Search

Summary Background Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates. Objectives To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin. Methods Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser. Results Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated (P

A novel fiber-optic-based method for simultaneous time- and wavelength-resolved fluorescence spectroscopy for the rapid diagnosis of diseased tissue is demonstrated. By combining multiple bandpass and dichroic filters (405/40, 460/50, and 550/50) with different lengths of optical fiber (1, 10, and 19 m) acting as an optical delay this system enables the near real-time acquisition and characterization of time-resolved fluorescence spectra using a single detector and excitation input. The recording of multiple fluorescence response pulses at selected wavelengths can be completed in hundreds of nanoseconds, which provides the capability of a real-time characterization of biological systems.

This paper presents an optical system for performing excitation resolved imaging of fluorescent dyes, tissue phantoms and ex vivo tissues. The excitation source was a supercontinuum generated in a highly nonlinear fibre, spectrally filtered using dispersive optics and a movable slit or digital micromirror device. This allowed excitation with multiple spectra, which may be chosen to optimize the fluorescence yield from a particular fluorophore, or to maximize discrimination between multiple labels. As an initial validation, the analysis of fluorescent dye solutions embedded in collagen gels in the presence of scatterers and absorbers by using fixed reference isomap (FR-IsoMap) is presented, followed by initial evaluation with human lung bronchial tissue. The proposed system has potential applications for in vivo fluorescence endoscopy of cancerous tissues through excitation spectral selection and analysis with FR-IsoMap.

Recent rapid developments in multi-modal optical imaging have created a significant clinical demand for its in vivo - in situapplication. This offers the potential for real-time tissue characterization, functional assessment, and intra-operative guidance. One of the key requirements for in vivoconsideration is to minimise the acquisition window to avoid tissue motion and deformation, whilst making the best use of the available photons to account for correlation or redundancy between different dimensions. The purpose of this paper is to propose a feature selection framework to identify the best combination of features for discriminating between different tissue classes such that redundant or irrelevant information can be avoided during data acquisition. The method is based on a Bayesian framework for feature selection by using the receiver operating characteristic curves to determine the most pertinent data to capture. This represents a general technique that can be applied to different multi-modal imaging modalities and initial results derived from phantom and ex vivotissue experiments demonstrate the potential clinical value of the technique.

We present a platform for fluorescence lifetime tomography utilising tuneable supercontinuum excitation and wide-field time-gated technology. Applied to optical projection and diffuse fluorescence tomography, we demonstrate 3-D time-resolved fluorescence reconstruction in transparent and scattering phantoms.

The use of the time gating technique for lifetime reconstruction in the Fourier domain is a novel technique. Time gating provides sufficient data points in the time domain for reliable application of the Fourier transform, which is essential for the time deconvolution of the system of the integral equations employed in the reconstruction. The Fourier domain telegraph equation is employed to model the light transport, which allows a sufficiently broad interval of frequencies to be covered. Reconstructed images contain enough information needed for recovering the lifetime distribution in a sample for any given frequency within the megahertz-gigahertz band. The use of this technique is essential for recovering time-dependent information in fluorescence imaging. This technique was applied in reconstruction of the lifetime distribution of four tubes filled with Rhodamine 6G embedded inside a highly scattering slab. Relatively accurate fluorescence lifetime reconstruction demonstrates the effectiveness and the potential of the proposed technique.

Current arc-lamp illumination systems have a number of technical and ergonomic limitations. White light-emitting diodes (LEDs) are energy-efficient solid-state lighting devices which are small, durable and inexpensive. Their use as an alternative to arc-lamp light sources in minimal access surgery has not been explored. This study aims to develop an LED-based endo-illuminator and to determine its lighting characteristics for use in minimal access surgery.We developed an LED endo-illuminator using a white LED mounted at the tip of a steel rod. Offline image analysis was carried out to compare the illuminated field using the LED endo-illuminator or an arc-lamp based endoscope in terms of uniformity, shadow sharpness and overall image intensity. Direct radiometric power measurements in light intensity and stability were obtained. Visual perception of fine details at the peripheral endoscopic field was assessed by 13 subjects using the different illumination systems.Illumination from the LED endo-illuminator was more uniform compared to illumination from an arc-lamp source, especially at the closer distance of 4 cm (0.0006 versus 0.0028 arbitrary units--lower value indicates more uniform illumination). The shadows were also sharper (edge widths of 16 versus 44 pixels for the first edge and 15 versus 61 pixels for the second edge). The overall mean image intensity was higher (127 versus 100 arbitrary units) when using the autoshutter mode despite the lower direct radiometric power, about one tenth of the arc-lamp endoscopic system. The illumination was also more stable with less flickering (0.02% versus 5% of total power in non-DC components). Higher median scores on visual perception was also obtained (237 versus 157, p0.001).The LED endo-illuminator provides more uniform illumination with sharper shadows, less flickering and better illumination for visual perception than the arc-lamp-based system currently used.

