Wide-field Autofluorescence Research Articles

EARLY STAGES OF COLORECTAL CANCER CHARACTERIZATION BY AUTOFLUORESCENCE 3D MICROSCOPY: A PRELIMINARY STUDY.

Colorectal cancer is one of the most prevalent pathologies worldwide whose prognosis is linked to early detection. Colonoscopy is the gold standard for screening, and diagnosis is usually made histologically from biopsies. Aiming to reduce the inspection and diagnostic time as well as the biopsies and resources involved, other techniques are being promoted to conduct accurate in vivo colonoscopy assessments. Optical biopsy aims to detect normal and neoplastic tissues analysing the autofluorescence spectrum based on the changes in the distribution and concentration of autofluorescent molecules caused by colorectal cancer. Therefore, the autofluorescence contribution analysed by image processing techniques could be an approach to a faster characterization of the target tissue. Quantify intensity parameters through digital processing of two data sets of three-dimensional widefield autofluorescence microscopy images, acquired by fresh colon tissue samples from a colorectal cancer murine model. Additionally, analyse the autofluorescence data to provide a characterization over a volume of approximately 50 µm of the colon mucosa for each image, at second (2nd), fourth (4th) and eighth (8th) weeks after colorectal cancer induction. Development of a colorectal cancer murine model using azoxymethane/dextran sodium sulphate induction, and data sets acquisition of Z-stack images by widefield autofluorescence microscopy, from control and colorectal cancer induced animals. Pre-processing steps of intensity value adjustments followed by quantification and characterization procedures using image processing workflow automation by Fiji's macros, and statistical data analysis. The effectiveness of the colorectal cancer induction model was corroborated by a histological assessment to correlate and validate the link between histological and autofluorescence changes. The image digital processing methodology proposed was then performed on the three-dimensional images from control mice and from the 2nd, 4th, and 8th weeks after colorectal cancer chemical induction, for each data set. Statistical analyses found significant differences in the mean, standard deviation, and minimum parameters between control samples and those of the 2nd week after induction with respect to the 4th week of the first experimental study. This suggests that the characteristics of colorectal cancer can be detected after the 2nd week post-induction. The use of autofluorescence still exhibits levels of variability that prevent greater systematization of the data obtained during the progression of colorectal cancer. However, these preliminary outcomes could be considered an approach to the three-dimensional characterization of the autofluorescence of colorectal tissue, describing the autofluorescence features of samples coming from dysplasia to colorectal cancer. • A new digital image processing method was developed to measure intensity in 3D autofluorescence images of colorectal samples using a CRC mouse model. • This method showed that autofluorescence intensity in colon mucosa is similar in healthy tissue but changes significantly in tumor development. • Statistical analysis revealed CRC traits detectable from the second week post-induction, aiding in early CRC detection. • The study provides a basis for 3D autofluorescence characterization in colorectal tissue from dysplasia to cancer, although variability in autofluorescence limits data systematization during cancer progression.

Fundus imaging features of congenital rubella retinopathy.

To evaluate the clinical characteristics of congenital rubella retinopathy (CRR) with modern fundus imaging. Single-center case series. Eleven patients (2005-2020) at the Emory Eye Center with known or presumed CRR. Trained image readers reviewed fundus imaging (color fundus photography, widefield pseudocolor imaging, near-infrared reflectance imaging, autofluorescence imaging, and spectral-domain optical coherence tomography) for pre-specified features suggestive of CRR. Eleven patients with confirmed (63.6%) or presumed (36.3%) CRR were identified. All were female with median (range) age of 53 (35-67) years. Six (54.5%) were born during the 1964-1965 United States rubella epidemic. All had congenital hearing loss. Two (18.2%) had a congenital heart defect. Eleven (50.0%) eyes had salt-and-pepper retinal pigmentary changes. Twenty-two eyes (100.0%) had irregularly distributed regions of speckled hypoautofluorescence. One eye (4.5%) had a presumed macular neovascularization. Modern fundus imaging demonstrates characteristic features of CRR, even when pigmentary changes are not readily apparent on examination. Widefield autofluorescence findings of irregularly distributed speckled hypoautofluorescence are particularly revealing. This series of newly diagnosed adults with CRR may represent the milder end of the phenotypic spectrum of this condition, highlighting imaging findings that may aid in diagnostically challenging cases of CRR.

Multimodal optical imaging with real-time projection of cancer risk and biopsy guidance maps for early oral cancer diagnosis and treatment.

