Early Upper Aerodigestive Tract Cancer Detection Using Electron Microscopy to Reveal Chromatin Packing Alterations in Buccal Mucosa Cells.

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself. Large-scale electron microscopy (nanotomy) images were acquired of cancer patients and controls. Within the nuclei, the chromatin packing of euchromatin and heterochromatin was characterized. Furthermore, the chromatin organization was quantified through chromatin packing density scaling. A significant difference was found between the cancer and control groups in the chromatin packing density scaling parameter for length scales below the optical diffraction limit (200 nm) in both the euchromatin (p = 0.002) and the heterochromatin (p = 0.006). The chromatin packing scaling analysis also indicated that the chromatin organization of cancer patients deviated significantly from the control group. They might allow for novel strategies for cancer risk stratification and diagnosis with high sensitivity. This could aid clinicians in personalizing screening strategies for high-risk patients and follow-up strategies for treated cancer patients.

Influence of sildenafil on blood oxygen saturation of the obstructed bladder.

BACKGROUND: Blood oxygen saturation (BOS) is decreased in a low-compliant, overactive obstructed bladder. The objective of this study is to determine the effect of Sildenafil (SC) on bladder function and BOS) in an in vivo animal model of bladder outlet obstruction. METHODS: Thirty-two guinea pigs; sham operated (n = 8), sham operated + SC (n = 8), urethrally obstructed (n = 8) and urethrally obstructed + SC (n = 8) were studied during an 8 week period. BOS of the bladder wall was measured by differential path-length spectroscopy (DPS) before obstruction, at day 0, and at week 8. The bladder function was evaluated by urodynamic studies every week. RESULTS: Before surgery and after sham operation all study parameters were comparable. After sham operation, bladder function and BOS did not change. In the obstructed group the urodynamic parameters were deteriorated and BOS was decreased. In the group obstruction + SC, bladder compliance remained normal and overactivity occurred only sporadic. BOS remained unchanged compared to the sham group and was significantly higher compared to the obstruction group. CONCLUSIONS: In an obstructed bladder the loss of bladder function is accompanied by a significant decrease in BOS. Treatment of obstructed bladders with SC yields a situation of high saturation, high bladder compliance and almost no overactivity. Maintaining the microcirculation of the bladder wall might result in better bladder performance without significant loss of bladder function. Measurement of BOS and interventions focussing on tissue microcirculation may have a place in the evaluation / treatment of various bladder dysfunctions.

Differentiation between nerve and adipose tissue using wide-band (350-1,830 nm) in vivo diffuse reflectance spectroscopy.

BACKGROUND: Intraoperative nerve localization is of great importance in surgery. In certain procedures, where nerves show visual resemblance to surrounding adipose tissue, this can be particularly challenging for the human eye. An example of such a delicate procedure is thyroid and parathyroid surgery, where iatrogenic injury of the recurrent laryngeal nerve can result in transient or permanent vocal problems (0.5-2.0% reported incidence). A camera system, enabling nerve-specific image enhancement, would be useful in preventing such complications. This might be realized with hyperspectral camera technology using silicon (Si) or indium gallium arsenide (InGaAs) sensor chips. METHODS: As a first step towards such a camera, we evaluated the performance of diffuse reflectance spectroscopy by analysing spectra collected during 18 thyroid and parathyroid resections. We assessed the contrast information present in two different spectral ranges, for respectively Si and InGaAs sensors. Two hundred fifty three in vivo, wide-band diffuse reflectance spectra (350-1,830 nm range, 1 nm resolution) were acquired on 52 tissue spots, including nerve (n = 22), muscle (n = 12), and adipose tissue (n = 18). We extracted 36 features from these spectroscopic data: 18 gradients and 18 amplitude differences at predefined points in the tissue spectra. Best distinctive feature combinations were established using binary logistic regression. Classification performance was evaluated in a cross-validation (CV) approach by leave-one-out (LOO). To generalize nerve recognition applicability, we performed a train-test (TT) validation using the thyroid and parathyroid surgery data for training purposes and carpal tunnel release surgery data (10 nerve spots and 5 adipose spots) for classification purposes. RESULTS: For combinations of two distinctive spectral features, LOO revealed an accuracy of respectively 78% for Si-sensors and 95% for InGaAs-sensors. TT revealed accuracies of respectively 67% and 100%. CONCLUSIONS: Using diffuse reflectance spectroscopy we have identified that InGaAs sensors are better suited for automated discrimination between nerves and surrounding adipose tissue than Si sensors.

Microscopic analysis of the localization of two chlorin-based photosensitizers in OSC19 tumors in the mouse oral cavity.

BACKGROUND AND OBJECTIVE: The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT. MATERIALS AND METHODS: In this paper we have investigated the distribution of the photosensitizers chlorin e6 and Bremachlorin in the oral squamous cell carcinoma cell-line OSC19-Luc-Gfp in a tongue tumor, tumor boundary, invasive tumor boundary, and normal tongue tissue by the use of confocal microscopy of frozen sections. Tongues were harvested at t = [3, 4.5, 6, 24, 48] hours after injection. RESULTS: Both photosensitizers showed a decreasing fluorescence with increasing incubation time, and at all time points higher fluorescence was measured in tumor boundary than in tumor itself. For short incubation times, a higher fluorescence intensity was observed in the invasive tumor border and normal tissue compared to tumor tissue. Bremachlorin showed a small increase in tumor to normal ratio at 24 and 48 hours incubation time. Ce6 was undetectable at 48 hours. We did not find a correlation between photosensitizer localization and the presence of vasculature. CONCLUSION: The modest tumor/tumor boundary to normal selectivity of between 1.2 and 2.5 exhibited by Bremachlorin 24 and 48 hours after administration may allow selective targeting of tongue tumors. Further studies investigating the relationship between Bremachlorin concentration and therapeutic efficacy PDT with long incubation times are warranted.

Theoretical and experimental determination of the confocal function of OCT systems for accurate calculation of sample optical properties.

The attenuation coefficient of biological tissue could serve as an indicator of structural and functional changes related to the onset or progression of disease. Optical coherence tomography (OCT) provides cross sectional images of tissue up to a depth of a few millimeters, based on the local backscatter properties. The OCT intensity also depends on the confocal function, which needs to be characterised to determine correctly the exponential decay of the intensity based on Lambert-Beer. We present a model for the confocal function in scattering media based on the illumination with a Gaussian beam and the power transfer into a single mode fibre (SMF) of the backscattered light for an incoherently back scattered Gaussian beam using the Huygens-Fresnel principle and compare that model with the reflection from a mirror. We find that, contrary to previous literature, the confocal functions characterised by the Rayleigh range in the two models are identical. Extensive OCT focus series measurements on a mirror, Spectralon and Intralipid dilutions confirm our model, and show that for highly scattering samples the confocal function characterised by the Rayleigh range becomes depth dependent. From the diluted Intralipid measurements the attenuation coefficients are extracted using a singly scatter model that includes the previously established confocal function. The extracted attenuation coefficients were in good agreement for weakly scattering samples (µ s < 2 mm-1).

Localization of liposomal mTHPC formulations within normal epithelium, dysplastic tissue, and carcinoma of oral epithelium in the 4NQO-carcinogenesis rat model.

