A high-speed and hardware-only algorithm using a center of mass method has been proposed for single-detector fluorescence lifetime sensing applications. This algorithm is now implemented on a field programmable gate array to provide fast lifetime estimates from a 32 × 32 low dark count 0.13 μm complementary metal-oxide-semiconductor single-photon avalanche diode (SPAD) plus time-to-digital converter array. A simple look-up table is included to enhance the lifetime resolvability range and photon economics, making it comparable to the commonly used least-square method and maximum-likelihood estimation based software. To demonstrate its performance, a widefield microscope was adapted to accommodate the SPAD array and image different test samples. Fluorescence lifetime imaging microscopy on fluorescent beads in Rhodamine 6G at a frame rate of 50 fps is also shown.
A new, simple, high-speed, and hardware-only integration-based fluorescence-lifetime-sensing algorithm using a center-of-mass method (CMM) is proposed to implement lifetime calculations, and its signal-to-noise-ratio based on statistics theory is also deduced. Compared to the commonly used iterative least-squares method or the maximum-likelihood-estimation-based, general purpose fluorescence lifetime imaging microscopy (FLIM) analysis software, the proposed hardware lifetime calculation algorithm with CMM offers direct calculation of fluorescence lifetime based on the collected photon counts and timing information provided by in-pixel circuitry and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. A real-time hardware implementation of this CMM FLIM algorithm suitable for a single-photon avalanche diode array in CMOS imaging technology is now proposed for implementation on field-programmable gate array. The performance of the proposed methods has been tested on Fluorescein, Coumarin 6, and 1,8-anilinonaphthalenesulfonate in water/methanol mixture.
A holographic optical tweezers system is constructed using time-shared multiplexing to generate multiple optical traps. Computer generated Fresnel zone plates are used to produce the required laser focuses for standard optical traps while helical zone plates are used to generate Laguerre-Gaussian (LG) laser modes that produce doughnut shaped focuses. Standard Fresnel zone plates are used for trapping non-biological matter whereas LG modes are used in trapping delicate biological matter that is susceptible to damage from excessive laser radiation. A reflective high speed ferroelectric spatial light modulator (SLM), which is used to display the zone plate images, is capable of multiplexing a maximum of 24 binary images at a refresh rate of up to 1440Hz. By programming the SLM to display one binary hologram per bit plane, a commercial 24 bit colour SLM is used to provide 24 multiplexed traps. The ferroelectric holographic system enables multiple independently movable traps using time-shared multiplexing without the need for mechanical movement within the system. The software developed to display the zone plates uses Open GL acceleration to allow fast smooth movement in real time. Open GL utilises the graphics processing unit (GPU) common on many computers today which greatly increases the frame rate of the images displayed on the SLM. Use of the apparatus is demonstrated by the trapping and manipulation of colloidal particles and yeast cells. Preliminary results indicate that the use of LG zone plates for trapping biological cells results in significantly less damage than standard Fresnel zone plates.
The length and time scales accessible to optical tweezers make them an ideal tool for the examination of colloidal systems. Embedded high-refractive-index tracer particles in an index-matched hard sphere suspension provide 'handles' within the system to investigate the mechanical behaviour. Passive observations of the motion of a single probe particle give information about the linear response behaviour of the system, which can be linked to the macroscopic frequency-dependent viscous and elastic moduli of the suspension. Separate 'dragging' experiments allow observation of a sample's nonlinear response to an applied stress on a particle-by particle basis. Optical force measurements have given new data about the dynamics of phase transitions and particle interactions; an example in this study is the transition from liquid-like to solid-like behaviour, and the emergence of a yield stress and other effects attributable to nearest-neighbour caging effects. The forces needed to break such cages and the frequency of these cage breaking events are investigated in detail for systems close to the glass transition.
Suspensions of motile E. coli bacteria serve as a model system to experimentally study the hydrodynamics of active particle suspensions. Colloidal probe particles are localised within a suspension of motile bacteria by use of optical tweezers and their
uctuations are monitored. The activity of the bacteria effects the
fluctuations of the probe particles and their correlation, revealing information about the hydrodynamics of the suspension. We highlight experimental problems that make the interpretation of 'single probe' experiments (as reported before in literature) diffcult and present some preliminary results for 'dual probe' cross-correlation experiments.
Optical tweezers are proving themselves useful and powerful tools in many areas of biological research, including fungal cell biology. Here we investigate their potential as a novel method for measuring the growth forces of fungal hyphae. We have calibrated the trapping forces produced by our tweezers system, and using beads have investigated whether growing hyphae can overcome this force and push beads out of the trap. Although 4 μm beads were pushed out of the trap by fungal hyphae, using 1 μm beads produced some more intriguing results. The problems encountered with this method of measurement and possible solutions are discussed.
Colloidal systems form the basis of many complex areas of academic and industrial research efforts. As well as contributing to the understanding of many industrially-produced substances such as paints and glues, they have also proved an excellent model thermodynamic system. Changing the properties of an ensemble of colloidal particles, chemically or otherwise, and observing the evolution of the many-body system gives insight into thermodynamic phenomena such as condensation and crystallisation. Some colloidal systems with a particle density that would place them around the dense end of the liquid-crystal coexistence region show a transition into an amorphous glassy state. Long range particle movement is prevented, while local diffusion is still allowed. Such systems are ideal candidates for exploration with optical force measurements, which allow the relatively non-intrusive manipulation of particles deep within colloidal suspensions. Careful use of refractive index matching has allowed an invisible colloidal suspension to be examined with probe particles of a similar size, but higher refractive index. The environment of particles in colloidal cages has been measured by studying the forces acting on a localised particle, as well as the forces needed to break one or more cages.
We describe a new filter that simultaneously achieves spectral filtering and image replication to yield a two-dimensional,
snapshot spectral imager. Filtering is achieved by spectral demultiplexing; that is without rejection of light; so optical throughput efficiency is, in principle, unity. The principle of operation can be considered as a generalisation of the Lyot filter to achieve multiple bandpasses. We report on the design and experimental implementation of an eight-band system for use in the visible. Proof-of-concept demonstrations are reported for imaging of the ocular fundus and microscopy of fluorescently labelled living cells.
Typical optical tweezers setups use high numerical aperture oil-immersion objectives to trap particles suspended in an aqueous medium. When trapping deep inside a sample or out of the imaging plane the quality of the trap in such a system deteriorates due to optical aberrations caused by the refractive index mismatch at the glass-water interface. We investigate this effect experimentally by monitoring the two-photon fluorescence of trapped dye-stained polystyrene spheres. We consider the effect of the numerical aperture on the trap quality and also partly corrected the aberrations by optimising the fluorescence signal using an adaptive deformable membrane mirror.
The stretching and unwinding of polymers under flow is important for understanding the rheological properties of dilute polymer solutions.
Scaling theory based on the blob picture of single polymer chains predicts several regimes for the overall shape of a hydrodynamically deformed macromolecule. We studied the shape of a DNA molecule stretched out by steady uniform flow at different velocities using optical trapping of single DNA molecules (tethered on polystyrene beads) and single molecule fluorescence imaging. The results show
a gradual transition from a non-draining regime at low velocities to
a free-draining regime at high velocities, thus verifying the predictions of the free draining-shell (F-shell) blob model.
We have observed a latency time of the order 10 s in the spermidine-induced condensation of YOYO-1-labelled DNA in flow. Higher flow speeds, longer DNA and higher salt all led to an increase in the latency time. We propose that two effects may be relevant to these observations (1) the competition between spermidine and YOYO-1 for DNA binding sites, and (2) the bending of DNA upon spermidine binding, so that flow-induced straightening of the DNA weakens spermidine's affinity for DNA. Moreover, literature data on the mechanical properties of condensed DNA chains suggest that the hydrodynamic drag forces in our experiments should indeed significantly perturb condensation kinetics.
Laguerre-Gaussian (LG) laser modes (annular shaped modes with helical phase fronts) are used to both manipulate and cut microscopic particles. We use holographically produced LG laser modes to manipulate microscopic bubbles. Interference patterns formed from LG modes of opposite phase helicity are used to create 3D structures and to continuously rotate glass rods. The technique of using and LG beam to create microscopic sections of chromosomes is described.
We demonstrate the use of the angular Doppler effect to obtain continuous motion of interference patterns. A small frequency shift between two beams can create such a moving pattern. By rotating a half wave plate in one arm of an interferometer, frequency shifts in the optical domain from less than 1 Hertz to kHz are achieved. We apply moving interference patterns in an optical tweezers set-up to enable controlled and continuous motion of optically trapped particles and structures.
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