Antonio Calcagni

Multispectral imaging of the ocular fundus using light emitting diode illumination

We present an imaging system based on light emitting diode (LED) illumination that produces multispectral optical images of the human ocular fundus. It uses a conventional fundus camera equipped with a high power LED light source and a highly sensitive electron-multiplying charge coupled device camera. It is able to take pictures at a series of wavelengths in rapid succession at short exposure times, thereby eliminating the image shift introduced by natural eye movements (saccades). In contrast with snapshot systems the images retain full spatial resolution. The system is not suitable for applications where the full spectral resolution is required as it uses discrete wavebands for illumination. This is not a problem in retinal imaging where the use of selected wavelengths is common. The modular nature of the light source allows new wavelengths to be introduced easily and at low cost. The use of wavelength-specific LEDs as a source is preferable to white light illumination and subsequent filtering of the remitted light as it minimizes the total light exposure of the subject. The system is controlled via a graphical user interface that enables flexible control of intensity, duration, and sequencing of sources in synchrony with the camera. Our initial experiments indicate that the system can acquire multispectral image sequences of the human retina at exposure times of 0.05 s in the range of 500-620 nm with mean signal to noise ratio of 17 dB (min 11, std 4.5), making it suitable for quantitative analysis with application to the diagnosis and screening of eye diseases such as diabetic retinopathy and age-related macular degeneration.

Sucrose cryo-protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo-preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12-µm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections.

Multispectral retinal image analysis: a novel non-invasive tool for retinal imaging

PurposeTo develop a non-invasive method for quantification of blood and pigment distributions across the posterior pole of the fundus from multispectral images using a computer-generated reflectance model of the fundus.MethodsA computer model was developed to simulate light interaction with the fundus at different wavelengths. The distribution of macular pigment (MP) and retinal haemoglobins in the fundus was obtained by comparing the model predictions with multispectral image data at each pixel. Fundus images were acquired from 16 healthy subjects from various ethnic backgrounds and parametric maps showing the distribution of MP and of retinal haemoglobins throughout the posterior pole were computed.ResultsThe relative distributions of MP and retinal haemoglobins in the subjects were successfully derived from multispectral images acquired at wavelengths 507, 525, 552, 585, 596, and 611 nm, providing certain conditions were met and eye movement between exposures was minimal. Recovery of other fundus pigments was not feasible and further development of the imaging technique and refinement of the software are necessary to understand the full potential of multispectral retinal image analysis.ConclusionThe distributions of MP and retinal haemoglobins obtained in this preliminary investigation are in good agreement with published data on normal subjects. The ongoing development of the imaging system should allow for absolute parameter values to be computed. A further study will investigate subjects with known pathologies to determine the effectiveness of the method as a screening and diagnostic tool.

Multispectral imaging of the ocular fundus using LED illumination

We present preliminary data from an imaging system based on LED illumination for obtaining sequential multi-spectral optical images of the human ocular fundus. The system is capable of acquiring images at speeds of up to 20fps and we have demonstrated that the system is fast enough to allow images to be acquired with minimal inter-frame movement. Further improvements have been identified that will improve both imaging speed and image quality. The long-term goal is to use the system in conjunction with novel image analysis algorithms to extract chromophore concentrations from images of the ocular fundus, with a particular emphasis on age-related macular degeneration. The system has also found utility in fluorescence microscopy.

Model-based parameter recovery from uncalibrated optical images

We propose a novel method for quantitative interpretation of uncalibrated optical images which is derived explicitly from an analysis of the image formation model. Parameters characterising the tissue are recovered from images acquired using filters optimised to minimise the error. Preliminary results are shown for the skin, where the technique was successfully applied to aid the diagnosis and interpretation of non-melanocytic skin cancers and acne; and for the more challenging ocular fundus, for mapping of the pigment xanthophyll.

Imaging of the human fundus in the clinical setting: past present and future

The human fundus is a complex structure that can be easily visualized and the world of ophthalmology is going through a golden era of new and exciting fundus imaging techniques; recent advances in technology have allowed a significant improvement in the imaging modalities clinicians have available to formulate a diagnostic and treatment plan for the patient, but there is constant on-going work to improve current technology and create new ideas in order to gather as much information as possible from the human fundus. In this article we shall summarize the imaging techniques available in the standard medical retina clinic (i.e. not limited to the research lab) and delineate the technologies that we believe will have a significant impact on the way clinicians will assess retinal and choroidal pathology in the not too distant future.

