We describe the effects created by selective oxidation of high aluminium content AlGaAs layers at the facets of 5-stack
quantum dot edge-emitting 50μm stripe lasers. The steam oxidation affects only the facet areas of the devices, where
unpumped sections are created. These unpumped regions alone enable reduction of the width of the lasing near-field
spatial profile of up to 65% and the reduction of threshold in long devices by up to 30%. These effects are attributed to
saturable absorber-type behaviour, where the absorber saturates first at the location of highest optical intensity, so
allowing lasing over a smaller spatial area. Secondly, a combination of self-heating at the facets and the saturable
absorption generates novel saw-toothed wavelength-time profiles. A model for the behaviour behind all of these results
is proposed and backed up with experimental data.
The development of an experimental setup capable of contrasting fluorescent materials by their recombinative lifetimes
in an imaging mode is discussed. Such materials might include molecular dyes and QDs. The system is comprised of a
standard upright microscope fitted with an imaging CCD, and a white light laser that illuminates a circular region within
the field of view with variable period excitation pulse trains. Different fluorescent species within this region absorb the
laser light and fluoresce with a recombination lifetime dependent on material composition and local environment.
Species with differing fluorescent lifetimes can be distinguished in an imaging mode by their contrasting intensity
response to the pulse train at the range of different pulse frequencies. The technique is discussed and applied to samples
containing both CdTe (705 nm) and CdSe (611 nm) QDs, showing contrast between long (70-100 ns) and (relatively)
short (25-35 ns) lifetime within an image.
KEYWORDS: Signal detection, Imaging systems, Luminescence, Light emitting diodes, Optical fibers, In vitro testing, Systems modeling, In vivo imaging, Biomedical optics, Scanning electron microscopy
The technical objective of this study has been to design, build and validate biocompatible hollow fiber implants based on
fluorescence with integrated biophotonics components to enable in fiber kinetic cell based assays. A human
osteosarcoma in vitro cell model fiber system has been established with validation studies to determine in fiber cell
growth, cell cycle analysis and organization in normal and drug treated conditions. The rationale for implant
development have focused on developing benchmark concepts in standard monolayer tissue culture followed by the
development of in vitro hollow fiber designs; encompassing imaging with and without integrated biophotonics.
Furthermore the effect of introducing targetable biosensors into the encapsulated tumor implant such as quantum dots for
informing new detection readouts and possible implant designs have been evaluated. A preliminary micro/macro
imaging approach has been undertaken, that could provide a mean to track distinct morphological changes in cells
growing in a 3D matrix within the fiber which affect the light scattering properties of the implant. Parallel engineering
studies have showed the influence of the optical properties of the fiber polymer wall in all imaging modes. Taken all
together, we show the basic foundation and the opportunities for multi-modal imaging within an in vitro implant format.
In this paper, we will discuss the necessary properties of the light source needed for high resolution and high penetration
OCT. We examine the performance of a self pulsating edge emitting quantum dot laser diode light source emitting at
~1050nm and configured as a split contact device with separate gain and absorber sections. The device can be configured
to operate with a reverse biased saturable absorber section or with the whole device forward biased into gain. With the
device operated without a saturable absorber section the laser emits a number of discrete narrow modes, which merge to
form a broad continuous lasing spectrum (width up to 10nm) on application of the saturable absorber. In the time domain
we observe continuous emission that becomes self pulsation, with pulse widths of 200-300ps and frequency of 0.6 -
1.5GHz depending on drive current and reverse bias, on application of the saturable absorber.
To produce a large increase in total throughput, a multi-stage microfluidics system (US Patent pending) is being
developed for flow cytometry and closed system cell sorting. The multi-stage system provides for sorting and re-sorting
of cohorts of cells beginning with multiple cells per sorting unit in the initial stages of the microfluidic device and
achieving single cell sorting at subsequent stages. This design theoretically promises increases of 2- or 3-orders of
magnitude in total cell throughput needed for cytomics applications involving gene chip or proteomics analyses of sorted
cell subpopulations.
Briefly, silicon wafers and CAD software were used with SU-8 soft photolithography techniques and used as a mold
to create Y-shaped, multi-stage microfluidic PDMS chips. PDMS microfluidic chips were fabricated and tested using
fluorescent microspheres driven through the chip by a microprocessor-controlled syringe drive and excited on an
inverted Nikon fluorescence microscope. Inter-particle spacings were measured and used as experimental data for
queuing theory models of multi-stage system performance.
A miniaturized electronics system is being developed for a small portable instrument. A variety of LED light sources,
waveguides, and APD detectors are being tested to find optimal combinations for creating an LED-APD configuration at
the entry points of the Y-junctions for the multi-stage optical PDMS microfluidic chips. The LEDs, APDs, and PDMS
chips are being combined into an inexpensive, small portable, closed system sorter suitable for operation inside a
standard biohazard hood for both sterility and closed system cell sorting as an alternative to large, expensive, and
conventional droplet-based cell sorters.
We have used flow-cytometry together with computational modeling of quantum dot portioning during cell division to
identify population distributions of proliferating cells. The objective has been to develop a robust assay of integrated
cellular fluorescence which reports the extent of cellular bifurcation within a complex population and potentially
provides profiles of drug resistance, cell clonality and levels of aneuploidy in tumour cells. The implementation of a data
analysis program based on genetic algorithms provides a complete description of the proliferation dynamics and gives
values for the inter-mitotic time, the partitioning ratio of quantum dots between daughter cells and their associated
deviations.
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
biochips.
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.
