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PubMed2001

Applications of spectral imaging: detection and analysis of human melanoma and its precursors.

Light-based imaging has extensive applications for medicine and biology, and recent advances in optical imaging modalities, such as confocal and multi-photon scanning fluorescence microscopy, bioluminescence, optical coherence tomography, and spectral imaging, have opened new avenues for visualizing and recording over time dynamic changes in genetic, developmental, and disease mechanisms that cannot be captured by conventional light microscopy. In the present article, we focus on spectral imaging, and using human melanoma and its precursor lesions as an example, we describe the ability of spectral imaging to detect early-stage disease, capture gene expression profiles in tissue specimens, and visualize gene functions in tumors growing in living animals.

Animals↗

PubMed2005

Real-time fusion of endoscopic views with dynamic 3-D cardiac images: a phantom study.

Minimally invasive robotically assisted cardiac surgical systems currently do not routinely employ 3-D image guidance. However, preoperative magnetic resonance and computed tomography (CT) images have the potential to be used in this role, if appropriately registered with the patient anatomy and animated synchronously with the motion of the actual heart. This paper discusses the fusion of optical images of a beating heart phantom obtained from an optically tracked endoscope, with volumetric images of the phantom created from a dynamic CT dataset. High quality preoperative dynamic CT images are created by first extracting the motion parameters of the heart from the series of temporal frames, and then applying this information to animate a high-quality heart image acquired at end systole. Temporal synchronization of the endoscopic and CT model is achieved by selecting the appropriate CT image from the dynamic set, based on an electrocardiographic trigger signal. The spatial error between the optical and virtual images is 1.4 +/- 1.1 mm, while the time discrepancy is typically 50-100 ms. Index Terms-Image guidance, image warping, minimally invasive cardiac surgery, virtual endoscopy, virtual reality.

Algorithms↗

PubMed1990

Direct observation of receptors and images in simple and compound eyes.

The relation between the quality of the optical image and the fineness of the retinal mosaic has been studied in eyes of three different optical types: the simple eyes of spiders, the superposition compound eyes of moths, and the apposition compound eyes of butterflies. In all three it is possible to observe both the receptor mosaic and the image in the living eye, using appropriate ophthalmoscopic techniques. Whereas in humans the retinal sampling frequency approaches the optical cut-off frequency quite closely, in diurnal insects of both types the image is undersampled by a factor of 2-3, and in crepuscular spiders this factor may be greater than 100. Reasons for these differences are discussed.

Animals↗

PubMed2000

Gaussian imaging transformation for the paraxial Debye formulation of the focal region in a low-Fresnel-number optical system

The Debye formulation of focused fields has been systematically used to evaluate, for example, the point-spread function of an optical imaging system. According to this approximation, the focal wave field exhibits some symmetries about the geometrical focus. However, certain discrepancies arise when the Fresnel number, as viewed from focus, is close to unity. In that case, we should use the Kirchhoff formulation to evaluate accurately the three-dimensional amplitude distribution of the field in the focal region. We make some important remarks regarding both diffraction theories. In the end we demonstrate that, in the paraxial regime, given a defocused transverse pattern in the Debye approximation, it is possible to find a similar pattern but magnified and situated at another plane within the Kirchhoff theory. Moreover, we may evaluate this correspondence as the action of a virtual thin lens located at the focal plane and whose focus is situated at the axial point of the aperture plane. As a result, we give a geometrical interpretation of the focal-shift effect and present a brief comment on the problem of the best-focus location.

Journal Article↗

PubMed2000

Automated analysis of normal and glaucomatous optic nerve head topography images.