We report a novel technique to reconstruct fluorescence lifetime distributions in turbid media by using Fourier domain reconstruction of time gated imaging data. The time gating provides sufficient temporal resolution to determine short fluorescence lifetimes while the use of the Fourier transform, which is essential for the time de-convolution of the system of the integral equations employed in the reconstruction, permits a relatively rapid reconstruction of 3-D tomographic data. This approach has been applied experimentally to reconstruct fluorescent lifetime distributions corresponding to phantoms with wells filled with fluorescent dyes embedded inside highly scattering slabs. In practice, the scattering medium can itself be fluorescent and we also suggest a simple iterative technique to account for background autofluorescence, which we have also tested experimentally.

Multidimensional fluorescence imaging is a powerful molecular imaging modality that is emerging as an important tool in the study of biological tissues. Due to the large volume of multi-spectral data associated with the technique, it is often difficult to find the best combination of parameters to maximize the contrast between different tissue types. This paper presents a novel framework for the characterization of tissue compositions based on the use of time resolved fluorescence imaging without the explicit modeling of the decays. The composition is characterized through soft clustering based on manifold embedding for reducing the dimensionality of the datasets and obtaining a consistent differentiation scheme for determining intrinsic constituents of the tissue. The proposed technique has the benefit of being fully automatic, which could have significant advantages for automated histopathology and increasing the speed of intraoperative decisions. Validation of the technique is carried out with both phantom data and tissue samples of the human pancreas.

We present the design, characterization and application of a novel, rapid, optically sectioned hyperspectral fluorescence lifetime imaging (FLIM) microscope. The system is based on a line scanning confocal configuration and uses a highspeed time-gated detector to extract lifetime information from many pixels in parallel. This allows the full spectraltemporal profiles of a fluorescence decay to be obtained from every pixel in an image. Line illumination and slit detection also gives the microscope a confocal optical sectioning ability. The system is applied to test samples and unstained biological tissue. In future, this microscope will be combined with recently-developed continuously electronically tunable, pulsed light sources based on tapered, micro-structured optical fibers. This will allow hyperspectral FLIM to be combined with the advantages of excitation spectroscopy to gain further insight into complex biological specimens including tissue and live cell imaging.

In this paper we demonstrate structured illumination microscopy, a cheap and flexible method of obtaining optical sectioning in wide-field microscopy, and we investigate line-scanning microscopy, both using the same microelement LED. In our first experiment we demonstrate structured illumination using a custom-designed microelement LED consisting of 120 individually addressable stripes emitting at 470 nm. An electrical driver was designed to produce a programmable grid pattern and the device was located in an Olympus BX41 fluorescence microscope in critical illumination configuration. By using an integrated solid-state alternative to a conventional piezo-actuated grid and separate illumination source, we improved the speed and accuracy of the system, reducing the artefacts due to the errors in the grid positions. Additionally, we investigated the use of the same LED device as a scanning source for confocal line-scanning microscopy. After each line scanning, an image was acquired using a CCD camera and the out-of-focus light was rejected by a post-processing method.

