.6B). Direct excitation of YFP and bleed-through of CFP fluorescence into the YFP detection channel have to be corrected for as shown in Chapters 7 and 8. The multiexponential fluorescence decay of all CFP variants complicates the quantification of FRET by donor lifetime methods. Altogether these factors make quantitative analysis of the FRET efficiency relatively difficult. 

Before using a fluorophore-labeled antibody from flow cytometry for immunocytochemistry, check the spectra of fluorescent labels to rule out the possibility of bleed-through. Remember that although flow cytometry and immunocytochemistry use fluorescent dyes, the characteristics of these dyes are optimized for the technique and might not translate well when moved from flow cytometry to immunocytochemistry. 

Fig. 13.4 Fluorescent bleed-through. A problem in fluorescent microscopy occurs when the filters cannot separate emission wavelengths, for example, when an emission from 488 fluorophore is detected in the 555 channel and in the 555 emission. The emission spectrum for the 488 fluorophore (black dashed line) is shown with a tail that extends into the 555 emission filter (gray solid line). If the intensity of the 555 fluorophore emission is low as seen by the maximum height at 40% and the 488 fluorophore has high-intensity emission, then the amount of overlap in emission can be significant (dark gray area). To compound the problem, if the detection system gain is increased in the red 555 emission, then the 488 fluorophore intensity is about 50% of the light as from the 555 fluorophore intensity.

Fluorescent filter set did not match the fluorophore - The filters and lasers were correct. The potential for bleed-through from the lower 488 nm channel into the higher 546 nm channel was checked, but the 546 nm fluorescence did not bleed through from the 488 nm laser. 

Penetrant Method. This method does not depend upon radiation interactions with the lest object and is essentially noninstrumental. A special penetrant substance is applied freely on the test object and allowed to work into tight cracks. See Fig. 3. The penetrant is removed from all surface areas and the piece is sprayed with a developer. The developer dries to an even white coating, while the penetrant bleeds up from any flaws through the developer, forming bright-red or fluorescent indications on the white surface. The size of the defect is indicated by the richness of color, speed of bleed-out, and dimensions ohserved. 

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