BACKGROUND AND OBJECTIVE: Foslip and Fospeg are liposomal formulations of the photosensitizer mTHPC (Foscan), which is used for photodynamic therapy (PDT) of malignancies. Literature suggests that liposomal mTHPC formulations have better properties and increased tumor uptake compared to Foscan. To investigate this, we used the 4NQO-induced carcinogen model to compare the localization of the different mTHPC formulations within normal, precancerous, and cancerous tissue. In contrast to xenograft models, the 4NQO model closely mimics the carcinogenesis of human oral dysplasia. MATERIALS AND METHODS: Fifty-four rats drank water with the carcinogen 4NQO. When oral examination revealed tumor, the rats received 0.15 mg/kg mTHPC (Foscan, Foslip, or Fospeg). At 2, 4, 8, 24, 48, or 96 hours after injection the rats were sacrificed. Oral tissue was sectioned for HE slides and for fluorescence confocal microscopy. The HE slides were scored on the severity of dysplasia by the epithelial atypia index (EAI). The calibrated fluorescence intensity per formulation or time point was correlated to EAI. RESULTS: Fospeg showed higher mTHPC fluorescence in normal and tumor tissue compared to both Foscan and Foslip. Significant differences in fluorescence between tumor and normal tissue were found for all formulations. However, at 4, 8, and 24 hours only Fospeg showed a significant difference. The Pearson's correlation between EAI and mTHPC fluorescence proved weak for all formulations. CONCLUSION: In our induced carcinogenesis model, Fospeg exhibited a tendency for higher fluorescence in normal and tumor tissue compared to Foslip and Foscan. In contrast to Foscan and Foslip, Fospeg showed significantly higher fluorescence in tumor versus normal tissue at earlier time points, suggesting a possible clinical benefit compared to Foscan. Low correlation between grade of dysplasia and mTHPC fluorescence was found.

Confocal corrected attenuation coefficient imaging in phantoms and in vivo using chromatic focal shift calibration.

Optical coherence tomography (OCT) is conventionally used for structural imaging of tissue. Calibrating the intensity values of OCT images can give information on the tissue's inherent optical properties, such as the attenuation coefficient, which can provide an additional parameter to quantify possible pathological changes. To obtain calibrated intensity values, the focus position and Rayleigh length of the incident beam need to be known. We explore the feasibility of extracting the focus position from an OCT scan acquired with a single focus setting using the chromatic aberration of the system. The chromatic focal shift of an OCT system is exploited to achieve different focus positions for sub-spectrum reconstructed OCT images. The ratios of these images are used to estimate the focus position. Reconstruction of a high-resolution B-scan from coherent addition of sub-spectrum confocal function corrected B-scans and subsequent high-resolution OCT attenuation coefficient imaging is demonstrated. Furthermore, we introduce a method to experimentally determine the chromatic focal shifts of an OCT system in phantoms and an in vivo human retina. These shifts are compared to the theoretically expected shifts calculated with ray tracing.

Extraction of intrinsic fluorescence from single fiber fluorescence measurements on a turbid medium.

This study utilizes Monte Carlo simulations of single fiber fluorescence to develop an empirical model that corrects for the influence of scattering and absorption on fluorescence intensity (F(SF)). The model expresses F(SF) in terms of the reduced scattering coefficient (µs') and absorption coefficient (µ(a)), each determined independently at excitation and emission wavelengths (λ(x) and λ(m)), and the fiber diameter (d(f)). This model returns accurate descriptions (mean residual <6%) of F(SF) across a biologically relevant range of µs' and µ(a) values and is insensitive to the form of the scattering phase function.

Changes in bladder wall blood oxygen saturation in the overactive obstructed bladder.

PURPOSE: Several studies suggest that hypoxia of the bladder wall contributes to bladder dysfunction but the exact relation between bladder function and blood oxygen saturation, a surrogate marker for hypoxia, is not known. We determined bladder wall blood oxygen saturation in vivo in an animal model of bladder outlet obstruction to establish the exact relation between blood oxygen saturation and bladder function. MATERIALS AND METHODS: In 8 sham operated and 8 urethrally obstructed guinea pigs we measured blood oxygen saturation of the bladder wall by differential path length spectroscopy before surgery and 8 weeks postoperatively. Urodynamic investigations performed during the whole 8-week period provided data on bladder function. RESULTS: Before surgery and 8 weeks after sham surgery blood oxygen saturation in the bladder wall was between 88% and 95% during filling. It decreased during voiding and returned to greater than 90% within 30 seconds. Eight weeks after obstruction saturation was significantly lower than in the sham operated group during filling and voiding. The decrease was positively related to bladder pressure during filling and voiding, and was more pronounced when overactivity was present. Local bladder contractions occurred without a measurable increase in bladder pressure but were associated with a decrease in saturation. CONCLUSIONS: A normal bladder maintains a high oxygen saturation level during filling. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder contractions without a measurable increase in bladder pressure were associated with a decrease in blood saturation.

