Cataracts are the leading cause of blindness worldwide. Current methods for discriminating cataractous lenses from healthy lenses of Sprague-Dawley rats during preclinical studies are based on either histopathological or clinical assessments which are weakened by subjectivity. In this work, both cataractous and healthy lens tissues of Sprague-Dawley rats were studied using multispectral imaging technique in combination with multivariate analysis. Multispectral images were captured in transmission, reflection and scattering modes. In all, five spectral bands were found to be markers for discriminating cataractous lenses from healthy lenses; 470 nm and 625 nm discriminated in reflection mode whereas 435 nm, 590 nm and 700 nm discriminated in transmission mode. With Fisher’s Linear discriminant analysis, the midpoints for classifying cataractous from healthy lenses were found to be 14.718 × 10 −14 and 3.2374 × 10 −14 for the two spectra bands in the reflection mode and the three spectral bands in the transmission mode respectively. Images in scattering mode did not show significant discrimination. These spectral bands in reflection and transmission modes may offer potential diagnostic markers for discriminating cataractous lenses from healthy lenses thereby promising multispectral imaging applications for characterizing cataractous and healthy lenses.
Cataracts are the leading cause of visual impairment worldwide, accounting for more than 50% of blindness in developing countries [
Over the years, histopathological evaluation of stained tissue biopsies and autopsies by pathologist has been the golden standard for discriminating cataractous lens from healthy lens in preclinical studies [
Multispectral imaging is a technique which has been used in various applications to extract detailed information about an image [
Ten-day-old Sprague-Dawley rat pups of either sex with mean weight of 24.29 g were used for this research work. The pups together with their mothers were housed in polyacrylic cages (34 cm × 47 cm × 18 cm) with soft wood shavings as bedding, under ambient laboratory conditions (temperature 28˚C ± 2˚C, relative humidity 60% - 70%, and a normal light?dark cycle at the husbandry of the School of Biological Sciences, University of Cape Coast. The mothers were fed on a normal commercial pellet diet (Agricare Ltd, Kumasi, Ghana) and had access to water ad libitum.
The ten-day old pup rats were put into 2 groups consisting of 45 pup rats per group. One group (Group A) was kept as control while the other group (Group B) was injected subcutaneously daily with 15 mol kg−1 sodium selenite in normal saline on the 11th and 12th day respectively. The two groups (A and B) were monitored till the 30th day. On the 31st day, the crystalline lens of Group B were assessed for cataract development using a Marco II-B Slit Lamp (Marco-Lombart Instrument, Japan) after the pupils’ of the pups had been dilated with 1% tropicamide ophthalmic solution (Akorn Inc., lake Forest, USA). The rats with developed cataract, from early to fully developed, were sacrificed by ether inhalation, followed by intracardiac injection of pentobarbital. After enucleation, the lenses of the rats (Group A and Group B) were extracted and kept in different containers with formalin at room temperature. Forty four percent (44%) of Group A were selected to match up the number of Group B lenses for tissue preparation. The lenses were fixed in 10% phosphate-buffered paraformaldehyde, and embedded in paraffin. Sections of 3-μm thickness were made and stained with hematoxylin and eosin [
The imaging system used was a Multispectral Light Emitting Diodes Imaging Microscope system (MSLEDIM) at Laser and Fibre Optics Centre (LAFOC), Department of Physics, University of Cape Coast, Cape Coast, as implemented in Opoku-Ansah et al. and Brydegaard et al. [
In acquiring the multispectral images of the of the Group A lens tissues, the 590 nm spectral band was used to adjust the camera settings with imaging parameters (i.e. the gain and the exposure time) for optimization. This spectral band was used as a standard due to the camera’s sensitivity. Grayscale images of each lens were captured in three modes (transmission, reflection and scattering) at all the 13 spectral bands and then saved as tagged image file format (TIFF). Thus, a total of 39 images were obtained for each lens. The image acquisition process was repeated for the Group B lens tissues. Pixel intensities of the images were extracted using Matlab codes for further analysis. A flow chart of the Matlab codes developed for pixel intensity extraction from the images, principal component and Fisher’s linear discriminant analysis is shown in
Principal Component Analysis (PCA) was applied in analysing the multispectral data using Matlab algorithm. The image intensity data, Z of m (4999) observations and n (5) variables was centered, Zc, such that the elements of the matrix of dimension m × n are around the sample mean of zero. The Zc was then converted into nonsingular covariant matrix, L, defined as
where
where
Based on the three PCs (PC1, PC2 and PC3), the groups (Group A and Group B) were classified using the linear discriminant function
where
A new observation PCxo is allocated to Group A, if
where
is the midpoint between two group averages, else PCxo is allocated to Group B if
The images shown in
Since the discrimination between the two (2) groups, in transmission, reflection and scattering modes, cannot be easily assessed by observation of the grayscale images, their averaged pixel intensities in each mode were extracted and plotted are shown in
and wrinkled nature of the Group B lenses. The average reflected intensities from 470 nm and 625 nm also show differences between the Group A and Group B lenses. All the other spectral bands could not show significant differences between the Group A and Group B lenses. This is an indication that both 470 nm and 625 nm can be used as markers to discriminate Group A from Group B lenses using averaged reflected pixel intensity values.
The averaged transmitted pixel intensity values from the Group A and Group B lenses are shown in
The average transmitted intensities from five (5) spectral bands, 435 nm, 590 nm, 700 nm, 750 nm and 810 nm are observed to be higher (>100 a.u) in both the Group A and Group B lenses compared to the other spectral bands which includes 375 nm, 400 nm, 470 nm, 525 nm, 625 nm, 660 nm, 850 nm and 940 nm. This indicates that the lenses can transmit enough light in all these spectral bands but much higher from 435 nm, 590 nm, 700 nm, 750 nm and 810 nm. The lower opacification of Group B lenses can be attributed to massive insolubilization of the soluble protein in the lens, which results in light scattering by the lens [
The averaged scattered pixel intensity values from all the thirteen (13) spectral bands, as shown in
Scatter plots of the first three (3) PCs (PC1, PC2 AND PC3) of both Group A and Group B lenses in transmission and reflection mode are shown in
values describing 99.6 % of the variability dataset. The first eigenvalue, the one describing the largest amount of variability on the dataset, 77.91%, describes the overall offset of the transmitted intensity data from the lenses. The second, 9.54% of the dataset’s variability was described by the second eigenvalue whiles the third, 7.08% of the dataset’s variability was describe by the third eigenvalue.
In the case of reflection, the PCs were obtained from 470 nm and 625 nm spectral bands with eigenvalues describing 99.5% of the variability of the dataset. As in the previous case, the first eigenvalue represented the overall reflection intensities from the lenses and described 85.07% of the dataset’s variability. The second, 5.56% of the dataset’s variability was described by the second eigenvalue whiles the third, 3.0% of the dataset’s variability was describe by the third eigenvalue. Transmission and reflection for the three and two spectral bands showed discrete classification between Group A and Group B lenses as shown in
Using trained transmission data from 435 nm, 590 nm and 700 nm, the allocation rule obtained from the Fisher’s linear discriminant function with equal cost and equal priors for the sample data of the Group A and Group B lenses and for maximum separation of the two stained sectioned lenses is given as
with a midpoints m = 3.2374 × 10−14, where r1, r2 and r3 representing PC1, PC2 and PC3 respectively with K1, K2 and K3 being the coefficient. In this case K1, K2 and K3 were found to be 0.99, −0.08 and −0.10 respectively. Thus, if Po ≥ m, then the lens is Group A (healthy), else it is Group B (cataractous). This can be seen in
Mode | Spectral Bands (nm) | Mid-point (m) |
---|---|---|
Reflection | 470; 625 | 14.718 × 10−14 |
Transmission | 435; 590; 700 | 3.2374 × 10−14 |
Group A and B lenses respectively. The black star in the middle is the classification midpoint between the Group A and Group B lenses. Evaluation of the Fishers’ linear discriminant function with ten (10) transmitted data showed 90% success of the discrimination function using the PCs of the Group A and the Group B lenses.
The lens data from the reflection mode in the coordinates of the first two Fisher’s discriminants is shown in
The values for L1, L2 and L3, which are the coefficients were found to be 0.97, −0.12 and −0.19 respectively. The midpoints values for discriminating Group A from Group B lenses in both transmission and reflection mode is shown in
Using extracted average pixel intensities from grayscale multispectral images of healthy lenses (Group A) and cataractous lenses (Group B) from rat, five (5) spectral bands were found to be markers for discriminating Group A from Group B: 470 nm and 625 nm discriminated in reflection mode whereas 435 nm, 590 nm and 700 nm discriminated in transmission mode. MSI technique has confirmed that Group A lenses transmit and reflect more light than Group B lenses. Upon further analysis with principal component and Fisher Linear discriminant, three (3) PCs confirm these five (5) spectral bands as markers for discriminating Group A from Group B lenses in the scatter plot. The Fisher’s linear discriminant analysis showed 87% and 90% success of the discrimination function for the two reflection spectral bands and for the three transmission spectral bands respectively. The midpoint for classifying Group A from Group B lenses for the two reflection spectral bands was found to be 14.718 × 10−14 whereas that from the three transmission spectral bands were found to be 3.2374 × 10−14. The five spectral bands in reflection and transmission modes offer potential diagnostic tools for discriminating cataractous lenses of Sprague-Dawley rats from healthy lenses for ophthalmic applications. MSI technique in combination with multivariate analysis has therefore been used to discriminate healthy (Group A) of Sprague-Dawley rats from cataractous lenses (Group B). MSI has more advantages in terms of rapidity, rigidity, objectivity and the ability to provide more information on a single sample. Any trained personnel would be able to implement this technique together with the analysis in this field especially in veterinary Ophthalmology.
The authors wish to express our appreciation to International Programme for Physical Sciences (IPPS), International Sciences Programme (ISP), Uppsala University, Sweden) for funding and donation of microscopes. We wish to express our appreciation to the Office of External Activities (OEA) and Associate Scheme of Abdus Salam ICTP, Trieste, Italy for our stay in Italy and not forgetting Mr. Daniel Portakey of Korle-Bu teaching hospital for assisting in sectioning of the lens tissues.
Adueming, P.O.-W., Eghan, M.J., Anderson, B., Kyei, S., Opoku-Ansah, J., Amuah, C.L.Y., Sackey, S.S. and Buah-Bassuah, P.K. (2017) Multispectral Imaging in Combination with Multivariate Analysis Discriminates Selenite Induced Cataractous Lenses from Healthy Len- ses of Sprague-Dawley Rats. Open Journal of Biophysics, 7, 145-156. https://doi.org/10.4236/ojbiphy.2017.73011