We have calculated recombination rates of an inhomogeneous ensemble of 106 dots by summing localized
recombination rates at individual dots, with occupation of dot states in the inhomogeneous distribution specified by
Fermi Dirac statistics. We assign the same single dot recombination lifetime (1 ns) to all recombination processes to
reveal the effect of localization on the overall rates. For the simplest system of the ground states alone deep state,
radiative and Auger recombination processes depend in a similar manner upon the population of electrons in the ground
states Consequently the light-current curves for the ground state are approximately linear and are not sensitive to the
dominant non-radiative process. When excited states are included Auger recombination becomes dominant at high
ensemble populations due to the higher degeneracy assigned to the excited states. While the form of the light-current
curves of the total dot system do depend upon the dominant recombination process, an analysis based on power law
relations with respect to the ensemble electron population are not appropriate.
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.
We have calculated the recombination rates of electrons and holes in a quantum dot ensemble. The calculation treats the various recombination mechanisms as localized events at the dot site and uses global population statistics, re-interpreted to provide integer occupancies of the ground and excited states of the dots. The results show that the localization means that unambiguous functional forms describing the recombination in terms of charge carrier density can no longer be determined. The evolution of the recombination rates with increasing injection is complex and the different radiative and non-radiative routes have a similar dependence. Experimentally this means that studies of the light output versus current can no longer be used to assess the dominant recombination process.
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.
This paper describes the properties of vertical-cavity semiconductor devices designed to emit light when driven in forward bias mode and detect optical radiation at wavelengths longer than that of emission when driven in reverse bias mode. The study of this type of devices is motivated by the miniaturization and integration into a single unit of the three functions that a microfluorimeter has to perform, optical pumping, optical detection, and optical filtering of weak light sources. The devices produced can generate fluorescence with a low output power since their emission wavelength can be tuned with that of maximum absorption of the fluorescent dye. We demonstrate also that they can detect low power fluorescence generated in a small volume of concentrated solution of a commercial dye. These devices can find useful application in microanalytical systems such as microfluidic devices or optical biochips.
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.
We present a novel vertical-cavity semiconductor device capable of generating in forward bias optical radiation to pump fluorescent labeling dyes and detecting their fluorescence emission when operated in reverse bias mode. The integration of a partially coherent light source and a sensitive detector within the same semiconductor wafer is a further step toward the realization of optical biochips for DNA analysis and cytometry. The structure and the criteria chosen to design these devices, their emission and detection properties are presented and discussed in detail.
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.
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 validate a microscopic laser model based on the quantum kinetic equations using experimentally determined threshold current, gain, spontaneous emission and quasi-Fermi level separation data taken on GaInP/AlGaInP lasers. By comparison of further experimental and calculated optical properties we find that there is a significant contribution to the threshold current from non-radiative recombination within the quantum wells.
The electroluminescence efficiency of InGaN LEDs is surprisingly high for structures which have high defect concentrations due to growth on mismatched substrates. We have measured the high-injection non-radiative lifetime in InGaN LEDs by analysis of the light current characteristics. We find that the values of (tau) nr decrease from 18ns at 200K to 5ns at 400K. This behavior is thermally activated with an activation energy of 40 meV which is compatible with the hypothesis that the temperature dependence is due to thermal delocalization of carriers form potential minima caused by modest fluctuations in In composition in the quantum well. We determine the internal quantum efficiency to lie between 52 percent and 65 percent at room temperature over the current range employed.
We describe a single-pass technique for the independent measurement of optical modal gain and internal mode loss in semiconductor lasers structures using a single, multi-section device which gives the loss and the gain spectrum in absolute units and over a wide current range. Comparison of the transverse electric and transverse magnetic polarized gain spectra also identifies the transparency point, provides the quasi-Fermi level energy separation and a second means for determination of the mode loss. Measurements are described for AlGaInP quantum well laser structures with emission wavelengths close to 670 nm, yielding an internal loss of 10 cm-1 and peak gain values up to 4000 cm-1 for current densities up to 4 kAcm-2. We have also made an independent measurement of the spontaneous emission spectrum through a top-contact window on the same device structure and have converted this to local gain using the usual thermodynamical relationship. By this means we have been able to confirm the validity of this relation between gain and emission for excited semiconductor structures of this type.
Spontaneous emission control has been achieved in GaAs/AlGaAs quantum well lasers by the use of Distributed Bragg microcavity perpendicular to the quantum wells. The room temperature emission is inhibited whilst below 130 K there is an enhancement. These changes to the spontaneous emission process directly effect the threshold current producing a 25% reduction at room temperature. Theoretical modeling of the lasers is in agreement with the experimental results and highlights the effect of the microcavity on the field-dipole interaction.
There is increasing interest in the use of visible emitting (formula available in paper) quantum well lasers for optical interconnections using polymer waveguides and this calls for the optimization of device structure for operation at a specific wavelength and usually at an elevated temperature. We concentrate on the mechanisms by which compressive strain modifies the threshold current in a regime where well composition (x) (strain) and quantum well width are adjusted to maintain a transition wavelength of 670 nm. In our model we assume a parabolic band structure, which is a reasonable approximation in this case since strain enhanced splitting of the valence bands is large, and we include the effects of monolayer fluctuations in well width and carrier-carrier scattering (where we calculate an energy and carrier density dependent lifetime). Using our model we examine the relative merits of various well composition (x)/well width combinations.
A series of strained AlGaInP quantum well lasers has been fabricated with GaxIn1-xP wells of a fixed width, Lz equals 65 angstrom, in which x was varied to give both compressive (x < 0.51) and tensile (x > 0.51) strain. By cooling these lasers to temperatures approximately 130 K we have been able to isolate the intrinsic effects of the strain from the extrinsic changes due to the quantum well structure and we see reductions in threshold current as compressive and tensile strain is applied.
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