Photobiomodulation (PBM) is a promising approach to treat Parkinson’s disease (PD) symptoms in cellular or animal models. Unfortunately, little information is available on the optical parameters playing a role in the light dosimetry during PBM. We conducted a study to determine the effective attenuation coefficient μeff of PD-relevant human deep brain tissues at 671 and 808 nm, using a multichannel fluence rate-meter comprising sub-millimeter isotropic detectors. The first step involved measurements of tissue modifications induced by postmortem situation and tissue storage on rabbit brains. The parameter μeff was measured using various tissue conditions (in vivo, immediately after sacrifice, after six weeks’ storage at −20°C or in 10 % formaldehyde solution) on eight female New Zealand white rabbits. In the second step, fluence rate was measured at various locations of a frozen human deep brain when the deep brain was illuminated from the sphenoidal sinus. The results were processed by an iterative Monte-Carlo algorithm to generate sets of optical parameters, and results collected on rabbit brains were used to extrapolate the μeff value that would be observed in human deep brain tissues in vivo. Under all tissue conditions, the value of μeff at 808 nm was smaller than that at 671 nm. After long-term storage for six weeks at −20°C, μeff decreased, on average by 15 to 25 % at all wavelengths, while it increased by 5 to 15 % at all wavelengths after storage in formaldehyde. Therefore, a reasonable estimate of in vivo human deep brain μeff values at 671 and 808 nm can be obtained by multiplying the data we report by 120 %.
KEYWORDS: Tissues, Brain, Optical properties, Scattering, In vivo imaging, Diffusers, Absorption, Signal attenuation, Monte Carlo methods, Tissue optics
The outcome of light-based therapeutic approaches depends on light propagation in biological tissues, which is governed by their optical properties. The objective of this study was to quantify optical properties of brain tissue in vivo and postmortem and assess changes due to tissue handling postmortem. The study was carried out on eight female New Zealand white rabbits. The local fluence rate was measured in the VIS/NIR range in the brain in vivo, just postmortem, and after six weeks’ storage of the head at −20°C or in 10% formaldehyde solution. Only minimal changes in the effective attenuation coefficient μeff were observed for two methods of sacrifice, exsanguination or injection of KCl. Under all tissue conditions, μeff decreased with increasing wavelengths. After long-term storage for six weeks at −20°C, μeff decreased, on average, by 15 to 25% at all wavelengths, while it increased by 5 to 15% at all wavelengths after storage in formaldehyde. We demonstrated that μeff was not very sensitive to the method of animal sacrifice, that tissue freezing significantly altered tissue optical properties, and that formalin fixation might affect the tissue’s optical properties.
Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420 nm, FWHM=50/100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525 nm, FWHM=80/50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.
Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.
During fluorescence cystoscopy, it is observed that the acquired images are sometimes blurred by a greenish background originating from the bladder washout fluid. Several fluorophores are involved in this overall liquid fluorescence, and their exact origin and relative contributions remain unknown. In this study, the bladder washout fluid is sampled at different times during fluorescence cystoscopy examinations. In total, 32 samples from 12 patients are analyzed with a spectrofluorimeter (excitation range: 350-445 nm, emission range 380-700 nm). This study shows clearly that the
position of the fluorescence peaks (excitation/emission wavelengths: 450/525 nm, 405/625 nm) and shoulder (440/525 nm) is reproducible between different patients. It also suggests that an excitation at wavelengths higher than 400 nm helps to suppress this solution background fluorescence. Additionally, the pH of the solution seems to influence the position of the fluorescence peaks, and this suggests that changing the pH of the examination liquid could help in
avoiding the greenish background.
Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer,
even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix) in the bladder for about an hour,
photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we
observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions
negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular
architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to
differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being
investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence
positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each
observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular
loops, vascular area and diameter) of the fluorescence positive sites was characterized in parallel with an in situ visual
grading and a dedicated software procedure. We describe here a simple image processing prototype that classifies the
HM images into two classes, according to their pixel distributions. For that purpose, we developed an algorithm in the
image spatial and frequency domain, so that the vascular architecture could be described objectively and quantitatively.
Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer,
even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix®) in the bladder for about an hour,
photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we
observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions
negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular
architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to
differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being
investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence
positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each
observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular
loops, vascular area and diameter) of the fluorescence positive sites was characterized in situ. Intrinsic contrast between
the vessels and the tissue was enhanced with an optimization of the spectral design. Preliminary results are presented
here.
Fluorescence detection of early superficial bladder cancer has been well established over the last years. This technique
exploits the selective production and accumulation within cancerous tissues of photoactive porphyrins (PaP), mainly
protoporphyrin IX (PpIX), after the instillation of hexaminolevulinic acid (Hexvix®) in the bladder. Although the
selective production of PpIX and the sensitivity of this procedure are outstanding, its specificity can be improved due to
false positive (FP) lesions. Therefore, our current research focuses on the Characterization of positive sites by high
magnification cystoscopy. Cancerization process often combines with changes in vascular architecture. It is likely that
the visualization of these modifications should allow us to differentiate false and true positive (TP). New methods, using
high magnification (HM) endoscopy, are being investigated by our group, and hopefully resulting in a reduced number
of biopsies. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" white light and fluorescence observation, as well as image acquisition with HM when the endoscope is in
contact with the tissue. This is realized by an optical setup directly integrated in the cystoscope. We describe here an offclinics
calibration procedure that will allow us to quantify the vessel structure and size once we use this optics to observe
the bladder mucosa.
Our long-term activity in the development of fluorescence imaging for the detection of early superficial bladder cancer
aimed at optimizing the selective production and accumulation of photoactivable porphyrins (PaP), mainly
protoporphyrin IX (PpIX), after the instillation of derivatives of aminolevulinic acid (ALA) within cancerous tissues.
This research eventually led to the approval of hexylaminolevulinate (HAL, Hexvix(R)) in 27 European countries.
Although the selective production of PpIX and the sensitivity of this procedure are outstanding, its specificity is limited
due to false positive lesions that are mainly associated with inflammations of the bladder mucosa. Therefore, our current
research focuses on the improvement of the specificity of this detection method. New methods, using high magnification
(HM) endoscopy, are being investigated by our group in order to discriminate false from true positive findings, and
hopefully resulting in a reduced number of biopsies. In this study, we are using a dedicated magnification cystoscope,
allowing conventional magnification during "macroscopic" white light and fluorescence observation, as well as image
acquisition with HM when the endoscope is in contact with the tissue. This is realized by an optical setup directly
integrated in the cystoscope. The diameter of the field of view of the images is 500 microns in the HM mode and the
resolution is about 3 microns. With this optical setup, our on going study is aimed at observing and characterizing the
neo*-vascularization of the flat fluorescing sites in order to distinguish (pre-)cancerous tissue from inflammation. Thirty
nine biopsies were taken on fluorescence-positive sites. The vascular patterns observed on CIS (n = 7) were significantly
altered in 5 of them (71%), as compared to normal and inflamed mucosa where such alterations were never observed.
Head and neck (H&N) cancer patients have a high incidence of second primary tumours in the tracheobronchial tree. Diagnostic autofluorescence bronchoscopy (DAFE) has shown promising results in the detection of early neoplastic and pre-neoplastic changes in the bronchi. We have investigated the medical impact of DAFE in a population of H&N cancer patients. The bronchoscopies were performed using a modified commercially available DAFE system. Endoscopic imaging of the tissue autofluorescence (AF) was combined with an online image analysis procedure allowing to discriminate between true and false positive results. White light (WL) bronchoscopy was performed as a control. Twenty-one patients with high lung cancer risk factors underwent WL and AF bronchoscopy with this improved system. Forty-one biopsies were taken on macroscopicall suspicious (WL or AF positive) sites. Seven patients were found to have second primary tumours in the bronchi. The sensitivity for the detection of these early lesions with the DAFE was 1.6 times larger than the sensitivity of WL bronchoscopy only. The positive predictive value (PPV) for AF is 79% (33% for WL alone). The PPV of both methods together is 100%. DAFE proved to be efficient for the detection of second primary lesions in H&N cancer patients and can be used as a simple addition to pre-operative work-up or follow-up in this patient population.
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