Abstract
Optical imaging is a most useful and widespread technique for the investigation of the structure and function of the cellular genomes. However, an analysis of immensely convoluted and irregularly compacted DNA polymer is highly challenging even by modern super-resolution microscopy approaches. Here we propose fluorescence lifetime imaging (FLIM) for the advancement of studies of genomic structure including DNA compaction, replication as well as monitoring of gene expression. The proposed FLIM assay employs two independent mechanisms for DNA compaction sensing. One mechanism relies on the inverse quadratic relation between the fluorescence lifetimes of fluorescence probes incorporated into DNA and their local refractive index, variable due to DNA compaction density. Another mechanism is based on the Förster resonance energy transfer (FRET) process between the donor and the acceptor fluorophores, both incorporated into DNA. Both these proposed mechanisms were validated in cultured cells. The obtained data unravel a significant difference in compaction of the gene-rich and gene-poor pools of genomic DNA. We show that the gene-rich DNA is loosely compacted compared to the dense DNA domains devoid of active genes.
Highlights
Studies of the cellular molecular structure have traditionally relied on fluorescence microscopy approaches including conventional laser scanning confocal microscopy[1] and multiphoton imaging[2,3]
We propose the development of two independent assays, wherein fluorescence-labeled nucleotides are directly incorporated into the DNA strands during the S-phase of the cell cycle and their lifetimes convey changes in DNA compaction
fluorescence lifetime imaging (FLIM) approaches to measurements of chromatin compaction (I) refractive index (RI) as a function of DNA compaction The electron and light microscopy observations have long identified that the distribution of proteins in genomic chromatin is non-uniform and correlates with the DNA compaction levels, producing densely packed heterochromatin domains with high RI43
Summary
Studies of the cellular molecular structure have traditionally relied on fluorescence microscopy approaches including conventional laser scanning confocal microscopy[1] and multiphoton imaging[2,3]. With the advent of powerful STORM (stochastic optical reconstruction microscopy), PALM (photo-activated localization microscopy), and STED (stimulated emission depletion microscopy) super-resolution imaging techniques[4,5,6], the cellular architecture was visualized in unprecedented details, bringing about a revolution in cell biology research. It is still challenging to study intracellular distribution and interactions of biomolecules due to the limited spatial resolution of fluorescence. An alternative strategy that overcomes existing limitations of conventional approaches for exploring subcellular structure at nanoscale involves fluorescence lifetime imaging (FLIM)[9,10,11,12]. FLIM is a technique that maps the spatial distribution of the fluorophore lifetimes within microscopic images. The most common application of FLIM in biology exploits the Förster resonance energy transfer (FRET)
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