Method to quantitate absorption coefficients from single fiber reflectance spectra without knowledge of the scattering properties.

This study presents a methodology to accurately extract the absorption coefficient from single fiber reflectance spectra measured in turbid media without a priori knowledge of either the reduced scattering coefficient or the phase function. This novel approach accounts for the interrelated effects these properties have on the photon path length, yielding estimates of an absorption coefficient on average within <7.5% of true values over a wide range of biologically relevant optical properties.

Method to quantitatively estimate wavelength-dependent scattering properties from multidiameter single fiber reflectance spectra measured in a turbid medium.

This study utilizes experimentally validated Monte Carlo simulations to identify a mathematical formulation of the reflectance intensity collected by a single fiber probe expressed in terms of the reduced scattering coefficient (µs'), fiber diameter d(fiber), and a property of the first two moments of the scattering phase function (γ). This model is then utilized to accurately obtain wavelength-dependent estimates of µs'(λ) and γ(λ) from multiple single fiber spectral measurements of a turbid medium obtained with different diameters. This method returns accurate descriptions (mean residual <3%) of both µs' and γ across the biologically relevant range.

Fluorescence localization and kinetics of mTHPC and liposomal formulations of mTHPC in the window-chamber tumor model.

BACKGROUND AND OBJECTIVE: Foslip® and Fospeg® are liposomal formulations of the photosensitizer mTHPC, intended for use in Photodynamic Therapy (PDT) of malignancies. Foslip consists of mTHPC encapsulated in conventional liposomes, Fospeg consists of mTHPC encapsulated in pegylated liposomes. Possible differences in tumor fluorescence and vasculature kinetics between Foslip, Fospeg, and Foscan® were studied using the rat window-chamber model. MATERIAL AND METHODS: In 18 rats a dorsal skin fold window chamber was installed and a mammary carcinoma was transplanted in the subcutaneous tissue. The dosage used for intravenous injection was 0.15 mg/kg mTHPC for each formulation. At seven time-points after injection (5 minutes to 96 hours) fluorescence images were made with a CCD. The achieved mTHPC fluorescence images were corrected for tissue optical properties and autofluorescence by the ratio fluorescence imaging technique of Kascakova et al. Fluorescence intensities of three different regions of interest (ROI) were assessed; tumor tissue, vasculature, and surrounding connective tissue. RESULTS: The three mTHPC formulations showed marked differences in their fluorescence kinetic profile. After injection, vascular mTHPC fluorescence increased for Foslip and Fospeg but decreased for Foscan. Maximum tumor fluorescence is reached at 8 hours for Fospeg and at 24 hours for Foscan and Foslip with overall higher fluorescence for both liposomal formulations. Foscan showed no significant difference in fluorescence intensity between surrounding tissue and tumor tissue (selectivity). However, Fospeg showed a trend toward tumor selectivity at early time points, while Foslip reached a significant difference (P < 0.05) at these time points. CONCLUSIONS: Our results showed marked differences in fluorescence intensities of Fospeg, Foslip, and Foscan, which suggest overall higher bioavailability for the liposomal formulations. Pegylated liposomes seemed most promising for future application; as Fospeg showed highest tumor fluorescence at the earlier time points.

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging.

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed.

Investigation of methods to extract confocal function parameters for the depth resolved determination of attenuation coefficients using OCT in intralipid samples, titanium oxide phantoms, and in vivo human retinas.

