Jason D. Riley

Fluorescence Lifetime Imaging System for In Vivo Studies

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.

Using noninvasive multispectral imaging to quantitatively assess tissue vasculature

This research describes a noninvasive, noncontact method used to quantitatively analyze the functional characteristics of tissue. Multispectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposis sarcoma lesions and the surrounding tissue before and during experimental therapies. The multispectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.

Direct curvature correction for noncontact imaging modalities applied to multispectral imaging.

Noncontact optical imaging of curved objects can result in strong artifacts due to the objects shape, leading to curvature biased intensity distributions. This artifact can mask variations due to the objects optical properties, and makes reconstruction of optical/physiological properties difficult. In this work we demonstrate a curvature correction method that removes this artifact and recovers the underlying data, without the necessity of measuring the objects shape. This method is applicable to many optical imaging modalities that suffer from shape-based intensity biases. By separating the spatially varying data (e.g., physiological changes) from the background signal (dc component), we show that the curvature can be extracted by either averaging or fitting the rows and columns of the images. Numerical simulations show that our method is equivalent to directly removing the curvature, when the objects shape is known, and accurately recovers the underlying data. Experiments on phantoms validate the numerical results and show that for a given image with 16.5% error due to curvature, the method reduces that error to 1.2%. Finally, diffuse multispectral images are acquired on forearms in vivo. We demonstrate the enhancement in image quality on intensity images, and consequently on reconstruction results of blood volume and oxygenation distributions.

Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

We have constructed a device that maximizes the probability of collecting all of the scattered and ballistic light isotropically generated at the focal spot of multiphoton excited emissions (MPE) to optimize the signal‐to‐noise ratio (SNR) for micro‐imaging. This was accomplished by optically coupling a parabolic reflector (that surrounds the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirects emitted light to a separate detector. These additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. Numerical simulations suggest an approximately 10‐fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in SNR (77% of predicted) for GFP‐labelled brain slices and similar results for fluorescent beads. This increase in SNR can be used to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, detection depth, for MPE imaging techniques.

Normative database of judgment of complexity task with functional near infrared spectroscopy – Application for TBI

The ability to assess frontal lobe function in a rapid, objective, and standardized way, without the need for expertise in cognitive test administration might be particularly helpful in mild traumatic brain injury (TBI), where objective measures are needed. Functional near infrared spectroscopy (fNIRS) is a reliable technique to noninvasively measure local hemodynamic changes in brain areas near the head surface. In this paper, we are combining fNIRS and frameless stereotaxy which allowed us to co-register the functional images with previously acquired anatomical MRI volumes. In our experiment, the subjects were asked to perform a task, evaluating the complexity of daily life activities, previously shown with fMRI to activate areas of the anterior frontal cortex. We reconstructed averaged oxyhemoglobin and deoxyhemoglobin data from 20 healthy subjects in a spherical coordinate. The spherical coordinate is a natural representation of surface brain activation projection. Our results show surface activation projected from the medial frontopolar cortex which is consistent with previous fMRI results. With this original technique, we will construct a normative database for a simple cognitive test which can be useful in evaluating cognitive disability such as mild traumatic brain injury.

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction‐limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330–337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8‐fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4‐fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2‐fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal‐to‐noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo‐damage).

Principal component model of multispectral data for near real-time skin chromophore mapping

Multispectral images of skin contain information on the spatial distribution of biological chromophores, such as blood and melanin. From this, parameters such as blood volume and blood oxygenation can be retrieved using reconstruction algorithms. Most such approaches use some form of pixelwise or volumetric reconstruction code. We explore the use of principal component analysis (PCA) of multispectral images to access blood volume and blood oxygenation in near real time. We present data from healthy volunteers under arterial occlusion of the forearm, experiencing ischemia and reactive hyperemia. Using a two-layered analytical skin model, we show reconstruction results of blood volume and oxygenation and compare it to the results obtained from our new spectral analysis based on PCA. We demonstrate that PCA applied to multispectral images gives near equivalent results for skin chromophore mapping and quantification with the advantage of being three orders of magnitude faster than the reconstruction algorithm.

Choice of data types in time resolved fluorescence enhanced diffuse optical tomography

In this paper we examine possible data types for time resolved fluorescence enhanced diffuse optical tomography (FDOT). FDOT is a particular case of diffuse optical tomography, where our goal is to analyze fluorophores deeply embedded in a turbid medium. We focus on the relative robustness of the different sets of data types to noise. We use an analytical model to generate the expected temporal point spread function (TPSF) and generate the data types from this. Varying levels of noise are applied to the TPSF before generating the data types. We show that local data types are more robust to noise than global data types, and should provide enhanced information to the inverse problem. We go on to show that with a simple reconstruction algorithm, depth and lifetime (the parameters of interest) of the fluorophore are better reconstructed using the local data types. Further we show that the relationship between depth and lifetime is better preserved for the local data types, suggesting they are in some way not only more robust, but also self-regularizing. We conclude that while the local data types may be more expensive to generate in the general case, they do offer clear advantages over the standard global data types.

Quantitative principal component model for skin chromophore mapping using multi-spectral images and spatial priors

We describe a novel reconstruction algorithm based on Principal Component Analysis (PCA) applied to multi-spectral imaging data. Using numerical phantoms, based on a two layered skin model developed previously, we found analytical expressions, which convert qualitative PCA results into quantitative blood volume and oxygenation values, assuming the epidermal thickness to be known. We also evaluate the limits of accuracy of this method when the value of the epidermal thickness is not known. We show that blood volume can reliably be extracted (less than 6% error) even if the assumed thickness deviates 0.04mm from the actual value, whereas the error in blood oxygenation can be as large as 25% for the same deviation in thickness. This PCA based reconstruction was found to extract blood volume and blood oxygenation with less than 8% error, if the underlying structure is known. We then apply the method to in vivo multi-spectral images from a healthy volunteer’s lower forearm, complemented by images of the same area using Optical Coherence Tomography (OCT) for measuring the epidermal thickness. Reconstruction of the imaging results using a two layered analytical skin model was compared to PCA based reconstruction results. A point wise correlation was found, showing the proof of principle of using PCA based reconstruction for blood volume and oxygenation extraction.

A hematoma detector-a practical application of instrumental motion as signal in near infra-red imaging.

Abstract: In this paper we discuss results based on using instrumental motion as a signal rather than treating it as noise in Near Infra-Red (NIR) imaging. As a practical application to demonstrate this approach we show the design of a novel NIR hematoma detection device. The proposed device is based on a simplified single source configuration with a dual separation detector array and uses motion as a signal for detecting changes in blood volume in the dural regions of the head. The rapid triage of hematomas in the emergency room will lead to improved use of more sophisticated/expensive imaging facilities such as CT/MRI units. We present simulation results demonstrating the viability of such a device and initial phantom results from a proof of principle device. The results demonstrate excellent localization of inclusions as well as good quantitative comparisons.