Elliot S. Wachman

Non-invasive image acquisition and advanced processing in optical bioimaging

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Near-simultaneous hemoglobin saturation and oxygen tension maps in mouse brain using an AOTF microscope

A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.

AOTF microscope for imaging with increased speed and spectral versatility

We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse.

Spatial Distribution of Calcium Entry Evoked by Single Action Potentials within the Presynaptic Active Zone

The nature of presynaptic calcium (Ca2+) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca2+ concentration within 1 msec of action potential invasion are attributable to Ca2+ entry through N-type Ca2+ channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca2+ signals recorded before and after experimental manipulations (ω-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca2+) support the hypothesis that there is a remarkably low probability of a single Ca2+ channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca2+ entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.

IMAGING ACOUSTO-OPTIC TUNABLE FILTER WITH 0.35-MICROMETER SPATIAL RESOLUTION

Image blur in acousto-optic tunable filters (AOTFs) has been a persistent problem. Here we describe the connection between transducer structure and image blur and experimentally measure it by using a 5-cm 12°-cut TeO(2) crystal of our design. With these quantitative results, we develop an image-processing method that minimizes AOTF-related image degradation. The combination of long crystal design and image processing results in substantially improved image contrast and spatial resolution relative to conventional AOTF imaging devices. We present high-magnification images of fluorescent actin fibers in cells in which we obtain a resolution of approximately 0.35 μm, representing the first successful use of an AOTF for ultra-high-resolution microscopy. Further improvements are also predicted.

Spectral imaging in biomedicine: a selective overview

We present a survey of spectral imaging for biological and medical applications. Brief philosophical and historical considerations are followed by an overview of the reasons for the modalities of achieving the fertile confluence of spectroscopy and imaging. Methods of wavelength selection at both the excitation and detection ends of an imaging system are listed and critically evaluated. A number of biological and medical applications of spectral imaging are discussed, highlighting microscopy and including our own work. We emphasize that the outlook for this research area critically depends on the further development of all component technologies, from reagents and optics to electronics and software.

Microscopic and mesoscopic spectral bioimaging

Light microscopy has become a versatile tool for investigating biological phenomena as they unfold, using cells as living microcuvettes. The progress is based on improvements in a number of technological fields, including optics, electronics, reagent chemistry and computer science. The non-destructiveness, spatial resolution and speed of optical imaging can provide high versatility for investigating biological structure and function at the tissue or even whole organism level as well. Having previously reported on some new approaches to meeting the challenges posed by the fluorescence-based imaging of cells and tissues, we concentrate here on imaging with increased spectral content and resolution. We improved and applied two techniques of spectral selection: (1) acousto-optic tunable filtering, allowing for multiwavelength fluorescence microscopy with diffraction-limited spatial imaging and sub- millisecond temporal resolution, and (2) 2D Sagnac interferometry-based Fourier spectroscopy, yielding advanced spectral imaging capabilities. Enhanced implementations of existing optical technologies, coupled with image processing, were key in these approaches. We present an overview of the methods, and summarize some of our results in applying these advances to imaging biological specimens. The extension of spectral selection approaches to the mesoscopic domain, suitable for in vivo imaging is also illustrated, by fluorescence-based tumor visualization in the near infrared spectral region. Finally, some future directions are discussed.

The hyperspectral imaging endoscope: a new tool for in vivo cancer detection

We developed a new endoscope that allows for non-contact, rapid (sub-second) acquisition of polarized spectral images of tissue in vivo. The intent was to enable exploration of a variety of optical contrast mechanisms (such as light absorption, reflectance, scattering, and fluorescence) in a search for new methods of early cancer detection in a clinical setting. Our first new implementation for cancer detection is based on a body of spectroscopic work that employs elastic scattering (Mie) theory to estimate the size of bulk scatterers in a given medium - in our case, the epithelial tissue of lungs. This paper describes the novel design of the Hyperspectral Imaging Endoscope, and our initial experiences with employing it for the early detection of dysplasia and cancer in lung epithelia.

Approaches to Spectral Imaging Hardware

Instruments used for spectral, multispectral, and hyperspectral imaging in the biosciences have evolved significantly over the last 15 years. However, very few are calibrated and have had their performance validated. Now that spectral imaging systems are appearing in clinics and pathology laboratories, there is a growing need for calibration and validation according to universal standards. In addition, some systems produce spectral artifacts that, at the very least, challenge data integrity if left unrecognized. This unit includes a comparison of the band‐pass and light‐transmission characteristics of electronic tunable filters, interferometers, and wavelength‐dispersive spectral imaging instruments, as well as a description of how they work. Methods are described to test wavelength accuracy and perform radiometric calibration. A real‐life example of spectral artifacts is dissected in detail in order to show how to detect, diagnose, verify, and work around their presence when they cannot be eliminated. Curr. Protoc. Cytom. 53:12.20.1‐12.20.40.

Near-simultaneous hemoglobin saturation and oxygen tension maps in the mouse cortex during amphetamine stimulation.

Changes in neuronal activity lead to changes in oxygen consumption, glucose uptake, and blood flow in the brain. While a regional coupling between blood flow and oxygen consumption is generally observed during resting conditions, focal increases in neuronal activity may result in disproportionately larger increases in blood flow to the activated region.1 This alters the oxygenation state of blood and is thought to be the basis of functional activation using nuclear magnetic resonance imaging (f-MRI)2,3 and optical imaging using intrinsic signals.4,5 While f-MRI has become increasingly popular for studying functional activation in the human cortex, the physiologic events underlying the observed signal changes remain poorly characterized and controversial.6,7 In particular, the important dynamic relationship between the oxygen saturation of hemoglobin (SO2) and the blood oxygen tension (PO2) during activation has not been adequately addressed. In this study, we describe, the application of a new type of intravital microscope, using acoustooptic tunable filters (AOTFs),8–10 to the generation of both SO2 and PO2 maps at high-resolution in the cerebral cortex of mice during global neuronal activation by amphetamine administration. SO2 maps are generated from the spectral analysis of reflected absorbance images collected at different wavelengths and PO2 maps are generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement.