Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells. We applied spectral phasors to isolate intracellular RNA species with similar spectral properties. We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level. Most notable were transcripts in the nucleoli which were spectrally similar to RNA clusters in the cytoplasm. We propose that these species represent ribosomal RNA and clustered ribonucleoprotein complexes. Further, we observed within this cluster Cajal bodies in the proximity of the nucleolus. In addition, transcripts in the cytoplasm undertook a filamentous morphology composed of multiple puncti structures which individually localized along and close to mitochondria but were distinct from mitochondria.

The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

Analysis of the cellular distributions of coenzymes including NADH may aid in understanding a cells metabolic status. We altered serum concentration (0, 2, and 10%) to induce living myoblast cells to undergo the early stages of differentiation. Through microscopy and phasor-FLIM, we spatially mapped and identified variations in the distribution of free and bound NADH. Undifferentiated cells displayed abundant free NADH within the nucleus along with specific regions of more bound NADH. Complete serum starvation dramatically increased the fraction of bound NADH in the nucleus, indicating heightened requirement for transcriptional processes. In comparison, cells exposed to 2% serum exhibited intermediate free nuclear NADH fraction. Overall our results suggest an order of events in which a cell metabolic status alters significantly during the early stages of serum induced differentiation.

Nicotinamide adenine dinucleotide (NADH) is an endogenous fluorescent molecule commonly used as a metabolic biomarker. Fluorescence lifetime imaging microscopy (FLIM) is a method in which the fluorescence decay is measured at each pixel of an image. While the fluorescence spectrum of free and protein-bound NADH is very similar, free and protein-bound NADH display very different decay profiles. Therefore, FLIM can provide a way to distinguish free/bound NADH at the level of single bacteria within biological samples. The phasor technique is a graphical method to analyse the entire image and to produce a histogram of pixels with different decay profile. In this study, NADH fluorescence decay profiles within Lactobacillus acidophilus samples treated using different protocols indicated discernible variations. Clear distinctions between fluorescence decay profiles of NADH in samples of artificially heightened metabolic activity in comparison to those of samples lacking an accessible carbon source were obtained.

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor-fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm.