Photoswitchable and photoactivable proteins Dronpa and PhotoActivable mCherry (PA-mCherry) respectively, were used in order to perform FRET (Förster Resonance Energy Transfer) imaging at the single molecule level, using a FRET standard construct consisting of Dronpa and PA-mCherry separated by seven amino acids expressed in cells. Given Dronpa’s complex photophysical properties and the existence of a preswitched emissive state, irradiation conditions at 491 and 405 nm were optimised. We discuss strategies for observing FRET at the single molecule level with photoactivatable proteins by monitoring modifications in the donor and acceptors emissive states.
Cancer studies require a thorough understanding of how human gene expressions and DNA modifications are translated
at the proteome level. In order to unravel the large and complex interactions between proteins, we have developed a
compact lifetime-based flow cytometer, utilising a commercial microfluidic chip, to screen large non-adherent cell
populations. Fluorescent signals from cells are detected using time correlated single photon counting (TCSPC) in the
burst integrated fluorescence lifetime (BIFL) mode and used to determine the lifetime of each cell. Initially, the system
was tested using 10 μm highly fluorescent beads to determine optical throughput and detection efficiency. The system
was validated with 293T monkey kidney adenocarcinoma cell line transiently transfected with a FRET standard,
consisting of eGPF and mRFP1 fluorescent proteins linked by a19 amino-acid chain. Analysis software was developed to
process detected signals in BIFL mode and chronologically save the transient burst data for each cell in a multi-dimensional
Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for
light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying
multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen
fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex
in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions
also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to
generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for
establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction
with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo.
For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we
show the application of this method for intravital imaging of direct protein-protein interactions.
An automated high-content screening microscope has been developed which uses fluorescence anisotropy imaging and fluorescence lifetime microscopy to identify Förster resonant energy transfer between eGFP and mRPF1 in drug screening assays. A wide-field polarization resolved imager is used to simultaneously capture the parallel and perpendicular components of both eGFP and mRFP1 fluorescence emission to provide a high-speed measurement of acceptor depolarization. Donor excited state lifetime measurements performed using laser scanning microscopy is then used to determine the FRET efficiency in a particular assay. A proof-of-principle assay is performed using mutant Jurkat human T-cells to illustrate the process by which FRET is first identified and then quantified by our high-content screening system.
The development of surface-active biochips for control of fluorescence within microscopy platforms is described. These
use surface-plasmon control to provide selective excitation of fluorescently labeled, live cell populations. These chips
effectively combine a number of commonly used techniques such as SPR, TIRF and epi-fluorescence within a single device and have the potential to provide sub-cellular discrimination of excitation in 3-D. Thus within a single field of view we can selectively excite membrane versus cytoplasm and localise the excitation within the lateral plane to an area of a few square microns.
Optical biochips may incorporate both optical and microfluidic components as well as integrated light emitting
semiconductor devices. They make use of a wide range of materials including polymers, glasses and thin metal films
which are particularly suitable if low cost devices are envisaged. Precision laser micromachining is an ideal flexible
manufacturing technique for such materials with the ability to fabricate structures to sub-micron resolutions and a
proven track record in manufacturing scale up.
Described here is the manufacture of a range of optical biochip devices and components using laser micromachining
techniques. The devices employ both microfluidics and electrokinetic processes for biological cell manipulation and
characterization. Excimer laser micromachining has been used to create complex microelectrode arrays and microfluidic
channels. Excimer lasers have also been employed to create on-chip optical components such as microlenses and
waveguides to allow integrated vertical and edge emitting LEDs and lasers to deliver light to analysis sites within the
Ultra short pulse lasers have been used to structure wafer level semiconductor light emitting devices. Both surface
patterning and bulk machining of these active wafers while maintaining functionality has been demonstrated. Described
here is the use of combinations of ultra short pulse and excimer lasers for the fabrication of structures to provide ring
illumination of in-wafer reaction chambers.
The laser micromachining processes employed in this work require minimal post-processing and so make them ideally
suited to all stages of optical biochip production from development through to small and large volume production.
An optical biochip is being developed for monitoring the sensitivity of biological cells to a range of environmental
changes. Such changes may include external factors such as temperature but can include changes within the suspending
media of the cell. The ability to measure such sensitivity has a broad application base including environmental
monitoring, toxicity evaluation and drug discovery. The device under development, capable of operating with both
suspension and adherent cell populations, employs electrokinetic processes to monitor subtle changes in the physicochemical
properties of cells as environmental parameters are varied. As such, the device is required to maintain cells in
a viable condition for extended periods of time.
The final device will employ integrated optical illumination of cells using red emitting LED or laser devices with light
delivery to measurement regions achieved using integrated micro-optical components. Measurements of electrokinetic
phenomena such as dielectrophoresis and electrorotation will be achieved through integrated optical detectors.
Environmental parameters can be varied while cells are actively retained within a measurement structure. This enables
the properties and sensitivity of a cell population to be temporally tracked.
The optical biochip described here uses a combination of microfabrication techniques including photolithographic and
laser micromachining processes. Here we describe the design and manufacturing processes to create the components of
the environmental monitoring strutures of the optical biochip.
We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices
encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport
and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a
incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation
wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated
microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell
proliferation status in living cells.
Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring
longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays.
QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a
range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional
organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the
added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing
their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing
labelling of division events, whereby over time the QD signal becomes diluted with each generation.
Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications
in drug screening and optical biochip environments.
We demonstrate complete integration of a fluorescence-based assay in that the analyte well is also an optical emitter.
Laser machining is used to create 'active micro-wells' within semiconductor light emitting diode and laser structures.
These are then used to optically excite fluorescently-labelled beads in solution within the well. The results show
efficient illumination on a par with traditional lamp-based excitation. This technology therefore provides active microwell
plates with completely localized excitation, confined to the analysis well, that can be engineered via the micro-well
geometry. The micro-wells have also been machined within the cavity of lasing semiconductor structures and coherent
emission maintained. Thus lasing multi-well plates are also realizable.
We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence
intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent
species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength
corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye
(Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in
contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the
excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical
modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly
contrasting regions of fluorescence excitation providing regions of high spatial selectivity.
Colloidal quantum dots (QDs) are now commercially available in a bio-functionalized form and Förster resonance
energy transfer (FRET) between bioconjugated dots and fluorophores within the visible range has been observed by
several groups of researchers. We are particularly interested in the far-red region, as from a biological perspective, there
are benefits in pushing to ~700 nm to minimize optical absorption (ABS) within tissue and avoiding cell
autofluorescence. We report on FRET between streptavidin (STV) conjugated CdTe quantum dots, Qdot705-STV, with
biotinylated Dy731-Bio fluorescent molecules in a donor-acceptor assay. We also highlight an unusual change in
Dy731-Bio absorptivity during the streptavidin-biotin binding process that can be attributed to the structural
reorientation. In moving to wavelengths beyond 700 nm, different alloy compositions are required for the quantum dot
core and these introduce associated changes in the physical shape. These changes directly affect the fluorescence decay
dynamics producing a marked biexponential decay with an extremely long lifetime component, a lifetime in excess of
100 ns. We compare and contrast the influence of the two QD relaxation processes upon the FRET dynamics in the
presence of Dy731-Bio.
In this paper we report on the development of an optical biochip to control both the excitation and resultant fluorescence
using grating coupled surface plasmons. Electron beam lithography is used to fabricate line gratings in thin layers of gold
on the surface of 150μm thick coverslips. Laser diodes operating at 630nm are close coupled to the coverslip resulting in
the excitation of surface plasmons. In the region of the grating light can radiate into the far-field, and both the angle of
emission and beam divergence can be controlled by the grating pitch and the number of lines included in the pattern. A
model is presented which treats the grating as an optical antenna array which shows how these characteristics can be
explained in terms of the wave vector matching between the surface plasmons and the grating. Fluorescence has also
been excited in standard organic dyes on-chip. When placed in close proximity to the surface of the sample strong
quenching of the fluorescence is seen in the region of the grating. In contrast an enhancement of the signal is seen when
the fluorophores are placed on a 200nm thick spacer layer.
