Phase-sensitive Images Research Articles

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca(2+)-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca(2+) concentration image using anin vitro-determined calibration curve. The Ca(2+) concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.

Fluorescence lifetime imaging

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created lifetime images of Y t-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.

Fluorescence lifetime imaging of free and protein-bound NADH.

We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe am imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.

Mapping of normal brain maturation in infants on phase-sensitive inversion-recovery MR images

It is illustrated that phase-sensitive inversion-recovery MR images are particularly well suited for the monitoring of brain maturation and myelination in the neonate and young infant. Provided appropriate timings are applied with the inversion-recovery MR pulse sequence, the myelinated areas show up as bright spots in the phase-sensitive images. The chronology of the appearance, and the location of these hyperintense zones correlate well with the chronology of brain maturation, as assessed by other means. In particular, the progressive functional capabilities of the infant correlate well with the progress of myelination, as exhibited by the MR images.

Intravascular signal in MR imaging: use of phase display for differentiation of blood-flow signal from intraluminal disease.

Intravascular signal from flowing blood is frequently observed on magnetic resonance (MR) images and may be indistinguishable from partial or complete vascular occlusion caused by thrombus or tumor. With a phase-display reconstruction method, qualitative assessment of large-vessel patency within the abdomen was undertaken in 15 healthy subjects and 12 patients with angiographically or surgically documented intravascular thrombus or tumor. Computed tomographic (CT) scans were available in all patients for correlation. MR studies were performed with a multisection spin-echo pulse sequence and two-dimensional Fourier transform spatial encoding. Data acquired from a single sequence was reconstituted in two ways to provide both routine anatomic images and a pictorial representation of large-vessel flow on a phase-sensitive image. With this method, reliable and easy differentiation of intraluminal thrombus and tumor from blood flow signal within large vessels was achieved. Information from these phase-display images compared favorably with findings from angiography and contrast-enhanced CT in the determination of luminal patency and obstruction.

Phase-insensitive pulse-echo imaging.

A method for reducing speckle in B-scan images using phase-insensitive detection is presented. This technique is based on the nonlinear processing of the detected output of an array of transducers. Using this method, phase-insensitive images of tissue phantoms have been compared to conventional phase-sensitive images of the same objects. The results of this comparison suggest that phase-insensitive imaging can reduce speckle in B-scan images.

Phase-insensitive pulse-echo imaging

A method for reducing speckle in B-scan images using phase-insensitive detection is presented. This technique is based on the nonlinear processing of the detected output of an array of transducers. Using this method, phase-insensitive images of tissue phantoms have been compared to conventional phase-sensitive images of the same objects. The results of this comparison suggest that phase-insensitive imaging can reduce speckle in B-scan images.

Ultrasonic Imaging Systems for Nondestructive Testing

Methods for detecting ultrasonic images are briefly reviewed, and discussed in light of their potential application to nondestructive test problems. In a typical nondestructive test system involving a water immersion tank, the ultrasonic image detector should respond to an ultrasonic intensity of 10−2 W/cm2 or less. This requirement eliminates from consideration number of detection methods involving photographic, chemical, or thermal mechanisms. Of the remaining image detection systems, only a few have been applied to nondestructive test problems. These include the surface relief technique, the Pohlman cell, and scanning methods involving piezoelectric transducers. The latter method has been most used for image applications, with mechanical movement of the transducer(s) in a C scan method. Electron scan of a single piezoelectric detector to provide a television presentation is coming into wider use. A description of one such system is given. For a thin, flat inspection object, the system offers good inspection speed, resolution, and sensitivity to inhomogeneities. Thick, irregularly shaped inspection objects present problems because lens systems are involved. Lenses for imaging are not well developed. Also, the use of a lens tends to slow the inspection of a thick object because one must examine the object by focusing at different depths. Pulsed television systems and holographic techniques offer some promise in that area. A new phase-sensitive color television ultrasonic image system offers hope for improved detection of subtle discontinuities.