Introduction Gold nanoparticles possess unique physiochemical properties due to their ability to target cancerous tissue and a strongly enhanced absorption of visible and near infra-red (NIR) light resulting in hyperthermia and photothermal therapy (PTT). Fluorescence emission from their functionalised surface provides diagnostic information regarding tumour location. Their biocompatibility in vivo is facilitated through surface polymer coating. We investigate these properties in human oesophageal adenocarcinoma by examining in vitro targeting of functionalised gold nanorods to human oesophageal adenocarcinoma cells (FLO-1) and evaluating the PTT of tumour xenografts in vivo . Method Multifunctional coated gold nanorods (GNRs) were fabricated using a seed-mediated method. Subsequent EDC/NHS chemistry functionalised the GNRs with a fluorophore (Cy 5.5) modified with anti-EGFR antibody. FLO-1 cells were fixed in paraformaldehyde in 0.1 mol/l PBS solution. Dilute functionalised GNRs were added and incubated overnight. Slides were then washed with PBS and stained with the nuclear dye DAPI prior to microscopic examination. Tumour xenografts were established in immunodeficient mice by subcutaneous inoculation of FLO-1 cells. When tumour size reached 5 mm, functionalized GNRs were administered either intratumourally (I. T.) or intravenously (I. V.). Fluorescence spectroscopy was performed followed by NIR irradiation of the tumour for 3 min using an 808 nm laser at 0.92 W. A thermal imaging camera measured the temperature changes that occurred. Inductively coupled plasma mass spectrometry (ICP-MS) measured gold concentrations in blood and organs remote to the tumour. Results Functionalised GNRs with a peak optical absorption at 808 nm were used. Fluorescence imaging of immunohistochemically stained FLO-1 cells demonstrated active in vitro targeting of GNRs. NIR irradiation of tumours established 12°C and 18°C temperature increases in mice receiving I. V. and I. T. GNRs respectively. Histological examination of tumour sites showed fully preserved tissue layers with no signs of proliferating tumour. ICP-MS revealed no evidence of toxic accumulation of gold in blood, kidneys, liver or spleen after 30 days. Conclusion Fluorescence detection of functionalised GNRs that localise to cancerous tissue may enhance oesophageal cancer diagnosis. When coupled with NIR irradiation, this is an effective method of inducing irreversible tumour photodestruction, with minimal collateral damage expected in healthy tissues. The strong absorption of GNRs of relatively low laser energies renders the therapy method minimally invasive. Disclosure of interest None Declared.

Shear wave speed is quantitatively related to tissue viscoelasticity. Previously we reported shear wave tracking at centimetre depths in a turbid optical medium using laser speckle contrast detection. Shear wave progression modulates displacement of optical scatterers and therefore modulates photon phase and changes the laser speckle patterns. Time-resolved charge-coupled device (CCD)-based speckle contrast analysis was used to track shear waves and measure the time-of-flight of shear waves for speed measurement. In this manuscript, we report a new observation of the laser speckle contrast difference signal for dual shear waves. A modulation of CCD speckle contrast difference was observed and simulation reproduces the modulation pattern, suggesting its origin. Both experimental and simulation results show that the dual shear wave approach generates an improved definition of temporal features in the time-of-flight optical signal and an improved signal to noise ratio with a standard deviation less than 50% that of individual shear waves. Results also show that dual shear waves can correct the bias of shear wave speed measurement caused by shear wave reflections from elastic boundaries.

We demonstrate a continuously electronically tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated by injecting infrared femtosecond pulses in to a microstructured fibre. We demonstrate this source applied to confocal and wide-field microscopy, as well as multiwell-plate imaging. We also report first application of a tunable supercontinuum source (TSS) to FLIM and to in situ measurements of relative excitation spectra. We are currently applying this source to hyperspectral imaging and working towards a system for acquiring the full fluorescence excitation-emission matrix. Fluorescence lifetime imaging (FLIM) can provide further information about fluorescent samples and requires a rapidly modulated or pulsed light source, in general, (tunable) ultrafast light sources, particularly in the visible, are complex and expensive and usually require a significant degree of adjustment to achieve tunability

The field of microfluidics has developed rapidly with promising applications such as 'lab on a chip' devices for micro-assays and efficient chemical reactions and processes, including e.g. DNA sequencing. The design of microfluidic devices requires an understanding of fluid flow and dynamics, which we demonstrate can be enhanced by direct observation using (time-resolved) multi-dimensional fluorescence imaging (MDFI). As well as providing opportunities to study fluid dynamics, MDFI provides new opportunities for read-out and analysis which may be applied to assay development and to studies of reaction kinetics e.g. stop-flow experiments. We present our first results using time-gated fluorescence imaging, resolved with respect to 2 or 3 spatial dimensions. Time-resolved imaging of fluorescence polarisation anisotropy may be used to image mixing in a microfluidic device via maps of the rotational correlation time, which is proportional to the local fluorophore solvent viscosity

We demonstrate an optically sectioned fluorescence lifetime imaging microscope with a wide-field detector, using a convenient, continuously tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated in a microstructured fiber.