Despite recent advances in multimodal optical imaging, oral imaging systems often do not provide real-time actionable guidance to the clinician who is making biopsy and treatment decisions. We demonstrate a low-cost, portable active biopsy guidance system (ABGS) that uses multimodal optical imaging with deep learning to directly project cancer risk and biopsy guidance maps onto oral mucosa in real time. Cancer risk maps are generated based on widefield autofluorescence images and projected onto the at-risk tissue using a digital light projector. Microendoscopy images are obtained from at-risk areas, and multimodal image data are used to calculate a biopsy guidance map, which is projected onto tissue. Representative patient examples highlight clinically actionable visualizations provided in real time during an imaging procedure. Results show multimodal imaging with cancer risk and biopsy guidance map projection offers a versatile, quantitative, and precise tool to guide biopsy site selection and improve early detection of oral cancers. The ABGS provides direct visible guidance to identify early lesions and locate appropriate sites to biopsy within those lesions. This represents an opportunity to translate multimodal imaging into real-time clinically actionable visualizations to help improve patient outcomes.

Comparison between standard and wide-field autofluorescence in detection of retinal displacement after rhegmatogenous retinal detachment surgery

PurposeTo analyse the differences between 30° blue autofluorescence (BAF30), 55° blue autofluorescence (BAF55) and 200° green Ultra-Wide Field autofluorescence (UWF200) imaging to detect retinal displacement (RD) after vitrectomy surgery for rhegmatogenous retinal detachment (RRD). MethodsThis cross-sectional study considers forty-nine consecutive patients who underwent RRD surgery in the time period 4–8 months previous to image acquisition. The exclusion criteria contemplate previous retinal pathology, under 18-year-olds and non-assessable images in any of the 3 devices. These images were analysed by two masked graders that assessed either the presence or absence of retinal displacement. A third observer reviewed the images that presented discordance. ResultsA total of forty-nine patients were analysed. 7 eyes were excluded due to poor quality in either of the imaging modalities. The final analysis included 42 eyes of forty-two patients with a mean age of 60.3 ​± ​11.9 years. All patients underwent a 3 port 23-gauge pars plana vitrectomy as the technique of choice. Any grade of RD was detectable in 45.2% of images. It was similar between BAF30 and UWF200 (42.9% of eyes). BAF55 showed RD to a lesser extent (38.1%). Agreement index between BAF30 and BAF55 was 0.901, 0.903 between BAF30 and UWF200 and 0.803 between BAF55 and UWF200. Kappa agreement index between graders was 0.775 for BAF30, 0.798 for BAF50 and 0.808 for UWF200 images. ConclusionsAll imaging modalities were able to detect RD after vitrectomy for RRD, with no inferiority of BAF30 and BAF55 over UWF200.

Three-dimensional morphological characterization of colorectal pits from label-free microscopy images.

The prognosis of colorectal cancer (CRC), one of the most prevalent pathologies worldwide, is linked to early detection. Kudo's pit pattern classification states morphological pit patterns of the Lieberkühn crypts by analyzing the superficial mucosa, predicting the histology of colorectal lesions. Its use as a highly accurate two-dimensional diagnostic criterion has increased, mostly involving expert endoscopists' judgment. The processing of autofluorescence images could allow the diagnostic, bypassing staining techniques and decreasing the biopsies, resources and times involved in the inspection. That criterion could be extended by data of the pit three-dimensional (3D) morphology. Thus, this work was aimed at obtaining 3D morphological information by quantifying geometrical and shape descriptors through software processing and analysis of widefield autofluorescence microscopy image stacks acquired by fresh colon tissue samples from a murine model of CRC. Statistical analyses included pits from control mice and from the second (2nd), fourth (4th), and eighth (8th) weeks of treatment. Statistically significant differences were found for almost all parameters between the pits from control and from the 4th treated week, stating that the major morphological changes begin after the 2nd week. In particular, pits from control or initial treatment time points were more tubular, straighter and less rough than the ones from later treatment points. Therefore, they may be more associated to normal or non-neoplastic crypt lumens than linked to adenomas or even cancer crypts. These preliminary outcomes could be considered an advance in 3D pit morphology characterization.

Detecting Progression of Treated Choroidal Melanomas: Is Ultrasonography Necessary?

Simple SummaryLong-term surveillance following radiotherapy for choroidal melanoma is important for detecting recurrence. There is a perceived notion that regular ultrasonography is required to detect recurrence. The skills required to perform ocular ultrasound are not widely available, which can prevent patients being seen close to home. We aimed to determine whether local treatment failure can reliably be detected with colour fundus photography alone. We found that in 74 out of 75 patients (98.7%), with a clear view of their fundus, recurrence could be detected using colour photography alone. One patient with a clear fundal view developed extraocular extension which was detected on ultrasound without visible change in the intraocular part of the tumour. We conclude that most treated choroidal melanomas can be monitored without ultrasonography if they can be adequately imaged with colour photography.Prompt detection and treatment of local treatment failure after radiotherapy for choroidal melanoma optimises any opportunities for conserving vision and the eye, possibly reducing an increased risk of metastatic disease. Long-term surveillance is therefore required but is hampered by the perceived need to perform ultrasonography, which may not be available at a patient’s local hospital. The aim of this study was to determine whether local treatment failure can reliably be detected with colour fundus photography alone, and, if so, in which patients. Patients were included in the study if diagnosed with local treatment failure between April 2016 and February 2021 after eye-conserving therapy for choroidal melanoma. Wide-field colour and fundal autofluorescence (FAF) images, optical coherence tomography (OCT), and ultrasonography (US) were analysed by two of the authors (GN and UH). The cohort included 87 patients with local treatment failure. In 75 patients with clear media, tumour progression was detected by colour photography alone in 74 (98.7%) patients. Sensitivity was not increased by the addition of either OCT or AF. One patient with clear media developed extraocular extension detected with US without visible change in the intraocular part of the tumour. In the other 12 patients, US was required because of opaque media and a consequently poor fundal view. Local treatment failure after radiotherapy for choroidal melanoma is detected in 98.7% of cases with colour photography when the media are clear. Ultrasonography is useful when photography is prevented by opaque media or tumours having locations in the far periphery.

A ROD-CONE DYSTROPHY IS SYSTEMATICALLY ASSOCIATED TO THE RTN4IP1 RECESSIVE OPTIC ATROPHY.

RTN4IP1 biallelic mutations cause a recessive optic atrophy, sometimes associated to more severe neurological syndromes, but so far, no retinal phenotype has been reported in RTN4IP1 patients, justifying their reappraisal. Seven patients from four families carrying biallelic RTN4IP1 variants were retrospectively reviewed, with emphasis on their age of onset, visual acuity, multimodal imaging including color and autofluorescence frames, spectral-domain optical coherence tomography with RNFL and macular analyses. Seven patients from four RTN4IP1 families developed in their first decade of life a bilateral recessive optic atrophy with severe central visual loss, and primary nystagmus developed in 5 of 7 patients. Six patients were legally blind. In a second stage, the seven individuals developed a rod-cone dystrophy, sparing the macular zone and the far periphery. This retinal damage was identified by 55° field fundus autofluorescence frames and also by spectral-domain optical coherence tomography scans of the temporal part of the macular zone in five of the seven patients. Full-field electroretinography measurements disclosed reduced b-wave amplitude of the rod responses in all patients but two. Family 4 with the p.R103H and c.601A > T (p.K201*) truncating mutation had further combined neurological signs with cerebellar ataxia, seizures, and intellectual disability. RTN4IP1 recessive optic atrophy is systematically associated to a rod-cone dystrophy, which suggests that both the retinal ganglion cells and the rods are affected as a result of a deficit in the mitochondrial respiratory chain. Thus, systematic widefield autofluorescence frames and temporal macular scans are recommended for the evaluation of patients with optic neuropathies.

Handheld swept-source optical coherence tomography guided by smartphone-enabled wide-field autofluorescence photography for imaging facial sebaceous glands.

We report a method to image facial sebaceous glands (SGs) using smartphone-enabled wide-field autofluorescence photography (AFP) and handheld swept-source optical coherence tomography (SS-OCT). Smartphone-enabled AFP provides a 2D wide-field fluorescence image that is used both as a functional mapping of the sebum and a positioning guidance for OCT imaging of the SG. Following the guidance, handheld SS-OCT conducts the volume scan to investigate depth-resolved conditions of the SG in the selected regions of interest. We show the results from smartphone-enabled AFP and handheld SS-OCT to demonstrate the ability of our method to image facial SGs, potentially useful for the assessment of skin conditions in dermatology and cosmetology.

