Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo.

In this Letter, we will discuss the development of a multifocal multiphoton fluorescent lifetime imaging system where four individual fluorescent intensity and lifetime planes are acquired simultaneously, allowing us to obtain volumetric data without the need for sequential scanning at different axial depths. Using a phase-only spatial light modulator (SLM) with an appropriate algorithm to generate a holographic pattern, we project a beamlet array within a sample volume of a size, which can be preprogrammed by the user. We demonstrate the capabilities of the system to image live-cell interactions. While only four planes are shown, this technique can be rescaled to a large number of focal planes, enabling full 3D acquisition and reconstruction.

Bimodal fluorogenic sensing of matrix proteolytic signatures in lung cancer.

Optical biosensing based on the activation of fluorescent reporters offers a powerful methodology for the real-time molecular interrogation of pathology. Here we report a first-in-class, bimodal fluorescent reporter strategy for the simultaneous and highly specific detection of two independent proteases (thrombin and matrix metalloproteases (MMPs)) pivotal in the fibroproliferative process surrounding lung cancer, based on a dual, multiplexing, peptide FRET system. This sophisticated synthetic smartprobe, with a molecular weight of 6 kDa, contains two independent fluorophores and quenchers that generate photonic signatures at two specific wavelengths upon activation by target enzymes within human lung cancer tissue.

Low-cost high sensitivity pulsed endomicroscopy to visualize tricolor optical signatures.

A highly sensitive, modular three-color fluorescence endomicroscopy imaging platform spanning the visible to near-infrared (NIR) range is demonstrated. Light-emitting diodes (LEDs) were sequentially pulsed along with the camera acquisition to provide up to 20 frames per second (fps) three-color imaging performance or 60 fps single color imaging. The system was characterized for bacterial and cellular molecular imaging in ex vivo human lung tissue and for bacterial and indocyanine green imaging in ex vivo perfused sheep lungs. A practical method to reduce background tissue autofluorescence is also proposed. The platform was clinically translated into six patients with pulmonary disease to delineate healthy, cancerous, and fibrotic tissue autofluorescent structures. The instrument is the most broadband clinical endomicroscopy system developed to date (covering visible to the NIR, 500 to 900 nm) and demonstrates significant potential for future clinical utility due to its low cost and modular capability to suit a wide variety of molecular imaging applications.

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

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

Electrophysiological and Anatomical Correlates of Spinal Cord Optical Coherence Tomography.

Despite the continuous improvement in medical imaging technology, visualizing the spinal cord poses severe problems due to structural or incidental causes, such as small access space and motion artifacts. In addition, positional guidance on the spinal cord is not commonly available during surgery, with the exception of neuronavigation techniques based on static pre-surgical data and of radiation-based methods, such as fluoroscopy. A fast, bedside, intraoperative real-time imaging, particularly necessary during the positioning of endoscopic probes or tools, is an unsolved issue. The objective of our work, performed on experimental rats, is to demonstrate potential intraoperative spinal cord imaging and probe guidance by optical coherence tomography (OCT). Concurrently, we aimed to demonstrate that the electromagnetic OCT irradiation exerted no particular effect at the neuronal and synaptic levels. OCT is a user-friendly, low-cost and endoscopy-compatible photonics-based imaging technique. In particular, by using a Fourier-domain OCT imager, operating at 850 nm wavelength and scanning transversally with respect to the spinal cord, we have been able to: 1) accurately image tissue structures in an animal model (muscle, spine bone, cerebro-spinal fluid, dura mater and spinal cord), and 2) identify the position of a recording microelectrode approaching and inserting into the cord tissue 3) check that the infrared radiation has no actual effect on the electrophysiological activity of spinal neurons. The technique, potentially extendable to full three-dimensional image reconstruction, shows prospective further application not only in endoscopic intraoperative analyses and for probe insertion guidance, but also in emergency and adverse situations (e.g. after trauma) for damage recognition, diagnosis and fast image-guided intervention.

Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.

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

An investigation of the potential of optical computed tomography for imaging of synchrotron-generated x-rays at high spatial resolution.

X-ray microbeam radiation therapy (MRT) is a novel form of treatment, currently in its preclinical stage, which uses microplanar x-ray beams from a synchrotron radiation source. It is important to perform accurate dosimetry on these microbeams, but, to date, there has been no accurate enough method available for making 3D dose measurements with isotropic, high spatial resolution to verify the results of Monte Carlo dose simulations. Here, we investigate the potential of optical computed tomography for satisfying these requirements. The construction of a simple optical CT microscopy (optical projection tomography) system from standard commercially available hardware is described. The measurement of optical densities in projection data is shown to be highly linear (r2=0.999). The depth-of-field (DOF) of the imaging system is calculated based on the previous literature and measured experimentally using a commercial DOF target. It is shown that high quality images can be acquired despite the evident lack of telecentricity and despite DOF of the system being much lower than the sample diameter. Possible reasons for this are discussed. Results are presented for a complex irradiation of a 22 mm diameter cylinder of the radiochromic polymer PRESAGE, demonstrating the exquisite 'dose-painting' abilities available in the MRT hutch of beamline ID-17 at the European Synchrotron Radiation Facility. Dose distributions in this initial experiment are equally well resolved on both an optical CT scan and a corresponding transmission image of radiochromic film, down to a line width of 83 microm (6 lp mm(-1)) with an MTF value of 0.40. A group of 33 microm wide lines was poorly resolved on both the optical CT and film images, and this is attributed to an incorrect exposure time calculation, leading to under-delivery of dose. Image artefacts in the optical CT scan are discussed. PRESAGE irradiated using the microbeam facility is proposed as a suitable material for producing phantom samples for quantitative characterization of optical CT microscopy systems.