Multidimensional fluorescence imaging is a powerful molecular imaging modality that is emerging as an important tool in the study of biological tissues. Due to the large volume of multi-spectral data associated with the technique, it is often difficult to find the best combination of parameters to maximize the contrast between different tissue types. This paper presents a novel framework for the characterization of tissue compositions based on the use of time resolved fluorescence imaging without the explicit modeling of the decays. The composition is characterized through soft clustering based on manifold embedding for reducing the dimensionality of the datasets and obtaining a consistent differentiation scheme for determining intrinsic constituents of the tissue. The proposed technique has the benefit of being fully automatic, which could have significant advantages for automated histopathology and increasing the speed of intraoperative decisions. Validation of the technique is carried out with both phantom data and tissue samples of the human pancreas.

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.

Polarisation imaging has the potential to provide enhanced contrast based on variations in the optical properties, such as scattering or birefringence, of the tissue of interest. Examining the signal at different wavebands in the visible spectrum also allows interrogation of different depths and structures. A stereo endoscope has been adapted to allow snapshot acquisition of orthogonal linear polarisation images to generate difference of linear polarisation images. These images are acquired in three narrow bands using a triple-bandpass filter and pair of colour cameras. The first in vivo results, acquired during a surgical procedure on a porcine subject, are presented that show wavelength dependent variations in vessel visibility and an increase in contrast under polarised detection.

Multi-beam confocal sectioned fluorescence lifetime imaging microscopy is demonstrated using a Yokogawa spinning disk. The single-photon excitation source is a supercontinuum generated from a Ti:sapphire seeded photonic crystalline fibre.

We report real-time (video-rate) fluorescence lifetime imaging and its application to tissue autofluorescence and endoscopy, demonstrating FLIM of unstained ex vivo tissue at update rates of 5.5 Hz through a flexible endoscope.

We investigate the temporal properties of the fluorescence emitted by such devices after excitation with an ultrashort optical pulse using a novel wide-field method, fluorescence lifetime imaging (FLIM). This has not previously been applied to PLEDs and provides a new technique for studying the processes leading to black spot formation. The FLIM system developed at Imperial College uses an ultrafast time-gated image intensifier based on microchannel plate technology. This (coupled together with a CCD) can stroboscopically record time-gated images of the fluorescence emitted by a sample after pulsed excitation by changing the trigger delay between optical excitation and signal collection by the CCD. The images from the CCD are then analysed computationally pixel-by-pixel using, for instance, a single exponential decay model so that an image of the fluorescence lifetimes can be created on a false colour scale (a FLIM map). PLED defects have also been studied with FLIM, including bubble formation in the polymer layer. The application of fluorescence lifetime imaging provides a novel method to achieve contrast in microscopic studies of the properties of PLEDs and to probe the mechanisms behind defect formation.

We present the application of fluorescence lifetime imaging (FLIM) and hyperspectral FLIM to autofluorescence of normal and arthritic cartilage tissue.

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance. © 2003 Optical Society of America.

The editors introduce the Biomedical Optics Express feature issue, “Advances in Optics for Biotechnology, Medicine and Surgery,” which includes 12 contributions from attendees of the 2011 conference Advances in Optics for Biotechnology, Medicine and Surgery XII.

Use of a blue picosecond laser diode allows a portable fluorescence lifetime imaging system. We show the application of the system to multi-well plate imaging of biological fluorophores and microscopic imaging of unstained tissue sections.

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and

Heat-induced tissue fusion is an important procedure in modern surgery and can greatly reduce trauma, complications, and mortality during minimally invasive surgical blood vessel anastomosis, but it may also have further benefits if applied to other tissue types such as small and large intestine anastomoses. We present a tissue-fusion characterization technology using laser-induced fluorescence spectroscopy, which provides further insight into tissue constituent variations at the molecular level. In particular, an increase of fluorescence intensity in 450- to 550-nm range for 375- and 405-nm excitation suggests that the collagen cross-linking in fused tissues increased. Our experimental and statistical analyses showed that, by using fluorescence spectral data, good fusion could be differentiated from other cases with an accuracy of more than 95%. This suggests that the fluorescence spectroscopy could be potentially used as a feedback control method in online tissue-fusion monitoring.

We report a whole-field fluorescence imaging microscope that combines 3-D spatial resolution by optical sectioning, using structured illumination, with fluorescence lifetime imaging and spectrally-resolved imaging. We show the potential of this technique in the elimination of common artefacts in fluorescence lifetime imaging and apply it to study the dependence of the lifetime on the emission wavelength in biological tissue.