Ocular albinism with mutation in GPR143: Findings in wide-field autofluorescence and optical coherence tomography

A 12 year-old boy who consulted due to nystagmus and low vision from birth. His mother also consulted for low vision of the right eye since she was a child, which worsened recently. The physical examination revealed no alterations in skin and hair pigmentation. In the examination of the anterior segment of the child, areas of slight circumferential hypopigmentation were observed in the iris in both eyes. The fundus examination revealed a choroidal fundus due to the absence of melanin in the retinal pigment epithelium. In the autofluorescence, an absence of physiological macular hypo-autofluorescence was observed and, in optical coherence tomography, foveal hypoplasia was observed in both eyes. In the ocular fundus examination of the mother, slight macular pigmentary changes were observed in the right eye, with hyperpigmented radiated spots in the retinal periphery of both eyes, which were hypo-autofluorescent in the wide-field autofluorescence. In the optical coherence tomography of the right eye, a cavitation of the outer retinal layers was observed in the fovea. The genetic study by nucleotide sequencing was performed on the mother and the child. In the mutation found in the GPR143 gene, the son was hemizygous and the mother was heterozygous. X-linked ocular albinism was diagnosed and the genetic counselling was carried out. Ocular albinism linked to X is the most frequent genetic variant of this disease. Peripheral pigment alterations in heterozygous mothers have been previously described in the literature, but there are no reports of cavitations in the external retinal layers using optical coherence tomography.

MORPHOLOGICAL CHARACTERIZATION OF COLORECTAL PITS USING AUTOFLUORESCENCE MICROSCOPY IMAGES.

Colorectal cancer is one of the most prevalent pathologies. Its prognosis is linked to the early detection and treatment. Currently diagnosis is performed by histological analysis from polyp biopsies, followed by morphological classification. Kudo's pit pattern classification is frequently used for the differentiation of neoplastic colorectal lesions using hematoxylin-eosin stained samples. Few articles have reported this classification with image software processing, using exogenous markers over the samples. The processing of autofluorescence images is an alternative that could allow the characterization of the pits from the crypts of Lieberkühn, bypassing staining techniques. Processing and analysis of widefield autofluorescence microscopy images obtained by fresh colon tissue samples from a murine model of colorectal cancer in order to quantify and characterize the pits morphology by measuring morphology parameters and shape descriptors. Adult male BALB/cCmedc strain mice (n=27), ranging from 20 to 30 g, were randomly assigned to four and five groups of treated and control animals. Colon samples were collected at day zero and at fourth, eighth, sixteenth and twentieth weeks after treatmentwith azoxymethane. Two-dimensional (2D) segmentation, quantification and morphological characterization of pits by image processing applied using macro programming from FIJI. Type I is the pit morphology prevailing between 53 and 81% in control group weeks. III-L and III-S types were detected in reduced percentages. Between the 33 and 56% of type I was stated as the prevailing morphology for the 4th, 8th and 20th weeks of treated groups, followed by III-L type. For the 16th week, the 39% of the pits was characterized as III-L type, followed by type I. Further, pattern types as IV, III-S and II were also found mainly in that order for almost all of the treated weeks. These preliminaries outcomes could be considered an advance in two-dimensional pit characterization as the whole image processing, comparing to the conventional procedure, takes a few seconds to quantify and characterize non-pathological colon pits as well as to estimate early pathological stages of colorectal cancer.

Albinismo ocular con mutación en GPR143: hallazgos en autofluorescencia de campo amplio y tomografía de coherencia óptica

A 12 year-old boy who consulted due to nystagmus and low vision from birth. His mother also consulted for low vision of the right eye since she was a child, which worsened recently. The physical examination revealed no alterations in skin and hair pigmentation. In the examination of the anterior segment of the child, areas of slight circumferential hypopigmentation were observed in the iris in both eyes. The fundus examination revealed a choroidal fundus due to the absence of melanin in the retinal pigment epithelium. In the autofluorescence, an absence of physiological macular hypo-autofluorescence was observed and, in optical coherence tomography, foveal hypoplasia was observed in both eyes. In the ocular fundus examination of the mother, slight macular pigmentary changes were observed in the right eye, with hyperpigmented radiated spots in the retinal periphery of both eyes, which were hypo-autofluorescent in the wide-field autofluorescence. In the optical coherence tomography of the right eye, a cavitation of the outer retinal layers was observed in the fovea. The genetic study by nucleotide sequencing was performed on the mother and the child. In the mutation found in the GPR143 gene, the son was hemizygous and the mother was heterozygous. X-linked ocular albinism was diagnosed and the genetic counselling was carried out.Ocular albinism linked to X is the most frequent genetic variant of this disease. Peripheral pigment alterations in heterozygous mothers have been previously described in the literature, but there are no reports of cavitations in the external retinal layers using optical coherence tomography.

