Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microsco...Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field. In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions, the increase of [Ca2+]i was still observable. It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.展开更多
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 i...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.展开更多
文摘Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field. In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions, the increase of [Ca2+]i was still observable. It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.
基金This work has been partially supported by the National Natural Science Foundation of China(61620106016/61835009/31771584)Shenzhen International Cooperation Project(GJHZ20180928161811821/GJHZ20190822095420249).
文摘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.