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.

Summary form only given. We have developed simple, intuitive semi-analytical ABCD technique. that allows transverse gain saturation effects in end-pumped lasers to be accounted for with minimal approximation. The new formalism makes use of an explicit solution to the homogeneous gain-saturation equation and, for most cases, is virtually indistinguishable from a full beam propagation code (BPC). Our new ABCD method correctly treats saturation of the beam in the wings, overcoming the limitations of the quadratic approximation to the gain. The work has been extended to include pump and laser beam divergence in the gain medium. The predictions of the model are compared with experimental results from a diode-pumped Cr:LiSAF laser.

Tissue viscosity is correlated with tissue pathological changes and provides information for tissue characterization. In this study, we report an optical method to track continuous shear-wave propagation at centimeter depths in an optically turbid medium. Shear-wave attenuation coefficients were measured at multiple frequencies using shear-wave laser speckle contrast analysis (SW-LASCA) to quantitatively estimate tissue viscosity using the Voigt model. Shear waves were generated within tissue-mimicking phantoms by an amplitude-modulated ultrasound (modulation frequency: 100 to 600 Hz) and tracked by time-resolved laser speckle contrast difference received on a charged-coupled device camera. Averaged contrast difference over a selected time window was related to shear-wave amplitude and used to calculate the shear-wave attenuation coefficient. Phantoms of varying viscosities (0.1 and 0.3 Pa s) were studied. Attenuation coefficients for different shear-wave frequencies (100 to 600 Hz) were calculated. Derived viscosity values had a maximum standard deviation of 9%, and these values were consistent with the independent measurements reported in a previous study using nonoptical methods.

Introduction Fluorescence lifetime imaging (FLIM) provides a contrast parameter for tissue components independent of wavelength, spectrum or polarization. It has been shown that contrast between autofluorescent matrix components such as collagen and elastin is available through FLIM (Siegel et al. 2003; Dowling et al. 1998), which is not clear using other fluorescent techniques. The lifetime of collagen has been studied whilst in solution; however, research is ongoing to quantify its fluorescence whilst in tissue. The aim of this study was to investigate the contrast available by applying fluorescence lifetime imaging (FLIM) to articular cartilage, which has extracellular matrix containing collagen type-II and proteoglycan. Materials and methods A time-gated FLIM system was used to analyse the articular cartilage from unstained sections of human femurs as well as from the knee joints young and old sheep. The sections were also observed under a conventional fluorescence microscope, and for the human femurs, adjacent H&E-stained sections were also obtained. Details of the FLIM system can be found elsewhere (Dowling et al. 1998), with the laser source being tuneable, allowing excitation over a range 350–600 nm. Fluorescence half-lives were obtained using single exponentials applied to the intensity decay data. Results It was found that younger cartilage was generally less fluorescent than older. For example, when exciting at 401 nm and observing above 450 nm, it was found that the young sheep samples provided a signal to noise ratio too low to obtain lifetime data, whereas other samples fluoresced brightly. It was also found that within the matrix, FLIM provided contrast unavailable with conventional fluorescence microscopy and, in the case of human femurs, with H&E stains. Discussion In the study of collagen, it is believed that the cross-linkages are responsible for fluorescence (Richards-Kortum & Sevick-Muraca 1996). Because the cross-linkages increase with age, the fluorescence intensity is also expected to increase. The lesser amount of fluorescence from the younger samples therefore suggests that the dominant fluorophore in cartilage is collagen. The contrast obtained with FLIM in the cartilage indicates nonuniformity in the matrix structure. Acknowledgements This work was supported by the EPSRC, BBSRC, Kentech Instruments, a DTI Beacon award and a Wellcome Trust Showcase award.

Elastic light scattering spectroscopy (LSS) is widely utilized to investigate cellular structures in cultured cells and various tissues. However, few imaging systems, especially endoscopic imaging systems, can implement LSS. It is the aim of this work to create a polarized multispectral imaging system based around a rigid endoscope to detect micrometer sized particles using LSS. The instrument first validated with different sized mono-disperse polystyrene microspheres, then an image is reconstructed based on LSS which shows the differentiation of different sized microspheres. Finally a preliminary experiment is conducted to demonstrate its capability to discriminate different types of cells.

