This PDF file contains the editorial “Guest Editorial: Special Section on Light Scatter and Fluorescence of the Eye Lens” for JBO Vol. 1 Issue 03

This paper reviews previous work done to demonstrate the clinical relevance of the measurement of bluegreen autofluorescence and light transmission of the ocular lens (l=450 to 550 nm). These can be determined quantitatively with fluorophotometry in a few seconds. Autofluorescence and transmission values are determined in healthy volunteers, in patients with insulin-dependent diabetes mellitus, and in patients with untreated glaucoma or untreated ocular hypertension. The lens autofluorescence of healthy volunteers increased linearly and transmission decreased exponentially with age. Each year of diabetes induced an increase of autofluorescence equal to one extra year of age. Untreated glaucoma or ocular hypertension had no significant effect on lens autofluorescence and transmission. Increased autofluorescence and decreased transmission values in comparison with values of a healthy population are proved to be indicative for an increased risk of developing cataract and the clinical usefulness of these measures is demonstrated. Diabetes is a risk factor for developing cataract while untreated glaucoma or ocular hypertension is not.

This critical survey of studies on the fluorescence of human eye lenses covers basic problems of metrology and their relation to comparative measurements on extracts, on in vivo, and on in vitro material. Attention is drawn to the effects of lenticular refraction and absorbance, and to potential photic involvement on the part of ambient illumination. The extent to which fluorescence may interfere with vision is also reviewed.

Recent data in the literature on psychophysically determined in vivo intraocular straylight, and on in vitro fluorescence of the eye lens are analyzed. From the psychophysical straylight data, light scattering changes in the lens due to normal aging and age-related cataract formation are derived in physical terms. The intensities of these light-scattering changes prove to follow approximately cu p (u=scatter angle) with p'−2 and c dependent on age and cataract. Both p and c are in accordance with recent in vitro studies on light scattering using donor lenses. Fluorescence of the lens causes light with wavelengths of 420 nm and lower to be in total visually much more effective: by a factor of 2.7 to 6.8 at 400 nm, and a factor of 71 to 151 at 380 nm. Because fluorescence adds a homogeneous veil to the point spread function, for some visual effects (e.g., glare) the increase can be (much) larger.

As the crystalline lens ages and as cataracts develop, highly concentrated proteins in lens fiber cells undergo rearrangements leading to substantial increases in scattered light. By exploiting a relationship between scattered light and refractive index, it is shown that a rearrangement of proteins necessarily leads to increased index as well. This change in refractive index is a second-order effect and must be quite small in most cases. However, it offers a possible explanation for the increase in index and refractive power associated with the development of nuclear cataract.

Rat lenses incubated in dimethyl sulfoxide (DMSO)–water binary mixtures of different compositions became turbid. A slight haziness developed up to 0.062 mole fraction of DMSO; at higher concentrations dense turbidity developed. Microscopic examination of the incubated rat lenses showed damage restricted to the epithelium and outer cortex at low DMSO concentrations; maximum damage occurred, in terms of fiber cell swelling, extracellular fluid (lake) formation and disintegration of epithelium, at 0.25 mole ratio of DMSO. Scanning electron microscopic observation showed that at high DMSO concentrations, especially at 0.25 mole fraction, the contents of the fiber cells were greatly damaged. Apparently a large part of the crystallins had coalesced around the cytoskeletal bodies. Polarized light-scattering intensity measurements and their analyses indicated that when dense opacities developed at high DMSO concentrations, the major contribution to the turbidity came from the optical anisotropy fluctuations. The change in the organization of the components within the fiber cells disturbs the balance between intrinsic birefringence and form birefringence necessary for transparency. Thus, the DMSO-caused opacification can be described as a good model for the involvement of optical anisotropy fluctuations in cataractogenesis.

We have developed a new lens measurement system (LMS) that simultaneously measures the intensities of fluorescence and Rayleigh components at various distances into the lens along the optical axis. The noninvasive measurement is performed through an undilated pupil, and with the assistance of a pupil tracking system that facilitates maintaining the x and y positions of the sample volume (ca. 300 mm in length, 600 mm high, and 80 mm wide) to within 6100 mm of any programmed "lock" position. The intensity of the Rayleigh component that is used to normalize the measured fluorescent signal serves to correct the attenuation effects due to absorption and lens light scatter (Mie–Tyndall type). This report, resulting from a SpectRx Site L clinical study using a refined instrumentation (SpecRx Scan V), presents analysis of fluorescence and Rayleigh data from the lenses of 923 controls and 239 diabetic subjects (45 Type I and 194 Type II) ranging from 23 to 75 years old. Fluorescence and Rayleigh data have been obtained via confocal mode from various locations nominally along the lens optical axis (the true trajectory of a sample volume could be shown in a figure) for controls and diabetics, at different ages, using three pairs of excitation and collection wavelengths: 364/495 nm, 434/495 nm, and 485/515 nm. For control subjects, there exists a strong, almost linear relationship between age and fluorescence, while diabetic subjects tend to deviate from this age fluorescence relationship. Our data show that the lenses of diabetic patients are subject to an accelerated aging process, presumably due to an elevated level of brown and fluorescent protein adducts and crosslinks from nonenzymatic glycosylation (Maillard-Amadori reactions). We have also shown that by using the measured Rayleigh profiles to normalize the measured fluorescence, most of the absorption effects are removed and therefore the separation between the fluorescence of diabetics and controls is greatly improved.

An in vivo human lens containing a cataract has been visualized by a series of orthogonal slices made through a three-dimensional volume reconstruction. Data acquisition was made with a transformed series of 60 rotated Scheimpflug digital images. Each digital image represents the light scatter from the lens in a plane that contains the optic axis. At each angular position of the camera, a digital image of the in vivo ocular lens was acquired. The set of 60 Scheimpflug digital images was mathematically transformed into a new data set in which the images were oriented perpendicular to the optic axis of the eye. The transformed set of optical sections was aligned to correct for small eye movements during the data collection process. In order to visualize the volume of the in vivo human lens, slices were projected through the lens volumetric data set. The use of orthogonal slices to visualize lenticular light scatter represents a new technique to visualize human cataracts in vivo in three dimensions.

Optical methods have always played an important role in the study of physiological phenomena. Light microscopes have been used for more than one hundred years to gain insight into the structure of biological systems in general and the nervous system in particular. Anatomical studies had given us a first understanding of shapes and types of nerve cells. Another example is the ubiquitous and rarely mentioned use of the light microscope to guide electrophysiological recording electrodes toward their targets. Only in the last two decades, however, has light microscopy become widely used to gather quantitative dynamic information about physiological processes. The list of examples includes: the study of diffusion in membranes with fluorescence methods;3–6 the detection of transmembrane potential changes using intrinsic signals7 or extrinsic fluorophores (for reviews see Refs. 8, 9, and 10); the measurements of nanometer displacements;11–14 and the quantification of changes in intracellular ion concentration using fluorescent indicator dyes (for a review see Ref. 15). More recently, light has also been used to trigger physiological processes by applying biological effector molecules with high spatial and temporal resolution via photochemical release, commonly called ‘‘uncaging.’’16–19 In this article, I review the contribution that the combination of two-photon absorption with laser scanning20 has made to functional biological imaging in recent years.

In this paper the sensitivity of a phased array and a continuous wave measurement setup are compared. Both techniques are used to image inhomogeneities hidden inside a tissuelike turbid medium. We have found that despite the higher technical complexity of the phased array measurement, its sensitivity in detecting inhomogeneities is virtually the same as the sensitivity of the continuous wave measurement.

A system is described and evaluated for noninvasive measurement of the scattering and absorption coefficients of tissue in the 600 to 850-nm range. An arc lamp/monochromator combination provides a tunable source which is intensity modulated at 135 MHz by a Pockel’s cell and coupled by fiber optics to the tissue surface. Diffusely reflected light is collected by optical fiber bundles at distances of 1 and 2 cm from the source and the intensity ratio and phase difference between these two signals are measured. These data are analyzed by means of a diffusion model of light to provide estimates of the scattering and absorption coefficients. The accuracy of these estimates is assessed by calculation of the influence of instrumental noise, tissue optical properties, and modulation frequency. Because the analysis is based on the assumption of a semi-infinite tissue geometry, systematic errors caused by idealized finite volumes (slabs, cylinders, and spheres) are also considered. The biomedical utility of the system is illustrated by noninvasive measurement of the uptake of an intravenously injected dye in rabbit leg muscle.

In ultrasound tomography the time-domain moment method is very promising in that it has been shown to yield a close agreement between the time-spatial moment expansion and the true field representation. This paper introduces a numerical technique to compute the analytical solution for forward scattering by using a Bessel function series and the inverse discrete Fourier transform, and shows that the artifacts that occur are due to convolution aliasing and undersampling aliasing. Computer simulation has reconstructed these two types of aliasing separately, and has shown that they can be removed by a properly designed algorithm. This alias-free numerical solution is used to verify Cavicchi’s moment-method formulation. A significant improvement in numerical verification is then obtained.

Previous investigation has proved time-resolved spectroscopy to be applicable to measurement of optical parameters in the human breast. To increase knowledge of these properties in vivo, the optical parameters of healthy breasts were measured using time-resolved reflectance spectroscopy. A time correlated single-photon counting method was used to obtain time-response curves for the breasts of 30 Japanese women. Values of ma (absorption coefficient) and ms8 (transport scattering coefficient) were analyzed by fitting the curves to the diffusion equation. The relationships of optical parameters to age, body mass index (BMI), thickness of the breast, number of pregnancies, and menstrual status were examined. The ma and ms8 ranged from 0.0024 to 0.0078/mm and from 0.63 to 1.08/mm, respectively. The values of ma and ms8 showed a high correlation with age and BMI, respectively. The range of the optical parameters of the healthy breasts was determined. These properties may be strongly influenced by changes in tissue components related to aging, menstrual status, and so on. This optical information will contribute to the investigation of photon migration in the human breast.

The aim of this study was to perform a preliminary evaluation of the diagnostic potential of laser induced autofluorescence spectroscopy (LIAFS) for urothelial tumors using fluorescence intensity ratios at different wavelengths. After testing three laser excitation wavelengths (308, 337, and 480 nm) in normal and malignant bladder cell lines, 308 nm appeared to be the most promising wavelength since two fluorescence bands were observed at 360 and 440 nm; these were attributed to tryptophan (Trp) and reduced nicotinamide adenine dinucleotide (NADH) respectively. This study was then performed on freshly removed normal bladder and bladder tumor specimens exclusively using the 308-nm excitation wavelength. The tumor spectra, regardless of stage and grade, were very similar to the malignant cell spectra. However, a marked reduction of overall intensity was observed for carcinoma in situ (CIS). Normal bladder mucosa exhibited a shift of the first fluorescence band to 380 nm, indicating an overlap of Trp urothelial cell emission and collagen fluorescence derived from the lamina propria. The intensity of the NADH emission band was markedly reduced in tumor tissues compared with normal mucosa, which could indicate different redox conditions in urothelial tumors. A fluorescence intensity ratio at 360 and 440 nm can accurately discriminate normal or inflammatory mucosa from all bladder tumors, including CIS. These findings support the use of LIAFS as a new diagnostic technique for occult urothelial tumors.

The use of near-infrared (NIR) measurements of photon migration has been recently demonstrated for the detection of breast cancer in Europe. Yet the clinical success of this potential screening tool depends upon consistent detection of the disease at earlier stages than is currently possible with conventional x-ray mammography. In this paper, we present the optical property measurements of 115 histologically classified breast tissue specimens in order to determine whether consistent and significant optical contrast exists for detection of the disease. Our in vitro optical properties measured with a double integrating sphere technique show consistent changes (yet statistically insignificant) in effective scattering coefficients, ms8, with tissue classification of infiltrating carcinoma (n=48), ductal carcinoma in situ (n=5), mucinous carcinoma (n=3), normal fatty (n=23), and normal fibrous tissues (n=35). However, there is little change in the in vitro tissue absorption coefficient, ma , measured at 749, 789, and 836 nm. For normal and diseased tissue specimens extracted from the same patient, we found differences in optical properties, indicating optical contrast. Using a finite-element prediction of light propagation, we evaluated this optical contrast for photon migration detection of ductal carcinoma in situ tissues using these optical properties measured in vitro.