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Purpose: VitreoDx is an experimental device enabling push-button collection of a neat vitreous liquid biopsy incidental to an intravitreal injection. We explored the ability of the device to collect a sample usable for proteomic biomarker discovery and testing., Design: Pilot study using ex vivo human eyes., Methods: Non-vitrectomized, human eyes from nine donors 75-91 years of age were refrigerated in BSS and used within 5 days of death. Four VitreoDx devices fitted with 25G needles, and four staked needle insulin syringes with 30G needles, were inserted at equal intervals through the pars plana of each eye and held in place by a fixture. The sampling mode of each VitreoDx device was triggered to attempt to acquire a liquid biopsy up to 70 μL. The plunger of each insulin syringe was retracted to attempt to obtain a liquid biopsy with a maximum volume of 50 μL. Samples acquired with the VitreoDx were extracted to polypropylene cryovials, refrigerated to -80 ºC, and sent for offsite proteomic analysis by proximity extension assay with a focus on panels containing approved and pipelined drug targets for neovascular disease and inflammatory factors., Results: Of the attempted liquid biopsies with the novel 25G VitreoDx, 92% (66 of 72) resulted in successful acquisition (>25 μL) while 89% (64 of 72) attempted by a traditional 30G needle resulted in a successful acquisition. Sample volume sufficient for proteomics array analysis was acquired by the VitreoDx for every eye. Detectable protein was found for 151 of 166 unique proteins assayed in at least 25% of eyes sampled by VitreoDx., Conclusions: The high acquisition rate achieved by the prototype was similar to that achieved in previous clinical studies where a standard syringe was used with a 25G needle to biopsy vitreous fluid directly prior to standard intravitreal injection. Successful aspiration rates were likewise high for 30G needles. Together, these suggest that it is possible to routinely acquire liquid vitreous biopsies from patients who typically receive intravitreal injections with an injection device using a standard size needle without a vitreous cutter. Protein analysis shows that proteins of interest survive the sampling mechanism and may have potential to direct care in the future., Competing Interests: All authors are employees or consultants of Twenty / Twenty Therapeutics, LLC which is developing the VitreoDx device and holds patents for technologies described. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Tumlinson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Low coherence laser interferometry has revolutionised quantitative biomedical imaging of optically transparent structures at cellular resolutions. We report the first optical recording of neuronal excitation at cellular resolution in the inner retina by quantifying optically recorded stimulus-evoked responses from the retinal ganglion cell layer and comparing them with an electrophysiological standard. We imaged anaesthetised paralysed tree shrews, gated image acquisition, and used numerical filters to eliminate noise arising from retinal movements during respiratory and cardiac cycles. We observed increases in contrast variability in the retinal ganglion cell layer and nerve fibre layer with flash stimuli and gratings. Regions of interest were subdivided into three-dimensional patches (up to 5-15 μm in diameter) based on response similarity. We hypothesise that these patches correspond to individual cells, or segments of blood vessels within the inner retina. We observed a close correlation between the patch optical responses and mean electrical activity of the visual neurons in afferent pathway. While our data suggest that optical imaging of retinal activity is possible with high resolution OCT, the technical challenges are not trivial.

Purpose: Chicks developing experimentally-induced myopia show profound thinning of the choroid. We observed a wide range of choroidal thicknesses in a sample of normal chicks prior to their use in a pedigree-based study of form-deprivation myopia. Hence, we tested whether pre-treatment choroidal thickness predicted susceptibility to myopia., Methods: Retinal, choroidal and scleral thickness were measured using A-scan ultrasonography in normal White Leghorn chicks (n= 891) aged 4 days old, and again (n=498) after 4 days of monocular form-deprivation at age 8 days of age. Refractive error was assessed by retinoscopy. Relationships between pre-treatment choroidal thickness and other variables were investigated using general linear models and variance components analysis., Results: Untreated 4 day-old male chicks had choroids approximately 10% thinner than females (p<0.001), but sex explained <2% of the overall variability in choroidal thickness. Axial eye length in these untreated chicks was not significantly associated with choroidal thickness (p=0.25). Moreover, pre-treatment choroidal thickness was not predictive of susceptibility to form-deprivation myopia (p=0.89). Heritability analysis suggested that at least 50% of the variation in pre-treatment choroidal thickness was determined by additive genetic effects (p<0.001)., Conclusions: Parental choroidal thickness is the major determinant of choroidal thickness in untreated 4-day old chicks. Despite choroidal thickness potentially being indicative of ongoing emmetropisation to innate refractive errors, in this study it was not predictive of subsequent susceptibility to form-deprivation myopia., (Ophthalmic & Physiological Optics © 2011 The College of Optometrists.)

Purpose: To demonstrate acquisition and analysis methods for depth-resolved observation of slow retinal physiology induced changes in infrared backscatter in vivo., Methods: A dark-adapted human was briefly subjected to a localized photobleach. For 20 min before and 30 min after the stimulus, volumetric optical coherence tomograms were collected partially overlapping the bleached region. Tomograms were segmented into retinal layers by a newly described algorithm exploiting information in adjacent B-scans. En face fundus images extracted from major intraretinal layers were laterally registered manually. Time series summarizing the observed backscatter in selected layers for the bleached and unbleached areas are shown with a variety of corrections and normalizations applied: tomograms were corrected for inherent sensitivity roll-off, and the ratio between other layers and an assumed unchanging layer (retinal pigment epithelium), as well as the ratio of the stimulated area to the unstimulated area, were calculated., Results: Adjacent B-scan information allows a simpler segmentation algorithm to be used. Sensitivity roll-off correction reduces signal variability due to eye motion. After normalizations, the signal correlated with the stimulus appears strongest at the photoreceptor inner-outer segment junction., Conclusions: Demonstrated methods manage data complexity and reduce uncorrelated signal variability. This single trial warrants further investigation of intrinsic optical signals to observe slow physiologic responses.

: Recent substantial developments in light source and detector technology have initiated a paradigm shift in retinal optical coherence tomography (OCT) performance. Broad bandwidth light sources in the 800 nm and 1060 nm wavelength region enable axial OCT resolutions of 2-3 mum and 5-7 mum, respectively. Novel high speed silicon based CMOS cameras at 800 nm and InGaAs based CCD cameras in combination with frequency domain OCT technology enable data acquisition speeds of up to 47,000 A-scans/s at 1060 nm and up to 312,500 A-scans/s at 800 nm. Combining ultrahigh axial resolution, ultrahigh speed OCT at 800 nm with pancorrected adaptive optics allows volumetric in vivo cellular resolution retinal imaging. Commercially available three-dimensional (3D) retinal OCT at 800 nm (20,000 A-scans/s, 6 mum axial resolution) is compared to ultrahigh speed 3D retinal imaging at 800 nm (160,000 A-scans/s, 2-3 mum axial resolution), high speed 3D choroidal imaging at 1060 nm (47,000 Ascan/ second, 6-7 mum axial resolution) and cellular resolution retinal imaging at 800 nm using adaptive optics OCT at 160,000 A-scans/second with isotropic resolution of ~2 mum. Analysis of the performance of these four imaging modalities applied in normal and pathologic eyes focusing on motion artifact free volumetric retinal imaging and revealing novel, complementary morphological information due to enhanced resolution, speed and penetration is presented.

Depth dependent broadening of the axial point spread function due to dispersion in the imaged media, and algorithms for postprocess correction, have been previously described for both time domain and frequency domain optical coherence tomography. We show that homogeneous media dispersion artifacts disappear when frequency domain samples are acquired with uniform spacing in circular wavenumber, as opposed to uniform sampling in optical frequency. We further explicate the source of this point spread broadening and simulate its magnitude in aqueous media. We experimentally demonstrate media dispersion compensation in high dispersion glass by choosing sample frequencies at equal intervals of media index of refraction divided by vacuum wavelength, and we recover unbroadened reflections without an additional postprocessing step.

Purpose: Optical coherence tomography (OCT) is a minimally invasive, depth-resolved imaging tool that can be implemented in a small diameter endoscope for imaging mouse models of colorectal cancer (CRC). In this study, we utilized ultrahigh resolution (UHR) OCT to serially image the lower colon of azoxymethane (AOM) treated A/J mouse models of CRC in order to monitor the progression of neoplastic transformations and determine if OCT is capable of identifying early disease., Experimental Design: Thirteen AOM treated A/J and two control A/J mice were surveyed at four timepoints (8, 14, 22 and 26 weeks post AOM treatment) using a 2.0 mm diameter UHR OCT endoscopic system with 3.2 microm axial and 4.4 microm lateral resolution. Histological samples obtained at the final timepoint served as the diagnostic reference. A blinded expert panel of mouse colon pathologists provided diagnoses from the OCT images based on criteria developed from a separate training set of OCT images. Panel results were compared to histological diagnoses assigned by a blinded pathologist., Results: At the final imaging timepoint, 95% of adenomas and 23% of gastrointestinal neoplasias (38% protruding GINs and 9% non-protruding GINs) were correctly diagnosed. The panel identified 68% of disease foci (95% adenoma, 76% protruding GINs and 13% non-protruding GINs). Over the OCT imaging timepoints, disease progression followed a typical succession, with normal or GIN preceding adenoma., Conclusions: Endoscopic UHR OCT enabled accurate diagnosis of adenomas, identification of protruding GIN and non-destructive visualization of CRC progression, providing a tool for cancer research in animal models.

Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy each have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models is unknown. In this study, we combined the 2 modalities to survey the GI tract of a variety of mouse strains and ages and to sample dysplasias and inflammatory bowel disease (IBD) of the intestines. Segments (length, 2.5 cm) of duodenum and lower colon and the entire esophagus were imaged ex-vivo with combined OCT and LIE We evaluated 30 normal mice (A/J and 10- and 21-wk-old and retired breeder C57BL/6J) and 10 mice each of 2 strains modeling colon cancer and IBD (Apc(Min) and IL2-deficient mice, respectively). Histology was used to classify tissue regions as normal, Peyer patch, dysplasia, adenoma, or IBD. Features in corresponding OCT images were analyzed. Spectra from each category were averaged and compared via Student t tests. OCT provided structural information that led to identification of the imaging characteristics of healthy mouse GI. With histology as the 'gold standard,' we developed preliminary image criteria for early disease in the form of adenomas, dysplasias, and IBD. LIF characterized the endogenous fluorescence of mouse GI tract, with spectral features corresponding to collagen, NADH, and hemoglobin. In the IBD sample, LIF emission spectra displayed potentially diagnostic peaks at 635 and 670 nm, which we attributed to increased porphyrin production by bacteria associated with IBD. OCT and LIF appear to be useful and complementary modalities for ex vivo imaging of mouse GI tissues.

Endoscopic ultrahigh-resolution optical coherence tomography (OCT) enables collection of minimally invasive cross-sectional images in vivo, which may be used to facilitate rapid development of reliable mouse models of colon disease as well as assess chemopreventive and therapeutic agents. The small physical scale of mouse colon makes light penetration less problematic than in other tissues and high resolution acutely necessary. In our 2-mm diameter endoscopic time domain OCT system, isotropic ultrahigh-resolution is supported by a center wavelength of 800 nm and full-width-at-half-maximum bandwidth of 150 nm (mode-locked titanium:sapphire laser) combined with 1:1 conjugate imaging of a small core fiber. A pair of KZFSN5/SFPL53 doublets provides excellent color correction to support wide bandwidth throughout the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. Our system achieves axial resolution of 3.2 microm in air and 4.4-microm lateral spot diameter with 101-dB sensitivity. Microscopic features too small to see in mouse tissue with conventional resolution systems, including colonic crypts, are clearly resolved. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

Optical coherence tomography (OCT), laser-induced fluorescence (LIF), and laser-scanning confocal microscopy (LSCM) were used for the task of multimodal study of healthy and adenomatous mouse colon. The results from each modality were compared with histology, which served as the gold standard. The Apc(Min/+) genetic mouse model of colon cancer was compared with wild-type mice. In addition, a special diet was used for the task of studying the origins of a 680 nm autofluorescent signal that was previously observed in colon. The study found close agreement among each of the modalities and with histology. All four modalities were capable of identifying diseased tissue accurately. The OCT and LSCM images provided complementary structural information about the tissue, while the autofluorescence signal measured by LIF and LSCM provided biochemical information. OCT and LIF were performed in vivo and nondestructively, while the LSCM and histology required extraction of the tissue. The magnitude of the 680 nm signal correlates with chlorophyll content in the mouse diet, suggesting that the autofluorescent compound is a dietary metabolite.

Background and Objectives: The diagnostic feasibility of optical coherence tomography (OCT) and laser-induced fluorescence (LIF) have been evaluated for human colorectal cancer. This study applies these technologies to a murine model of colorectal adenoma., Study Design/materials and Methods: The lower colon of 10 Apc(Min) and two C57BL/6J mice was surveyed over five 4-week intervals using a prototype 2.0 mm diameter OCT-LIF endoscope-based system. Four categories were histologically classified: control C57BL/6J, adenomatous, non-diseased regions of adenomatous, and non-diseased Apc(Min). OCT images were compared to histology. Spectra from the four categories were compared via the Student's t-test., Results: Three Apc(Min) and two control mice completed the study. One adenoma was histologically identified; OCT visualized mucosal thickening/abnormal mass development over the imaging timepoints. LIF spectral comparisons revealed decreased 405 nm intensity and the presence of a peak at 680 nm in the adenomatous Apc(Min)., Conclusions: These preliminary data indicate endoscopic OCT-LIF has the potential to identify colorectal adenomas in murine models., (Copyright 2006 Wiley-Liss, Inc.)

Frequency domain optical coherence tomography (FD-OCT) allows interferometer topologies with simplified system construction and handling. Problems of dispersion and polarization matching between the sample and reference arms, as well as beamsplitter spectral non-uniformity, are mitigated when the interferometer is wholly contained in the endoscope tip. A common path set-up, using a reference reflection originating from the inside surface of the glass envelope at the distal end of the endoscope, and an alternative approach with more efficient collection of the reference light using a novel beamsplitter design have been developed. High speed (20,000 A-lines/s) ultrahigh axial resolution (2.4 mum) tomograms of mouse colon have been acquired using a 2 mm outer diameter endoscope in vivo. The FD-OCT system uses a compact mode-locked Ti:Al(2)O(3) laser emitting a broad spectrum (160 nm full-width-half-maximum) centered at 800 nm in combination with a CCD based, spectrally sensitive detector.

We have designed a multimodality system that combines optical coherence tomography (OCT) and laser-induced fluorescence (LIF) in a 2.0-mm-diameter endoscopic package. OCT provides approximately 18-microm resolution cross-sectional structural information over a 6-mm field. LIF spectra are collected sequentially at submillimeter resolution across the same field and provide histochemical information about the tissue. We present the use of a rod prism to reduce the asymmetry in the OCT beam caused by a cylindrical window. The endoscope has been applied to investigate mouse colon cancer in vivo.