Femtosecond Laser Interactions In The Condensed Phase: Application to Transient Absorption and Materials Processing

Solid State laser systems are now capable of generating temporal pulses of sub-10 fs duration and facilitate the study of ultrafast phenomena in chemical, biological and physical systems. The high field intensities inherent in these pulses, means that highly non-linear processes may be observed. Studies of solution phase transient absorption and femtosecond pulse laser ablation have been undertaken and in each case an experimental apparatus has been developed. Ultrafast pump-probe studies of room temperature solutions of 3-hydroxyflavone and a number of its derivatives have been undertaken. Measurement of transient absorption spectra attributable to the excited tautomer arising from ESIPT was observed throughout the visible wavelength range (400 - 700 nm). In cyclohexanea nd acetonitriles olutions, ESIPT was found to be so rapid that it was only possible to assign a time constant < 35 fs to the process. In ethanol, however, a time constant of 60 fs was determined. The slower ESIPT in this solvent is attributed to the greater strength of the solute-solvent interactions. The influence of substituents in the 4' position of the phenyl ring of 3-hydroxyflavone has also been investigated and we observe unusual behaviour for a cyano substituent in polar solvents and for dimethylamino and aza-Crown substituents. We attribute this behaviour to charge transfer followed by rapid solvent re-organisation. The possibility of the formation of TICT states is also discussed. Ultrafast laser ablation has been studied for a variety of dielectric, semiconducting and metallic substrates with emphasis on the development of the technique for rapid prototyping of micro-fluidic devices. Some fundamental observations of the ablation mechanisms and residual structures is presented along with results for prototyped devices for molecular diffusion and macro-filter applications

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications

NDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription

Myosin VI (MVI) has been found to be overexpressed in ovarian, breast and prostate cancers. Moreover, it has been shown to play a role in regulating cell proliferation and migration, and to interact with RNA Polymerase II (RNAPII). Here, we find that backfolding of MVI regulates its ability to bind DNA and that a putative transcription co-activator NDP52 relieves the auto-inhibition of MVI to enable DNA binding. Additionally, we show that the MVI–NDP52 complex binds RNAPII, which is critical for transcription, and that depletion of NDP52 or MVI reduces steady-state mRNA levels. Lastly, we demonstrate that MVI directly interacts with nuclear receptors to drive expression of target genes, thereby suggesting a link to cell proliferation and migration. Overall, we suggest MVI may function as an auxiliary motor to drive transcription

A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo

Imaging the spatio-temporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using FRET measured by fluorescence lifetime imaging with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo

New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime

We demonstrate an implementation of a centre-of-mass method (CMM) incorporating background subtraction for use in multifocal fluorescence lifetime imaging microscopy to accurately determine fluorescence lifetime in live cell imaging using the Megaframe camera. The inclusion of background subtraction solves one of the major issues associated with centre-of-mass approaches, namely the sensitivity of the algorithm to background signal. The algorithm, which is predominantly implemented in hardware, provides real-time lifetime output and allows the user to effectively condense large amounts of photon data. Instead of requiring the transfer of thousands of photon arrival times, the lifetime is simply represented by one value which allows the system to collect data up to limit of pulse pile-up without any limitations on data transfer rates. In order to evaluate the performance of this new CMM algorithm with existing techniques (i.e. Rapid lifetime determination and Levenburg-Marquardt), we imaged live MCF-7 human breast carcinoma cells transiently transfected with FRET standards. We show that, it offers significant advantages in terms of lifetime accuracy and insensitivity to variability in dark count rate (DCR) between Megaframe camera pixels. Unlike other algorithms no prior knowledge of the expected lifetime is required to perform lifetime determination. The ability of this technique to provide real-time lifetime readout makes it extremely useful for a number of applications

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging