A microarray-image model is used that takes into account many factors, including spot morphology, signal strength, background fluorescent noise, and shape and surface degradation. The model yields synthetic images whose appearance and quality reflect that of real microarray images. The model is used to link noise factors to the fidelity of signal extraction with respect to a standard image-extraction algorithm. Of particular interest is the identification of the noise factors and their interactions that significantly degrade the ability to accurately detect the true gene-expression signal. This study uses statistical criteria in conjunction with the simulation of various noise conditions to better understand the noise influence on signal extraction for cDNA microarray images. It proposes a paradigm that is implemented in software. It specifically considers certain kinds of noise in the noise model and sets these at certain levels; however, one can choose other types of noise or use different noise levels. In sum, it develops a statistical package that can work in conjunction with the existing image simulation toolbox.

A method for polarization filtering, correlation processing, and wavelet analysis of coherent images of physiologically normal and necrotically changed (myocardium infarct) muscle tissue is presented. A technique for early optical diagnosis of the appearance of these biological tissues and the course of their degenerative-dystrophic changes is proposed.

Optical methods are becoming commonplace in investigations of the physical and mechanical behavior of biological tissues. Digital image correlation (DIC) is a versatile optical method that shows tremendous promise for applications involving biological tissues and biomaterials. We present the fundamentals of DIC with an emphasis on the application to biological materials. An approach for surface preparation is described that facilitates its application to hydrated substrates. Three examples are presented that highlight the use of DIC for biomedical research. The first example describes the use of DIC to study the mechanical behavior of arterial tissues up to 40% elongation. The second example describes an evaluation of the mechanical properties of bovine hoof horn in the dehydrated and fully hydrated states. Uniaxial tension experiments are performed to determine the elastic modulus (E) and Poisson's ratio (ν) of both the arterial and dermal tissues. Spatial variations in the mechanical properties are evident from the full-field characterization of both tissues. Finally, an application of DIC to study the evolution of loosening in cemented total hip replacements is described. The noncontact analysis enables measurement of the relative displacement between the bone/bone cement and bone cement/prosthesis interfaces. Based on the elementary optical arrangement, the simple surface preparation, and the ability to acquire displacement/strain measurements over a large range of deformation, DIC should serve as a valuable tool for biomedical research. Further developments will enable the use of DIC for in vivo applications.

To estimate the concentrations of melanin and blood and the oxygen saturation in human skin tissue, we propose a method using a multiple regression analysis aided by a Monte Carlo simulation for diffuse reflectance spectra from the skin tissue. By using the absorbance spectrum as a response variable and the extinction coefficients of melanin, oxygenated hemoglobin, and deoxygenated hemoglobin as predictor variables, the multiple regression analysis gives regression coefficients. The concentrations of melanin and blood are determined from the regression coefficients using conversion vectors that are estimated numerically in advance, while the oxygen saturation is obtained directly from the regression coefficients. Numerical and experimental investigations were performed for layered skin tissue models and phantoms. Measurements of human skin were also carried out to monitor variations in the melanin and blood contents and oxygenation during cuff occlusion. The results confirmed the usefulness of the proposed method.

Laser-induced heating in an ocular phantom is measured with magnetic resonance thermography (MRT) using temperature-dependent phase changes in proton resonance frequency. The ocular phantom contains a layer of melanosomes isolated from bovine retinal pigment epithelium. The phantom is heated by the 806-nm output of a continuous wave diode laser with an irradiance of 2.4 to 21.6 W/cm² in a beam radius of 0.8 or 2.4 mm, depending on the experiment. MRT is performed with a 2T magnet, and a two-turn, 6-cm-diam, circular radio frequency coil. Two-dimensional temperature gradients are measured within the plane of the melanin layer, as well as normal to it, with a temperature resolution of 1°C or better. The temperature gradients extending within the melanin layer are broader than those orthogonal to the layer, consistent with the higher optical absorption and consequent heating in the melanin. The temperature gradients in the phantom measured by MRT closely approximate the predictions of a classical heat diffusion model. Three-dimensional temperature maps with a spatial resolution of 0.25 mm in all directions are also made. Although the temporal resolution is limited in the prototype system (22.9 s for a single image "slice"), improvements in future implementations are likely. These results indicate that MRT has sufficient spatial and temperature resolution to monitor target tissue temperature during transpupillary thermotherapy in the human eye.

The feasibility of ultrahigh resolution optical coherence tomography (UHR OCT) to image ex vivo and in vitro brain tissue morphology on a scale from single neuron cells to a whole animal brain was investigated using a number of animal models. Sub-2-μm axial resolution OCT in biological tissue was achieved at different central wavelengths by separately interfacing two state-of-the-art broad bandwidth light sources (titanium:sapphire, Ti:Al₂O₃ laser, λ_c = 800 nm, Δλ= 260 nm, P_out = 50 mW and a fiber laser light source, λ_c = 1350 nm, Δλ = 470 nm, P_out = 4 mW) to free-space or fiber-based OCT systems, designed for optimal performance in the appropriate wavelength regions. The ability of sub-2-μm axial resolution OCT to visualize intracellular morphology was demonstrated by imaging living ganglion cells in cultures. The feasibility of UHR OCT to image the globular structure of an entire animal brain as well as to resolve fine morphological features at various depths in it was tested by imaging a fixed honeybee brain. Possible degradation of OCT axial resolution with depth in optically dense brain tissue was examined by depositing microspheres through the blood stream to various depths in the brain of a living rabbit. It was determined that in the 1100 to 1600-nm wavelength range, OCT axial resolution was well preserved, even at depths greater than 500 μm, and permitted distinct visualization of microspheres 15 μm in diameter. In addition, the OCT image penetration depth and the scattering properties of gray and white brain matter were evaluated in tissue samples from the visual cortex of a fixed monkey brain.

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 µs and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 μs, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.

For complex biological systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components cannot readily provide a true representation of the underlying fluorescence dynamics. We investigate an alternative nonparametric method for the analysis of time-resolved fluorescence data from biochemical and biological systems based on the expansion of fluorescence decay in a discrete Laguerre basis. We report that a unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution significantly faster than conventional multiexponential approximation methods. The Laguerre expansion coefficients are shown to be highly correlated with intrinsic fluorescence lifetimes and allow direct characterization of the fluorescence dynamics. A novel method for prediction of concentrations in mixtures of biochemical components using these coefficients is developed and successfully tested (prediction error <2%) using data from different mixtures of fluorescence lifetime standards. These findings suggest that the use of Laguerre expansion coefficients is a fast approach for the characterization and discrimination of complex biological systems such as tissues and cells, and that the method has potential for applications of fluorescence lifetime techniques to tissue diagnostics and imaging microscopy of living cells.

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein–protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein–protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches.

High-contrast imaging of bladder cancer is demonstrated using near-infrared autofluorescence under long-wavelength laser excitation in combination with cross-polarized elastic light scattering. Fresh unprocessed surgical specimens obtained following cystectomy or transurethral resection were utilized and a set of images for each tissue sample was recorded. These images were compared with the histopathology of the tissue sample. The experimental results indicate that the intensity of the near-infrared emission as well as that of the cross-polarized backscattered light was considerably different in cancer tissues than in that of the contiguous nonneoplastic tissues, allowing an accurate delineation of a tumor's margins.

The effect of photodynamic therapy (PDT) treatment on the metabolic state of tumor mitochondria is investigated by imaging of tumor redox status. PDT is performed using the photosensitizer pyropheophorbide-2-deoxyglucosamide (Pyro-2DG), which utilizes the glucose import pathway. It is found that Pyro-2DG-induced PDT resulting in a highly oxidized state of tumor mitochondria. This is determined from the redox ratio changes derived from the intrinsic oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) [i.e., reduced nicotinamide adenine dinucleotide (NADH)] fluorescence signals observed using a cryoimager. Thus, the redox ratio is a sensitive indicator for providing reliable and informative measurements of PDT-induced tissue damage. In the PDT treated region of the tumor, highly oxidized flavoprotein and diminishing NADH fluorescence is detected, suggesting that flavoprotein and NADH are oxidized by singlet oxygen produced in the photosensitization process.

One hallmark of cell death resulting from prolonged ischemia is cell membrane disruption. We apply optical spectroscopy to gauge membrane disruption in isolated rat hearts by monitoring (1) the washout of myoglobin (Mb) and (2) the accumulation of an exogenous contrast agent in permeabilized cells. The contrast agent, a neodymium (Nd) chelate, has several absorptions in the visible and near-IR, and when present in the perfusate, it cannot penetrate cellular membranes. When membrane integrity is disrupted, however, it is expected to accumulate within the intracellular space; moreover, cellular Mb is expected to wash out. To test this hypothesis, rat hearts (n = 12) are perfused with Krebs-Henseleit buffer (KHB), followed by perfusion with KHB in which a 5 mM Nd-DTPA solution is present. Membrane damage is then induced by infusion of digitonin into the Nd-KHB perfusate to provide a digitonin concentration of 2.5, 5, or 10 μg/mL. After 30 min of infusion, Mb levels fall to 46±14% of baseline levels and Nd-DTPA rises to 161±19% of predigitonin levels. No apparent dependence of total membrane disruption on digitonin concentration over the concentration range studied is found, although higher concentrations do lead to more rapid membrane disruption.

Near-infrared spectroscopy (NIRS) was used to measure frontal regional cerebral blood volume (rCBV) in a person whose brain was under the influence of pharmacological agents while the person was performing a complex task. Fourteen healthy participants were administered Uchida-Kraepelin psychodiagnostic (UKP) tests before and after caffeine intake, and the concentration of caffeine in the urine was measured. The average number of answers and the average number of correct answers given by the participants improved significantly following caffeine intake. During the UKP testing, changes in the rCBV in the inferior frontal cortex were continuously measured using NIRS. The volume during the rest periods decreased as a result of caffeine-induced constriction of the cerebral arteriola. The volume increased during the mental work, but the degree of the increase was the same before and after caffeine intake. Although the performance of the mental work improved following caffeine intake, the improvement was not reflected in the rCBV in the inferior frontal cerebral cortex. These results suggest that caffeine helps to protect the brain from excessive hyperemia in addition to activating the neurons in the prefrontal cortex.

There is increasing attention to prevention as a means to reduce cancer incidence. Prevention interventions or therapies in turn rely on risk assessment programs to identify those women most likely to benefit from education and lifestyle changes. These programs are usually based either on interviews to identify ethnic, genetic, and lifestyle factors contributing to risk or on physical examination of the breast. For the latter it has been shown that the parenchymal density pattern observed in X-ray mammography can be used to assess an individual's risk. Extensive areas of dense, glandular tissue that are relatively radio-opaque are associated with higher breast cancer risk, with an odds ratio of 4 to 6 compared with women in whom the breast density is low owing to an abundance of adipose tissue. Near-infrared optical transillumination spectroscopy has been used previously to investigate the physiological properties of breast tissue. In this study, women were recruited who underwent recently X-ray mammography. The tissue density was assessed by a radiologist. The women then underwent optical transillumination spectroscopy, for which an instrument was developed that delivered visible and near-infrared light to the breast. After being transmitted through the breast craniocaudally in one of four quadrants, the spectrum from 625 to 1050 nm was measured. The spectra were used as input to a Principal Component Analysis (PCA) that used the corresponding mammographic density as the reference standard. The study group consisted of 92 women aged 39 to 72 years. Without further stratification for age, menopausal status, or measurement position, the PCA numerical model predicted the radiological assessment of tissue density in the mid 80% to low 90%.