PURPOSE: To classify images of optic nerve head (ONH) topography obtained by scanning laser ophthalmoscopy as normal or glaucomatous without prior manual outlining of the optic disc. METHODS: The shape of the ONH was modeled by a smooth two-dimensional surface with a shape described by 10 free parameters. Parameters were adjusted by least-squares fitting to give the best fit of the model to the image. These parameters, plus others derived from the image using the model as a basis, were used to discriminate between normal and abnormal images. The method was tested by applying it to ONH topography images, obtained with the Heidelberg Retina Tomograph, from 100 normal volunteers and 100 patients with glaucomatous visual field damage. RESULTS: Many of the parameters derived from the fits differed significantly between normal and glaucomatous ONH images. They included the degree of surface curvature of the disc region surrounding the cup, the steepness of the cup walls, the goodness-of-fit of the model to the image in the cup region, and measures of cup width and cup depth. The statistics of the parameters were analyzed and were used to construct a classifier that gave the probability, P(G), that each image came from the glaucoma population. Images were classified as abnormal if P(G) > 0.5. The probabilities assigned to each image were in most cases close to 0 (normal) or 1 (abnormal). Eighty-seven percent of the sample was confidently classified with P(G) < 0.3 or P(G) > 0.7. Within this group, the overall classification accuracy was 92%. The overall accuracy of the method (the mean of sensitivity and specificity, which were similar) in the whole sample was 89%. CONCLUSIONS: ONH images can be classified objectively and dependably by an automated procedure that does not require prior manual outlining of disc boundaries.

Adult↗

PubMed2002

Computer technique for digital radiographic images correction based on the digitizer characteristic curve.

This article presents a computer correction technique for radiographic digital images based on digitization process inaccuracy in pixel gray levels relative to the respective image optical densities. The technique consists of determining the digitizer characteristic curve by digitizing a step-wedge radiographic image with known optical densities. Calibration is done by determining the mathematical function that automatically adjusts the pixel gray levels to the expected values according to the manufacturer. Two laser film digitizers were investigated for which discrepancies between measured and expected pixel values were reported. For these cases, the proposed algorithm automatically adjusted the pixel gray levels of the digitized images. Using this method, the digital images show more accurate values of gray levels compared to the original radiographic image. In addition, it allows the development of uniform images databases.

Image Processing, Computer-Assisted↗

PubMed2005

Perfusion-based fMRI: insights from animal models.

Modern functional neuroimaging techniques, including functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and optical imaging of intrinsic signals (OIS), rely on a tight coupling between neural activity and cerebral blood flow (CBF) to visualize brain activity using CBF as a surrogate marker. Because CBF is a uniquely defined physiological parameter, fMRI techniques based on CBF contrast have the advantage of being specific to tissue signal change, and the potential to provide more direct and quantitative measures of brain activation than blood oxygenation level-dependent (BOLD)- or cerebral blood volume (CBV)-based techniques. The changes in CBF elicited by increased neural activity are an excellent index of the magnitude of electrical activity. Increases in CBF are more closely localized to the foci of increased electrical activity, and occur more promptly to the stimulus than BOLD- or CBV-based contrast. In addition, CBF-based fMRI is less affected by confounds from venous drainage common to BOLD. Animal studies of brain activation have yielded considerable insights into the advantages of CBF-based fMRI. Based on results provided by animal studies, CBF fMRI may offer a means of better assessing the magnitude, spatial extent, and temporal response of neural activity, and may be more specific to tissue state. These properties are expected to be particularly useful for longitudinal and quantitative fMRI studies.

Animals↗

PubMed2001

Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein.

Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high-speed imaging, intrinsic or extrinsic dye-mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein-based and DNA-encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage-sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time-scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.

Action Potentials↗

PubMed1990

[A new laser scan system for video ophthalmoscopy. Initial clinical experiences also in relation to digital image processing].

The clinical advantages of a scanning laser ophthalmoscope (SLO) and video imaging of fundus pictures are described. Image quality (contrast, depth of field) and imaging possibilities (confocal stop) are assessed. Imaging with different lasers (argon, He-Ne) and changes in imaging rendered possible by confocal alignment of the imaging optics are discussed. Hard copies from video images are still of inferior quality compared to fundus photographs. Methods of direct processing and retrieval of digitally stored SLO video fundus images are illustrated by examples. Modifications for a definitive laser scanning system - in regard to the field of view and the quality of hard copies - are proposed.

