Abstract
A UV confocal laser microspectrofluorimeter prototype has been adapted for fluorescence lifetime measurements by using a frequency-domain phase/modulation method (modulation frequency 1 to 200 MHz, lifetime resolution: tenth of a ns). This technique enables excited state lifetimes of several fluorescent components to be resolved and determined. Through a global analysis, specific spectral contribution of each species can be monitored with no need to use model spectra. This approach is efficient to distinguish strongly overlapping components (e.g., intracellular multicomponent fluorescence signal) which otherwise cannot easily be discriminated from each other. Experimental set‒up is first described. Application dealing with an antisense oligonucleotide (a synthetic dT15oligomer analogue containing isopolar, non-isosteric, phosphonate [3'-O-P-CH2-O-5'] internucleotide linkages) bound to a fluorescent label (tetramethylrhodamine dye), in various solutions and interacting with living cells, is then presented. This is of a major interest for antisense and/or antigene strategies which have recently been developed as efficient ways to cure viral and/or malignant diseases. The frequency-domain phase/modulation technique enabled oligonucleotide stability inside the cells to be checked.
Highlights
An efficient and universal strategy for curing viral and malignant diseases is widely investigated in the recent years
Application dealing with an antisense oligonucleotide bound to a fluorescent label, in various solutions and interacting with living cells, is presented
This is of a major interest for antisense and/or antigene strategies which have recently been developed as efficient ways to cure viral and/or malignant diseases
Summary
An efficient and universal strategy for curing viral and malignant diseases is widely investigated in the recent years. Novel approaches targeting the very first events of the gene expression, i.e. early enough to prevent cellular enzymatic machinery from starting production of pathogens, are under development In this framework, the use of synthetic oligonucleotides in the regulation of the gene expression by antisense and antigene strategies brings new promising therapeutic opportunities [1,2,3,4,5,6]. A parallel extensive technological advance in the area of electronics and imaging devices enabled a remarkable development of the phase/modulation time-resolved fluorescence spectroscopy to be achieved over the past decade [8] This approach was employed on our confocal laser microspectrofluorometer. Cross-correlation detection was performed by optical multichannel detector hardware modification as described in [9] This technique, used in a multifrequency mode, enables multiple excited state lifetimes of intracellular fluorescent components to be resolved. By means of a global analysis, specific spectral contribution of each species can be determined with no need of model spectra [10]
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