The attenuation coefficient provides a quantitative parameter for tissue characterization and can be calculated from optical coherence tomography (OCT) data, but accurate determination requires compensation for the confocal function. We present extensive measurement series for extraction of the focal plane and the apparent Rayleigh length from the ratios of OCT images acquired with different focus depths and compare these results with two alternative approaches. By acquiring OCT images for a range of different focus depths the optimal focus plane difference is determined for intralipid and titanium oxide (TiO2) phantoms with different scatterer concentrations, which allows for calculation of the attenuation coefficient corrected for the confocal function. The attenuation coefficient is determined for homogeneous intralipid and TiO2 samples over a wide range of concentrations. We further demonstrate very good reproducibility of the determined attenuation coefficient of layers with identical scatter concentrations in a multi-layered phantom. Finally, this method is applied to in vivo retinal data.

Digital Resilience Biomarkers for Personalized Health Maintenance and Disease Prevention.

Health maintenance and disease prevention strategies become increasingly prioritized with increasing health and economic burden of chronic, lifestyle-related diseases. A key element in these strategies is the empowerment of individuals to control their health. Self-measurement plays an essential role in achieving such empowerment. Digital measurements have the advantage of being measured non-invasively, passively, continuously, and in a real-world context. An important question is whether such measurement can sensitively measure subtle disbalances in the progression toward disease, as well as the subtle effects of, for example, nutritional improvement. The concept of resilience biomarkers, defined as the dynamic evaluation of the biological response to an external challenge, has been identified as a viable strategy to measure these subtle effects. In this review, we explore the potential of integrating this concept with digital physiological measurements to come to digital resilience biomarkers. Additionally, we discuss the potential of wearable, non-invasive, and continuous measurement of molecular biomarkers. These types of innovative measurements may, in the future, also serve as a digital resilience biomarker to provide even more insight into the personal biological dynamics of an individual. Altogether, digital resilience biomarkers are envisioned to allow for the measurement of subtle effects of health maintenance and disease prevention strategies in a real-world context and thereby give personalized feedback to improve health.

Noninvasive measurement of oxygen saturation of the microvascular blood in Barrett's dysplasia by use of optical spectroscopy.

BACKGROUND: Current investigations into endoscopic screening for the early detection of Barrett's esophageal adenocarcinoma have focused on visualization of the microvascular morphology by using narrow-band imaging (NBI). Adjustment of the center wavelength, particularly of the NBI blue imaging filter, may lead to improved image contrast, depending on the oxygen saturation of the microvascular blood of dysplastic and early cancerous Barrett's mucosa. OBJECTIVE: To perform in vivo, noninvasive measurements of the oxygen saturation of the microvascular blood for different pathologic grades of Barrett's mucosa by using differential path-length spectroscopy (DPS). DESIGN: DPS measurements were made on normal (n = 7), low-grade dysplastic (n = 10), high-grade dysplastic (n = 7), and cancerous (n = 4) Barrett's mucosa by using a fiber-optic probe, and were correlated to the histologic outcome of biopsy specimens taken from the same location. SETTING: Academic medical center. PATIENTS: Fifteen patients with Barrett's esophagus who were undergoing gastroscopy. INTERVENTIONS: Biopsy specimens were taken from suspicious areas in the esophagus. MAIN OUTCOME MEASUREMENTS: The oxygen saturation of the microvascular blood of different pathologic grades of Barrett's mucosa was assessed. RESULTS: The oxygen saturation of the microvascular blood remains high (approximately 90%) throughout the metaplasia-dysplasia-adenocarcinoma sequence. LIMITATION: The small number of patients. CONCLUSIONS: The current NBI blue imaging filter, centered on the peak absorption of oxyhemoglobin (415 nm), is well chosen, and little improvement in image contrast is to be expected from changes in this center wavelength.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy.

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; approximately 0.2 mm(3) and approximately 10(2) mm(3), respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Monitoring interstitial m-THPC-PDT in vivo using fluorescence and reflectance spectroscopy.