Quantitative interpretation of multi-spectral fundus images

Multi-spectral imaging of the ocular fundus suffers from three main problems: the image must be taken through an aperture (the pupil), meaning that the absolute light intensity at the fundus cannot be known; long acquisition times are not feasible due to patient discomfort; patient movement can lead to loss of image quality. These difficulties have meant that multi-spectral imaging of the fundus has not yet seen wide application. We have developed a new method for optimizing the multi-spectral imaging process which also allows us to derive semi-quantitative information about the structure and properties of the fundus. We acquire images in six visible spectral bands and use these to deduce the concentration and distribution of the known absorbing compounds in the fundus: blood haemoglobins in the retina and choroid, choroidal melanin, RPE melanin and xanthophyll. The optimisation process and parameter recovery uses a Monte Carlo model of the spectral reflectance of the fundus, parameterised by the concentrations of the absorbing compounds. The model is used to compute the accuracy with which the values of the model parameters can be deduced from an image. Filters are selected to minimise the error in the parameter recovery process. Theoretical investigations suggest that parameters can be recovered with RMS errors of less than 10%. When applied to images of normal subjects, the technique was able to successfully deduce the distribution of xanthophyll in the fundus. Further improvement of the model is required to allow the deduction of other model parameters from images.

Macular Pigment Quantification with Multispectral Retinal Image Analysis

Objective: Variations in macular pigment (MP) have been linked with changes in the risk of visual loss secondary to macular pathology. MP density can be modified by diet; however there doesn’t appear to be a direct link between dietary intake of MP components and MP density in the retina and clinicians therefore need a reliable and objective method for MP measurement to establish if any intervention is yielding the required results. The objective of this study was to investigate whether multispectral retinal image analysis (MRIA), a new technique for mapping retinal pigments, is useful for measuring levels and distribution of MP to find differences between individuals with no clinical evidence of macular pathology and those diagnosed with Age-related Macular Degeneration (AMD). Methods: The study involved 90 volunteers from three subject groups: aged under 50 without AMD, aged 50 and over without AMD and aged 50 and over with AMD. The experiments yielded 607 usable data sets that were used for analysis. Multispectral image data was acquired at six selected wavelengths using a modified fundus camera. MRIA maps of MP were computed from 3 × 3 mm regions of interest (approximately 10 degrees of visual angle) centred at the fovea. Indices characterising MP distribution were computed both for individuals and for the three subject groups. For comparison Macular Pigment Optical Density (MPOD) measurements were acquired from the Macular Pigment Screener 9000 (MPS) based on the heterochromatic flicker photometry (HFP). Correlations for MP quantities measured with the two methods were computed between MP quantity and age, MP quantity and AMD diagnosis, and between the two methods. Results: MP maps obtained from MRIA were consistent with known histology and in agreement with expectations based on previous studies. Pooled results from the three groups suggest that the overall levels of MP across both the fovea and the parafovea are on average higher in healthy under-50 individuals than that over-50 with or without AMD. MP distribution might be more irregular in the over-50 groups than in the younger group. The correlation between age and MP levels was weak as measured individually by both techniques. The MRIA indices were not correlated with HFP-MPOD measurements for individuals, but high correlation was found between mean HFP-MPOD and mean MRIA peak value for pooled results. Conclusion: MRIA has potential to offer an objective, fast and reliable method of measuring MP throughout the posterior pole.

The importance of optical coherence tomography examination of normal fellow eyes in neovascular age-related macular degeneration monitoring clinics

Purpose: To demonstrate the importance of OCT examination of fellow, normal eyes in unilateral nAMD follow up clinics. Methods: The authors present three cases of unilateral nAMD who were undergoing treatment with ranibizumab, in whom OCT evaluation of the previously unaffected, asymptomatic fellow eye allowed early diagnosis, treatment and preservation of vision. Fundus examination had previously failed to demonstrate abnormality. Results: Intravitreal anti-VEGF treatment for nAMD has caused a sharp increase in the number of subjects attending macular clinics, frequently overburdening the system. It may sometimes be tempting for hospitals to reduce the workload by for example, concentrating only on OCT examination of the affected eye in cases of unilateral nAMD. The three reported cases demonstrate that OCT scanning of the fellow, previously unaffected eye is essential in detecting asymptomatic nAMD, which gives a better chance of preservation of vision. Conclusions: Patients with unilateral neovascular AMD undergoing review in macular clinics should always undergo OCT scanning of normal, fellow eyes, as otherwise asymptomatic, “invisible” choroidal neovascular membranes may be missed.