Nonintrusive, noncontacting frequency-domain photothermal radiometry (FD-PTR or PTR) and frequency-domain luminescence (FD-LUM or LUM) have been used with 659-nm and 830-nm laser sources to detect artificial and natural subsurface defects in human teeth. The major findings of this study are (1) PTR is sensitive to very deep (>5 mm) defects at low modulation frequencies (5 Hz). Both PTR and LUM amplitudes exhibit a peak at tooth thicknesses of ca. 1.4 to 2.7 mm. Furthermore, the LUM amplitude exhibits a small trough at ca. 2.5 to 3.5 mm. (2) PTR is sensitive to various defects such as a deep carious lesion, a demineralized area, an edge, a crack, and a surface stain, while LUM exhibits low sensitivity and spatial resolution. (3) PTR frequency scans over the surface of a fissure into demineralized enamel and dentin show higher amplitude than those for healthy teeth, as well as a pronounced curvature in both the amplitude and phase signal channels. These can be excellent markers for the diagnosis of subsurface carious lesions. (4) PTR amplitude frequency scans over the surface of enamels of variable thickness exhibit strong thickness dependence, thus establishing depth profilometric sensitivity to subsurface interfaces such as the dentin/enamel junction.

The results of an experimental study of the possibilities of monitoring erbium yttrium aluminum garnet laser-mediated ablation of human epidermis with the use of Fourier transform infrared (FTIR) spectroscopy and spectral polarization techniques are presented. The attenuated total reflection (ATR) method was used for FTIR spectroscopic measurements. Spectral polarization monitoring of the ablation was carried out by analyzing the spectra of the degree of residual linear polarization of a probe light diffusely reflected from the laser-treated region of skin. It was found that the analysis of FTIR spectra allows monitoring of the water and protein contents in the subsurface layers of the treated skin, while the degree of residual polarization measured at the wavelengths of maximal absorption of hemoglobin is sensitive to changes in the epidermis thickness and the blood content in the dermal layer (the degree of erythema).

Near infrared (NIR) diode laser low-intensity (soft) phototherapy with the topical application of indocyanine green (ICG) has been suggested for treatment of acne vulgaris. Twelve volunteers with acne lesions on their faces and/or backs were enrolled in the experiment. Skin areas of the subjects that were 4×5 cm² were stained with ICG solution for 5 min before laser irradiation (803 nm) at a power density up to 50 mW/cm² for 5 to 10 min. For 75% of the subjects, a single treatment was provided and for the other 25%, eight sequential treatments over a period of a month were carried out. Observations a month after the completion of the treatment showed that only the multiple treatments with a combination of ICG and NIR irradiation reduced inflammation and improved the state of the skin for a month without any side effects. A month after treatment, the improvement was about 80% for the group receiving multiple treatments. Single treatments did not have a prolonged effect.

Hearing aid shells (or earmolds) must couple the hearing aid with the user's ear. Earmolds have to fit the subject's outer ear canal properly to ensure a good performance of the aid. Because of the great variability in the anatomical pattern of the ear, earmolds are custom made. At present, an impression of the subject's ear canal is taken and used to fabricate the silicon-made mold. The postimpression activities that typically are performed during the fabrication process modify the physical dimensions of the resulting earmold and thus affect the fit of the product. A novel system for 3-D laser scanning and mesh reconstruction of the surface of ear canal impressions is presented. The reconstructed impression can be digitally stored and passed directly to dedicated CAD 3-D printing machines to model the silicon earmold and thus achieve the best possible fit. The proposed system is based on a couple of cameras and a commercial laser for the surface digitization and on a straightforward algorithm, based on the deformation of a geometric model, for the reconstruction of the acquired surface. Measurements on objects of well-known geometric features and dimensions are performed to assess the accuracy and repeatability levels of this 3-D acquisition system. Robustness to noise of the proposed reconstruction algorithm is determined by simulations with a synthetic test surface. Finally, the first measurements (acquisition+reconstruction) of closed surfaces from ear canal impressions are reported.

This PDF file contains the editorial “Book Review: Applied Laser Medicine” for JBO Vol. 9 Issue 04