Beam-profile compensation for quantum yield characterisation of Yb-Tm codoped upconverting nanoparticles emitting at 474 nm, 650 nm and 804 nm.

Upconverting nanoparticles (UCNPs) have found widespread applications in biophotonics and energy harvesting due to their unique non-linear optical properties arising from energy transfer upconversion (ETU) mechanisms. However, accurately characterising the power density-dependent efficiency of UCNPs using the internal quantum yield (iQY) is challenging due to the lack of methods that account for excitation beam-profile distortions. This limitation hinders the engineering of optimal UCNPs for diverse applications. To address this, this work present a novel beam profile compensation strategy based on a general analytical rate-equations model, enabling the evaluation of iQY for ETU processes of arbitrary order, such as ETU2, ETU3, and beyond. The method was applied to characterise the ETU2 and ETU3 processes corresponding to the main emission peaks (474 nm, 650 nm, and 804 nm) of a Yb-Tm codoped core-shell ß-UCNP. Through this approach, the transition power density points (which delimit the distinct non-linear regimes of the upconversion luminescence (UCL)), and the saturation iQY values (which are reached at high excitation power densities above the transition points) were determined. The ETU2 process exhibits a single transition power density point, denoted as ρ2, while the ETU3 processes involve two transition points, ρ2 and ρ3. By compensating for the beam profile, we evaluate the iQY of individual lines across a wide dynamic range of excitation power densities (up to 105 W cm-2), encompassing both non-linear and linear regimes of UCL. This study introduces a valuable approach for accurately characterising the iQY of UCNPs, facilitating a deeper understanding of the upconversion and its performance. By addressing excitation beam-profile distortions, this method provides a comprehensive and reliable assessment of the power density-dependent iQY. The results highlight the applicability and effectiveness of this beam profile compensation strategy, which can be employed for a wide range of UCNPs. This advancement opens new avenues for the tailored design and application of UCNPs in various fields, especially for biophotonics.

Generalised analytical model of the transition power densities of the upconversion luminescence and quantum yield.

The quantum yield (QY) evaluation of upconverting nanoparticles (UCNPs) is an essential step in the characterisation of such materials. The QY of UCNPs is governed by competing mechanisms of populating and depopulating the electronic energy levels involved in the upconversion (UC), namely linear decay rates and energy transfer rates. As a consequence, at low excitation, the QY excitation power density (ρ) dependence obeys the power law ρn-1, where n represents the number of absorbed photons required for the emission of a single upconverted photon and determines the order of the energy transfer upconversion (ETU) process. At high power densities, the QY transits to a saturation level independent of the ETU process and the number of excitation photons, as a result of an anomalous power density dependence present in UCNPs. Despite the importance of this non-linear process for several applications (e.g., living tissue imaging and super-resolution-microscopy), little has been reported in the literature regarding theoretical studies to describe the UC QY, especially for ETUs with order higher than two. Therefore, this work presents a simple general analytical model, which introduces the concept of the transition power density points and QY saturation to characterise the QY of an arbitrary ETU process. The transition power density points determine where the power density dependence of the QY and the UC luminescence changes. The results provided in this paper from fitting the model to experimental QY data of a Yb-Tm codoped ß-UCNP for 804 nm and 474 nm emissions (ETU2 and ETU3 processes, respectively) exemplify the application of the model. The common transition points found for both processes were compared to each other showing strong agreement with theory, as well as, compared to previous reports when possible.

Evaluation of relative beam-profile-compensated quantum yield of upconverting nanoparticles over a wide dynamic range of power densities.

The presented work uses a discrete strategy of beam profile compensation to evaluate the local internal quantum yield (iQY) of upconverting nanoparticles (UCNPs) at the pixel level of the beam profile using a compact CMOS camera. The two-photon process of upconversion with a central emission peak at 804 nm was studied for a ß-phase core-shell Tm-codoped UCNP under 976 nm excitation. At the balancing power density point, ρb, found to be 44 ± 3 W cm-2, the iQY, ηb, was obtained as 2.3 ± 0.1%. Combining the power density dynamic range provided by the pixel depth of the camera with the dynamic range achieved using two distinct beam profiles to excite the UCNPs, the iQY was evaluated throughout a range of 104 in the iQY scale (from 0.0003% to 4.6%) and 106 in power densities of excitation (from 0.003 W cm-2 to 1050 W cm-2). To the best of our knowledge, these are the lowest values ever obtained as QY results have never been reported under 0.02% or at excitation power densities below 0.01 W cm-2.