In situ spectral analysis can be used to understand the targeting and interaction of agents in cellular compartments. A range of novel red excitable fluorescent probes, related to the anthraquinone family of anti-cancer agents, were designed for their DNA affinic properties and their ability to enter and penetrate living cells. We report on the spectral features of these probes, both in solution and bound within intact cells, to identify unique fluorescent signatures that exploit their use in bioassays on optical biochip devices.
The probes demonstrated red shifted emission spectra and increased 2 photon lifetime, with minimal fluorescent enhancement, upon binding to DNA. Spectral confocal laser scanning microscopy revealed complex emission profiles representing the bound (nuclear) and unbound (cytoplasmic) fractions of the DNA probes within live interphase, mitotic and apoptotic cells.
Analysis of the emission peaks encoded the spectra to provide cell compartment recognition and profiles for cells in different cell states. Sampling the entire emission spectra of these probes for cell locating, even in the presence of unbound molecules, provides good signal-to-noise in biochip devices. Furthermore, by sampling the fluorescence output at specific spectral windows we can obtain high spatial information without imaging.
The technological challenge is to integrate these fluorophores and appropriate detection capacity onto an optical biochip platform with microfluidic systems for cell handling.
We report on the development of a simple technique for obtaining time-domain information using dc detection of fluorescence. We show that this is feasible for assays where a change in lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer for example, and could be particularly useful for assays performed in the scaled-down environment of a "lab-on-a-chip". A rate equation model is presented which allows an objective analysis of the relative importance of the key measurement parameters: optical saturation of the fluorophore and excitation pulse characteristics. We present a comparison of the model with a cuvette based analysis of a carbocyanine dye where the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode.
We report on the development of a stroboscopic excitation technique using a self-pulsing laser, and show that it is a feasible method for obtaining fluorescence lifetime information from a biochip format. The self-pulsing lasers described here are versatile devices which have been used for one photon excitation measurements to determine the lifetime of cyanine 5 in water and ethanol. The same devices have been used to develop a technique whereby the emphasis for time-resolution of a lifetime measurement can be transferred to the excitation source from the detector and processing electronics by virtue of the multiple-pulse, variable frequency nature of the laser output.
A full evaluation of the performance of InGaAs quantum dots as saturable absorbers in multi-contact
lasers emitting at a wavelength of 1μm has been carried out. The light-current curves of the two-section
quantum dot laser have been measured at 300K with varying levels of reverse bias applied to the
absorber section and are compared with a quantum well control sample. This measurement indicates
that the quantum confined stark effect (QCSE) is very different for the quantum dots, and this is
confirmed by measurements of differential loss spectra as a function of reverse bias. Up to voltages of -
6V there is no shift in the absorption edge of the quantum dots showing that the QCSE is weak for this
0D system. Dynamic measurements show that self-pulsation in these lasers is highly temperature
dependent, and completely ceases below 150K. We have also measured the absorber recovery time,
which is found to increase from 40ps at 300K to 600ps at 50K, demonstrating that a high loss condition
cannot be achieved quickly enough at low temperature for self-pulsation to occur.
We examine the mechanisms that lead to a low value of saturated modal gain in both 1μm emitting InGaAs based and ≈ 700nm emitting InP/GaInP quantum dot laser systems. We explain the observation that the value of the saturated modal gain increases as the temperature decreases using a simple model of the filling of the available dot and wetting layer states according to a Fermi-Dirac distribution. We show that it is the relatively large number of available wetting layer valence states and their proximity in energy to the dot states that limits the modal gain. We measure the population inversion factor for samples containing different numbers of layers of dots and for samples where the dots are grown in a quantum well (DWELL) and for dots grown in bulk layers of either GaAs or Al0.15Ga0.85As (non-DWELL). Comparison of this data with that calculated for a Fermi-Dirac distribution of carriers in the available states demonstrates that for most of the samples the carriers in the ground states of the quantum dots are not in thermal equilibrium with those in higher lying energy states - the excited states or wetting layer.