We investigated the effect of amplitude-modulated (AM) ultrasound (US) on acousto-optic (AO) signals. A phantom was exposed to both AM US and a green laser, and CCD measurements of speckle contrast were made with various exposure times. The results show that the AO signal oscillates at the AM frequency when the CCD exposure time is a fraction of the AM period and stops oscillating when the CCD exposure time is a multiple of the AM period. The AO signal decreases quickly as the AM frequency increases or peak-peak (pk-pk) amplitude decreases. With 4ms exposure time, 250Hz AM frequency and 1.27MPa pk-pk acoustic pressure, there is an ~30% increase in the AO signal compared with that of CW US. The increase in the signal is likely to be due to the particle oscillation and the induced shear wave as a result of the radiation force generated by the AM US.

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.2 ns. We demonstrate a significant contrast in fluorescence lifetime between tumor (1.77+/-0.26 ns) and normal (2.50+/-0.36 ns) tissues at 450 nm and an over 80% intensity decrease at 390 nm emission in tumor versus normal areas. The time-resolved images were minimally affected by tissue morphology, endogenous absorbers, and illumination. These results demonstrate the potential of FLIM as an intraoperative diagnostic technique.

We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.

High-speed (video-rate) fluorescence lifetime imaging (FLIM) is reported using two different time-domain approaches based on gated optical image intensifier technology. The first approach utilizes a rapidly switchable variable delay generator with sequential image acquisition, while the second employs a novel segmented gated optical imager to acquire lifetime maps in a single shot. Lifetimes are fitted using both a non-linear least-squares fit analysis and the rapid lifetime determination method. Monte Carlo simulations were used to optimize the acquisition parameters and a comparison between theory and experiment is presented. The importance of single-shot imaging to minimize the deleterious impact of sample movements is highlighted. Real-time FLIM movies of multi-well plate samples and tissue autofluorescence are presented.

We report the development of a high-speed wide-field fluorescence-lifetime imaging (FLIM) system that provides fluorescence-lifetime images at rates of as many as 29 frames/s. A FLIM multiwell plate reader and a potentially portable FLIM endoscopic system operating at 355-nm excitation have been demonstrated.

Summary form only given. Fluorescence is widely used in biomedicine and other applications both to track specific fluorophores and study anatomical features. Fluorescence lifetime measurements add functional information because they are dependent on the local environment of the fluorophores (e.g. oxygen, [Ca/sup 2+/], pH etc.) e.g. Fluorescence Lifetime Imaging (FLIM) is particularly exciting since it can exploit this feature to provide non-invasive functional/diagnostic imaging. Complementary functional information may be obtained using spectrally-resolved fluorescence imaging. In order to acquire both spectral and lifetime information from an optical section in a reasonable time, it is preferable to acquire-all the image information in parallel, rather than to scan pixel by pixel at different emission wavelengths. We report, for the first, time to our knowledge, the combination of depth-resolved FLIM, by structured illumination, and hyperspectral imaging, thus realising 5-D fluorescence imaging. The recent development of user-friendly and relatively portable ultrafast laser technology and the availability of ultrafast gated optical image intensifiers (GOI's) mean that this technology can be readily applied to almost any optical imaging modality including microscopy and endoscopy. We report a whole-field optically sectioning. fluorescence imaging system with fluorescence lifetime and spectral contrast, based on a GOI and an all-solid-state diode pumped Cr:LiSAF oscillator-amplifier system. Diode-pumped laser technology potentially provides a portable and relatively inexpensive FLIM instrument.

Recovering tissue deformation during robotic-assisted minimally invasive surgery procedures is important for providing intra-operative guidance, enabling in vivo imaging modalities and enhanced robotic control. The tissue motion can also be used to apply motion stabilization and to prescribe dynamic constraints for avoiding critical anatomical structures.Image-based methods based independently on salient features or on image intensity have limitations when dealing with homogeneous soft tissues or complex reflectance. In this paper, we use a triangular geometric mesh model in order to combine the advantages of both feature and intensity information and track the tissue surface reliably and robustly.Synthetic and in vivo experiments are performed to provide quantitative analysis of the tracking accuracy of our method, and we also show exemplar results for registering multispectral images where there is only a weak image signal.Compared to traditional methods, our hybrid tracking method is more robust and has improved convergence in the presence of larger displacements, tissue dynamics and illumination changes.

Fluorescence lifetime imaging (FLIM) provides a robust functional imaging modality for biomedicine. Recent advances in ultrafast laser and detector technology make FLIM increasingly accessible for life scientists and point the way to new clinical instrumentation.

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.