BACKGROUND AND OBJECTIVE: In order to understand the mechanisms of photodynamic therapy (PDT) it is important to monitor parameters during illumination that yield information on deposited PDT dose. The aim of this study is to investigate the possibility of monitoring implicit parameters, such as photobleaching, in addition to monitoring explicit parameters (fluence (rate), oxygenation, photosensitizer concentration) directly or indirectly. These parameters are monitored during PDT without interrupting the therapeutic illumination. MATERIALS AND METHODS: Rats were injected with 0.3 mg kg(-1) m-THPC. Sixteen hours after administration the abdominal muscle in rats was irradiated for 1,500 seconds using clinically relevant fluence rates of 50, 100, and 250 mW cm(-1) of diffuser length at 652 nm. In addition to the linear diffuser for delivering treatment light, isotropic fiber-optic probes and fiber-optic probes for differential path-length spectroscopy (DPS) were placed on both sides of the muscle to monitor tissue physiological parameters, fluence rate, and fluorescence. RESULTS: The m-THPC treatment groups show a decrease in fluence rate throughout PDT of 16%, 19%, and 27% for the 50, 100, and 250 mW cm(-1) groups, respectively. Both during and post-PDT differences in vascular response between treatment groups and animals within the same treatment group are observed. Furthermore we show fluence rate dependent bleaching of m-THPC up to a measured fluence rate of 100 mW cm(-1). CONCLUSION: The data presented in this study show the possibility of simultaneously monitoring fluence (rate), fluorescence, hemoglobin oxygen saturation, and blood volume during PDT without interruptions to the therapeutic illumination. Differences in saturation profiles between animals and treatment groups indicate differences in vascular response during illumination. Furthermore, the relationship between fluence rate and m-THPC fluorescence photobleaching is complex in an interstitial environment.

In vivo subdiffuse scanning laser oximetry of the human retina.

Scanning laser ophthalmoscopes (SLOs) have the potential to perform high speed, high contrast, functional imaging of the human retina for diagnosis and follow-up of retinal diseases. Commercial SLOs typically use a monochromatic laser source or a superluminescent diode for imaging. Multispectral SLOs using an array of laser sources for spectral imaging have been demonstrated in research settings, with applications mainly aiming at retinal oxygenation measurements. Previous SLO-based oximetry techniques are predominantly based on wavelengths that depend on laser source availability. We describe an SLO system based on a supercontinuum (SC) source and a double-clad fiber using the single-mode core for illumination and the larger inner cladding for quasi-confocal detection to increase throughput and signal-to-noise ratio. A balanced detection scheme was implemented to suppress the relative intensity noise of the SC source. The SLO produced dual wavelength, high-quality images at 10 frames / s with a maximum 20 deg imaging field-of-view with any desired combination of wavelengths in the visible spectrum. We demonstrate SLO-based dual-wavelength oximetry in vessels down to 50 µm in diameter. Reproducibility was demonstrated by performing three different imaging sessions of the same volunteer, 8 min apart. Finally, by performing a wavelength sweep between 485 and 608 nm, we determined, for our SLO geometry, an approximately linear relationship between the effective path length of photons through the blood vessels and the vessel diameter.

Confidence intervals on fit parameters derived from optical reflectance spectroscopy measurements.

We validate a simple method for determining the confidence intervals on fitted parameters derived from modeling optical reflectance spectroscopy measurements using synthetic datasets. The method estimates the parameter confidence intervals as the square roots of the diagonal elements of the covariance matrix, obtained by multiplying the inverse of the second derivative matrix of chi2 with respect to its free parameters by chi2/v, with v the number of degrees of freedom. We show that this method yields correct confidence intervals as long as the model used to describe the data is correct. Imperfections in the fitting model introduces a bias in the fitted parameters that greatly exceeds the estimated confidence intervals. We investigate the use of various methods to identify and subsequently minimize the bias in the fitted parameters associated with incorrect modeling.