Advanced Registration and Analysis of MALDI Imaging Mass Spectrometry Measurements through Autofluorescence Microscopy.

The correlation of imaging mass spectrometry (IMS) with histopathology can help relate novel molecular findings obtained through IMS to the well-characterized and validated histopathology knowledge base. The quality of correlation between these two modalities is limited by the quality of the spatial mapping that is obtained by registration of the two image types. In this work, we develop novel workflows for MALDI IMS-to-microscopy data registration and analysis using nondestructive IMS-compatible wide field autofluorescence (AF) microscopy combined with computational image registration. First, a substantially automated procedure for high-accuracy registration between IMS and microscopy data of the same section is described that explicitly links the MALDI laser ablation pattern imaged by microscopy to its corresponding IMS pixel. Subsequent examination of the registered data allows for high-confidence colocalization of image features between the two modalities, down to single-cell scales within tissue. Building on this IMS-microscopy spatial mapping, we furthermore demonstrate the automated spatial correlation between IMS measurements from serial sections. This AF-registration-driven inter-section analysis, using a combination of nonlinear AF-to-AF and IMS-to-AF image registrations, can be applied to tissue sections that are prepared and imaged with different sample preparations (e.g., lipids vs proteins) and/or that are measured using different spatial resolutions. Importantly, all registrations, whether within a single section or across serial sections, are entirely independent of the IMS intensity signal content and thus unbiased by it.

Wide-field autofluorescence-guided TUR-B for the detection of bladder cancer: a pilot study.

The aim of this pilot study was to assess the feasibility and value of wide-field autofluorescence imaging (AFI) for the detection of bladder cancer during transurethral resection of the bladder (TUR-B). For imaging, the D-Light/AF System (Karl Storz GmbH, Tuttlingen, Germany) and a customized band pass filter (≈480-780nm) at the eyepiece of the endoscope were used. The excitation light wavelength was 440nm. Representative spectral measurements of tissue autofluorescence (AF) were performed using a spectrometer attached behind the AF band pass filter in selected patients. During TUR-B, cystoscopy was performed in white light (WL) followed by wide-field AFI. Lesions were classified as suspicious or normal using either modality. Representative spectral measurements using excitation at a wavelength of 440nm resulted in significantly lower fluorescence intensity of malignant versus non-malignant tissue. Overall, 56 lesions (30 cancerous and 26 non-malignant) in 25 patients were assessed and classified by wide-field AFI. Papillary tumors as well as flat lesions lacked the green fluorescence seen in normal urothelium, thus emerging as "brown-reddish" areas. When compared with histopathological findings, the pooled per-lesion sensitivity and specificity for AF were 96.7 and 53.8%, respectively. For WL these values were 86.7 and 69.2%, respectively. Wide-field AFI imaging during TUR-B is simple and easy to use. Our preliminary data suggest that AFI has the potential to increase the detection rates of bladder tumors compared with WL without the need of intravesical instillation prior to the procedure.

Noninvasive Quantification of Retinal Microglia Using Widefield Autofluorescence Imaging.

To validate widefield autofluorescence (AF) in vivo imaging of the retina in mice expressing green fluorescent protein (gfp) in microglia, and to monitor retinal microglia reconstitution in vivo after lethal irradiation and bone marrow transplantation. Transgenic Cx3cr1gfp/gfp and wildtype Balb/c mice were used in this study. A confocal scanning laser ophthalmoscope was used for AF imaging with a 55° and a widefield 102° lens. Intrasession reproducibility was assessed for each lens. To investigate reconstitution in vivo, bone marrow from Cx3cr1gfp/gfp mice was used to rescue lethally irradiated wildtype mice. Data were compared to confocal microscopy of retinal flat mounts. Both the 55° and the 102° lens produced high resolution images of retinal microglia with similar microglia density. However, compared to the 55° lens, the widefield 102° lens captured approximately 3.6 times more microglia cells (1515 ± 123 cells versus 445 ± 76 cells [mean ± SD], for 102° and 55°, respectively, P < 0.001). No statistical difference in the number of gfp positive cells within corresponding areas was observed within the same imaging session. Imaging of microglia reconstitution showed a similar time course compared to flat mount preparations with an excellent correlation between microglia cell numbers in AF and gfp-stained flat mounts (R = 0.92, P < 0.0001). Widefield AF imaging of mice with gfp expressing microglia can be used to quantify retinal microglia. In vivo microglia counts corresponded very well with ex vivo counts on retinal flat mounts. As such, AF imaging can largely replace ex vivo quantification.