Computer Systems↗

PubMed2002

Magnetic resonance diffractive imaging.

Magnetic resonance (MR) diffractive imaging is proposed as a new approach to MR angiography. The expression of the nuclear MR signal is similar to the equation for the Fresnel diffraction of a three-dimensional (3-D) object in light or sound waves. The proposed technique offers the possibility of fast angiographic imaging and the on-line reconstruction of 3-D volumetric images using the holographic technique. Static imaging experiments using an ultra-low-field MRI system are performed to verify the feasibility of the technique. It is shown that the images focused on an arbitrary plane can be reconstructed from data scanned in two dimensions, even though blurred image data is superimposed on the image. Moreover, the 3-D image can be observed in a coherent optical imaging system. This study demonstrates the possibility of the proposed method as a fast imaging technique for MR angiography.

Feasibility Studies↗

PubMed2006

High-resolution ultrasound reflex transmission imaging and digital photography: potential tools for the quantitative assessment of pigmented lesions.

BACKGROUND/AIMS: High-resolution ultrasound (HRU) is a relatively cheap imaging method that shows small quantitative differences between benign naevi and melanoma. Previous studies using B-mode display suggest that these arise from their differing attenuating properties. Attenuation characteristics, however, are better evaluated using reflex transmission imaging (RTI). White light clinical (WLC) photography is an even cheaper imaging method that is routinely used for monitoring but less frequently in everyday diagnosis. As features from each method may have an independent origin, two such modalities may be of greater diagnostic value than either method alone. However, although quantitative analysis of digital photographs is being developed to aid tumour diagnosis, in vivo RTI for the evaluation of pigmented skin lesions has not previously been described. This paper presents the feasibility of performing RTI in vivo and evaluates the reliability of the objective features used. The potential of the combination of quantitative RTI and white light (WL) digital photography data for the classification of pigmented lesions was assessed. METHODS: Randomly selected patients were recruited via a skin cancer screening clinic. RTI data were acquired from each index lesion with a 20 MHz single-element scanner. WL images were taken using a high-resolution (2.8 Mpixels) digital camera. Quantitative features calculated from both images were used to derive a discriminant rule. This equation was then applied to reclassify each case based on its quantitative criteria. The resultant classification was compared with histological diagnosis. RESULTS: Twenty-four lesions (10 melanoma and 14 naevi) were studied. On RTI, no subjective differences were observed between benign naevi and melanoma. Many lesions were either not visible on RTI or lacked clearly definable borders. Consequently, the WL photographs were used to draw lesion boundaries on RT images for feature calculation. Melanoma were less attenuating than naevi on RTI (P=0.026) and had greater red colour variegation on WL imaging (P=0.016). The combination of quantitative parameters (two from RTI and four from photographs) improved sensitivity for this sample without compromising the specificity of 100% compared with either modality alone. The procedure is highly reproducible (r=0.85 between two operators). CONCLUSIONS: Pigmented skin lesions can be quantitatively defined from RTI data acquired in vivo and a significant difference in attenuation is shown. However, accurate registration of the RT image with a corresponding photograph was crucial for this purpose and only possible when corresponding points could be reliably identified on both images. Combination of features from ultrasound and optical images may synergistically improve diagnostic accuracy and a larger study is warranted to investigate this.

Dermoscopy↗

PubMed1992

The HOME microscope workstation. A new tool for cervical cancer screening.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope design for assisting pathologists and cytotechnologists in routine clinical tasks. The prototype system consists of an IBM PC-compatible computer and a light microscope in which a built-in high-resolution computer display image is super-imposed on the optical image of the specimen. Also, an encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on the stage to be uniquely defined. Software, written in C language and running under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor. A specific application has been developed for cervical cancer screening, taking into account the needs and constraints of microscopists performing this task. Informatics tools offered by the HOME system provide them with precise flagging and relocation of objects on the slide, control of the scanning pathway, and ability to write and print the report directly through the microscope. The computer files generated by microscopic examination are stored and contain information available for quality control assessment and laboratory management.

