DAPI-fluorescent fading: a problem in microscopy or a way to measure nuclear DNA content?

Cristian Gallardo-Escárate; Josué Álvarez-Borrego; V. Kober; Miguel Ángel del Río-Portilla

doi:10.1117/12.667178

2 February 2006 DAPI-fluorescent fading: a problem in microscopy or a way to measure nuclear DNA content?

Cristian Gallardo-Escárate, Josué Álvarez-Borrego, V. Kober, Miguel Ángel del Río-Portilla

Proceedings Volume 6026, ICO20: Biomedical Optics; 60260T (2006) https://doi.org/10.1117/12.667178
Event: ICO20:Optical Devices and Instruments, 2005, Changchun, China

Abstract

In observation by confocal or conventional fluorescence microscopy, the retardation of the lost in fluorescence, from highest signal of fluorescence to lowest intensity are important factors in order to obtain accurate images. This problem is very common in fluorochromes for nuclear DNA and especially for DAPI stain. The fluorescence of DAPI is rapidly lost when it is exposure to excitation by ultra violet (UV) light, and especially under optimal condition of observation. Although the fading process could be retardate by using of mounting medium with antifading solutions, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addiction, neither relationship has been tested between the fluorescence fading and nuclear DNA content. However, the capacity of the DNA to absorb UV light is knows. In order to test this relationship we measured by means of image analysis the fluorescence intensity in several nuclei types during a fading period. The analysis was performed by an algorithm specifically built in MATLAB software. The relationship between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility for estimates genome size by quantification of fluorescence fading. In this context, the present method allows to measure nuclear DNA content in several medical applications (cancer, HIV, organ transplants, etc). Nowadays, for measuring DNA content, flow cytometry is widely used; however, with the flow cytometry method it is not possible to select a specific group of cells, such as from a specific region of a tumor. Moreover, the using of image analysis allows automatizing diagnostics procedures.

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Cristian Gallardo-Escárate, Josué Álvarez-Borrego, V. Kober, and Miguel Ángel del Río-Portilla "DAPI-fluorescent fading: a problem in microscopy or a way to measure nuclear DNA content?", Proc. SPIE 6026, ICO20: Biomedical Optics, 60260T (2 February 2006); https://doi.org/10.1117/12.667178

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KEYWORDS

Luminescence

Image analysis

Flow cytometry

Microscopy

Confocal microscopy

Ultraviolet radiation

Image processing

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