The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis.

Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tightly bound via its GPI-anchor to the gut membrane. A function in defense against gut microbes is discussed.

Role of wide-field autofluorescence imaging and scanning laser ophthalmoscopy in differentiation of choroidal pigmented lesions.

To evaluate the diagnostic properties of wide-field fundus autofluorescence (FAF) scanning laser ophthalmoscope (SLO) imaging for differentiating choroidal pigmented lesions. A consecutive series of 139 patients were included, 101 had established choroidal melanoma with 13 untreated lesions and 98 treated with radiotherapy. Thirty-eight had choroidal nevi. All patients underwent a full ophthalmological examination, undilated wide-field imaging, FAF and standardized US examination. FAF images and imaging characteristics from SLO were correlated with the structural findings in the two patient groups. Mean FAF intensity of melanomas was significantly lower than the FAF of choroidal nevi. Only 1 out of 38 included eyes with nevi touched the optic disc compared to 31 out of 101 eyes with melanomas. In 18 out of 101 melanomas subretinal fluid was seen at the pigmented lesion compared to none seen in eyes with confirmed choroidal nevi. In "green laser separation", a trend towards more mixed FAF appearance of melanomas compared to nevi was observed. The mean maximal and minimal transverse and longitudinal diameters of melanomas were significantly higher than those of nevi. Wide-field SLO and FAF imaging may be an appropriate non-invasive diagnostic screening tool to differentiate benign from malign pigmented choroidal lesions.

This issue of ACTA, printed and electronic

This issue of <i><scp>ACTA</scp></i>, printed and electronic

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

Relevance of wide‐field autofluorescence imaging in Birdshot retinochoroidopathy: descriptive analysis of 76 eyes

To study and classify retinal lesions in patients with birdshot disease using wide-field autofluorescence imaging and correlate them according to patients' visual status. A multicentre study was carried out on 76 eyes of 39 patients with birdshot disease, analysing colour images and under autofluorescence using the wide-field Optomap(®) imaging system. This was combined with a complete clinical exam and analysis of the macula with OCT. In over 80% of the eyes, a chorioretinal lesion has been observed under autofluorescence with a direct correlation between the extent of the lesion and visual status. The presence of macular hypo-autofluorescence was correlated with a decreased visual acuity, due to the presence of a macular oedema, active clinical inflammation or an epiretinal membrane. The hypo-autofluorescence observed correlated with the duration of the disease and the degree of inflammation in the affected eye, indicating a secondary lesion in the pigment epithelium in relation to the choroid. The pigment epithelium was affected in a diffuse manner, as in almost 50% of the eyes the wider peripheral retina was affected. Wide-field autofluorescence imaging could appear to be a useful examination when monitoring patients, to look for areas of macular hypo-autofluorescence responsible for an irreversible loss of vision.

Observation of Patients with Oral Potentially Malignant Disorders Using Autofluorescence Imaging and Spectroscopy

Objectives: 1) Describe 2 noninvasive optical techniques, widefield autofluorescence imaging (AFI) and optical spectroscopy, that may be used in conjunction with conventional oral examination (COE) for long-term surveillance of patients with oral potentially malignant disorders (OPMD). 2) Present images and spectra collected from patients with OPMD using AFI and spectroscopy over a 33-month surveillance period, and compare results to clinical observations and patient outcomes over that time period. Methods: Patients undergoing routine follow-up surveillance for OPMD were enrolled in a longitudinal optical imaging study. The patients were monitored over time with COE, AFI, and spectroscopy. AFI was used to observe natural tissue fluorescence over a 4-5 cm field of view. Spectroscopy was used to obtain reflectance and fluorescence spectra from selected sites within the oral cavity. The images and spectra collected from these patients were evaluated for changes over time and compared to clinical observations and (where available) histopathology results. Results: During the 33-month period from May 2010 to February 2013, more than 140 patients were enrolled in the study. To date, more than 1,000 optical measurements have been performed during more than 400 clinical surveillance visits. During the surveillance period 26 patients were judged clinically to have progressed from OPMD to cancer. Representative images and spectra from some of these patients will be presented. Conclusions: These results indicate the potential for simple optical techniques, when used in combination with COE, to assist clinicians in monitoring patients with OPMD for early detection of cancer.