Cytodiagnosis↗

PubMed1994

Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.

Light scattering by brain tissue and phototoxicity are major obstacles to the use of high-resolution optical imaging and photo-activation ('uncaging') of bioactive compounds from inactive ('caged') precursors in intact and semi-intact nervous systems. Optical methods based on 2-photon excitation promise to reduce these obstacles (Denk, 1994; Denk et al., 1990, 1994). Here we show a range of imaging modes based on 2-photon laser scanning microscopy (TPLSM) as applicable to problems in neuroscience. Fluorescence images were taken of neurons labeled with ion-sensitive and voltage-sensitive dyes in invertebrate ganglia, mammalian brain slices, and from the intact mammalian brain. Scanning photochemical images with whole-cell current detection (Denk, 1994) show how the distribution of neurotransmitter receptors on the surface of specific cells can be mapped. All images show strong optical sectioning and usable images can be obtained at depths greater than 100 microns below the surface of the preparation.

Animals↗

PubMed2000

Consistency behind trial-to-trial variation in intrinsic optical responses to single-whisker movement in the rat D1-barrel cortex.

We examined consistent characteristics behind the trial-to-trial variati on in intrinsic optical imaging of single barrel cortical responses to D 1-whisker movement in 2-5-week postnatal (2-5 W) and adul t (>9-weeks) Wistar rats, and we identified the effective are a of the neural response. The extent/size, configuration and orientation of the intrinsic optical response area varied from trial-to-trial with the same whisker stimulation. We argue that the trial-to-trial variation was due to cortical blood circulation related to the barrel neural activity. Subsequently, interpolating a family of the traces of the optical response area imaged with repeated stimulation for each animal, we extracted a centered circular area from the trial-to-trial response for each animal. Although the trial-to-trial variation decreased gradually with age, the spatial extent of the interpolated response area was consistently about 660 microm in diameter, in agreement with that measured morphologically and/or histochemically. A possible interpretation is that the optically defined area appears to image the actual effective single-barrel response area, as a first approximation. Furthermore, the constancy of the extracted area independent of age suggests that the barrel cortex is, in fact, virtually mature by 2 weeks of age. The extracted area was also nearly independent of the frequency (>/=5 Hz) of whisker movement.

Animals↗

PubMed2006

In vivo measurement of retinal physiology with high-speed ultrahigh-resolution optical coherence tomography.

Noninvasive in vivo functional optical imaging of the intact retina is demonstrated by using high-speed, ultrahigh-resolution optical coherence tomography (OCT). Imaging was performed with 2.8 microm resolution at a rate of 24,000 axial scans per second. A white-light stimulus was applied to the dark-adapted rat retina, and the average reflectivities from different intraretinal layers were monitored as a function of time. A 10%-15% increase in the average amplitude reflectance of the photoreceptor outer segments was observed in response to the stimulus. The spatial distribution of the change in the OCT signal is consistent with an increase in backscatter from the photoreceptor outer segments. To our knowledge, this is the first in vivo demonstration of OCT functional imaging in the intact retina.

Animals↗

PubMed2001

Second-harmonic imaging microscopy of living cells.

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. SHG shares many of the advantageous features for microscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce three-dimensional images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It can be used to image membrane probes with high membrane specificity and displays extraordinary sensitivity in reporting membrane potential; it also has the ability to image highly ordered structural proteins without any exogenous labels.

Animals↗

PubMed2003

A two-photon and second-harmonic microscope.

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.

Animals↗

PubMed2000

High-density fiber-optic DNA random microsphere array.

A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a custom-built imaging system. Hybridization was visualized using fluorescent-labeled DNA targets with a detection limit of 10 fM. Hybridization times of seconds are required for nanomolar target concentrations, and analysis is performed in minutes.

Base Sequence↗