Characterization of a parallel-beam CCD optical-CT apparatus for 3D radiation dosimetry.

3D measurement of optical attenuation is of interest in a variety of fields of biomedical importance, including spectrophotometry, optical projection tomography (OPT) and analysis of 3D radiation dosimeters. Accurate, precise and economical 3D measurements of optical density (OD) are a crucial step in enabling 3D radiation dosimeters to enter wider use in clinics. Polymer gels and Fricke gels, as well as dosimeters not based around gels, have been characterized for 3D dosimetry over the last two decades. A separate problem is the verification of the best readout method. A number of different imaging modalities (magnetic resonance imaging (MRI), optical CT, x-ray CT and ultrasound) have been suggested for the readout of information from 3D dosimeters. To date only MRI and laser-based optical CT have been characterized in detail. This paper describes some initial steps we have taken in establishing charge coupled device (CCD)-based optical CT as a viable alternative to MRI for readout of 3D radiation dosimeters. The main advantage of CCD-based optical CT over traditional laser-based optical CT is a speed increase of at least an order of magnitude, while the simplicity of its architecture would lend itself to cheaper implementation than both MRI and laser-based optical CT if the camera itself were inexpensive enough. Specifically, we study the following aspects of optical metrology, using high quality test targets: (i) calibration and quality of absorbance measurements and the camera requirements for 3D dosimetry; (ii) the modulation transfer function (MTF) of individual projections; (iii) signal-to-noise ratio (SNR) in the projection and reconstruction domains; (iv) distortion in the projection domain, depth-of-field (DOF) and telecentricity. The principal results for our current apparatus are as follows: (i) SNR of optical absorbance in projections is better than 120:1 for uniform phantoms in absorbance range 0.3 to 1.6 (and better than 200:1 for absorbances 1.0 to 3.5 with the test target and a novel absorbance range extension method), (ii) the spatial resolution is shown to be at worst 0.5 mm (and often better than this) with an associated DOF of 8 cm, (iii) the SNR of uniform phantoms in reconstruction domain is above 80:1 (one standard deviation) over an absorbance dynamic range of 0.3 to 1.6, (iv) the apparatus is telecentric and without distortion. Finally, a sample scan and reconstruction of a scan of a PRESAGE dosimeter are shown, demonstrating the capabilities of the apparatus.

Fast laser scanning optical-CT apparatus for 3D radiation dosimetry.

Optical computed tomography (optical-CT) of 3D radiation dosimeters is a promising avenue for delivering an economic and reliable quality control of radiotherapy treatments such as intensity modulated radiotherapy, brachytherapy and stereotactic radiosurgery. The main problems in transferring 3D dosimeters to clinical setting have been in (1) the complexity of manufacture and behaviour of 3D dosimeters and (2) time-consuming readout and analysis of 3D dosimeters. This paper addresses the readout problem by showing that fast (20 min tomography scan), precise (projection absorbance signal-to-noise ratio is greater than 100:1 across the absorbance range 0.2 to 1.5) and accurate (good linearity in the calibration curve) measurements are possible using a novel method of optically scanning a laser beam across the 3D dosimeter.

Focusing optics of a parallel beam CCD optical tomography apparatus for 3D radiation gel dosimetry.

Optical tomography of gel dosimeters is a promising and cost-effective avenue for quality control of radiotherapy treatments such as intensity-modulated radiotherapy (IMRT). Systems based on a laser coupled to a photodiode have so far shown the best results within the context of optical scanning of radiosensitive gels, but are very slow ( approximately 9 min per slice) and poorly suited to measurements that require many slices. Here, we describe a fast, three-dimensional (3D) optical computed tomography (optical-CT) apparatus, based on a broad, collimated beam, obtained from a high power LED and detected by a charged coupled detector (CCD). The main advantages of such a system are (i) an acquisition speed approximately two orders of magnitude higher than a laser-based system when 3D data are required, and (ii) a greater simplicity of design. This paper advances our previous work by introducing a new design of focusing optics, which take information from a suitably positioned focal plane and project an image onto the CCD. An analysis of the ray optics is presented, which explains the roles of telecentricity, focusing, acceptance angle and depth-of-field (DOF) in the formation of projections. A discussion of the approximation involved in measuring the line integrals required for filtered backprojection reconstruction is given. Experimental results demonstrate (i) the effect on projections of changing the position of the focal plane of the apparatus, (ii) how to measure the acceptance angle of the optics, and (iii) the ability of the new scanner to image both absorbing and scattering gel phantoms. The quality of reconstructed images is very promising and suggests that the new apparatus may be useful in a clinical setting for fast and accurate 3D dosimetry.