Determination of reference values for optical properties of liquid phantoms based on Intralipid and India ink.

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable.

MADSTRESS: a linear approach for evaluating scattering and absorption coefficients of samples measured using time-resolved spectroscopy in reflection.

Time-resolved spectroscopy is a powerful technique permitting the separation of the scattering properties from the chemical absorption properties of a sample. The reduced scattering coefficient and the absorption coefficient are usually obtained by fitting diffusion or Monte Carlo models to the measured data using numerical optimization techniques. However, these methods do not take the spectral dimension of the data into account during the evaluation procedure, but evaluate each wavelength separately. A procedure involving multivariate methods may seem more appealing for people used to handling conventional near-infrared data. In this study we present a new method for processing TRS spectra in order to compute the absorption and reduced scattering coefficients. This approach, MADSTRESS, is based on linear regression and a two-dimensional (2D) interpolation procedure. The method has allowed us to calculate absorption and scattering coefficients of apples and fructose powder. The accuracy of the method was good enough to provide the identification of fructose absorption peaks in apple absorption spectra and the construction of a calibration model predicting the sugar content of apples.

In vitro measurements of optical properties of porcine brain using a novel compact device.

Knowledge of the optical properties of tissues can be applied in numerous medical and scientific fields, including cancer diagnostics and therapy. There are many different ways of determining the optical properties of turbid media. The paper describes measurements of the optical properties of porcine brain tissue using novel instrumentation for simultaneous absorption and scattering characterisation of small turbid samples. Integrating sphere measurements are widely used as a reference method for determination of the optical properties of relatively thin turbid samples. However, this technique is associated with bulky equipment, complicated measuring techniques, interference compensation techniques and inconvenient sample handling. It is believed that the sphere for some applications can be replaced by a new, compact device, called the combined angular and spatially resolved head sensor, to measure the optical properties of thin turbid samples. The results compare very well with data obtained with an integrating sphere for well-defined samples. The instrument was shown to be accurate to within 12% for microa and 1% for micro's in measurements of intralipid-ink samples. The corresponding variations of data were 17% and 2%, respectively. The reduced scattering coefficient for porcine white matter was measured to be 100 cm(-1) at 633 nm, and the value for coagulated brain tissue was 65 cm(-1). The corresponding absorption coefficients were 2 and 3 cm(-1), respectively.

Changes in tissue optical properties due to radio-frequency ablation of myocardium.

The optical properties of pig heart tissue were measured after in vivo ablation therapy had been performed during open-heart surgery. In vitro samples of normal and ablated tissue were subjected to measurements with an optically integrating sphere set-up in the region 470-900 nm. Three independent measurements were made: total transmittance, total reflectance and collimated transmittance, which made it possible to extract the absorption and scattering coefficients and the scattering anisotropy factor g, using an inverse Monte Carlo model. Between 470 and 700 nm, only the reduced scattering coefficient and absorption could be evaluated. The absorption spectra were fitted to known tissue chromophore spectra, so that the concentrations of haemoglobin and myoglobin could be estimated. The reduced scattering coefficient was compared with Mie computations to provide Mie equivalent average radii. Most of the absorption was from myoglobin, whereas haemoglobin absorption was negligible. Metmyoglobin was formed in the ablated tissue, which could yield a spectral signature to distinguish the ablated tissue with a simple optical probe to monitor the ablation therapy. The reduced scattering coefficient increased by, on average, 50% in the ablated tissue, which corresponded to a slight decrease in the Mie equivalent radius.

Kinetics of the superficial perfusion and temperature in connection with photodynamic therapy of basal cell carcinomas using esterified and non-esterified 5-aminolaevulinic acid.

BACKGROUND: Photodynamic therapy (PDT) is a local treatment modality with increasing indications for various malignant and non malignant diseases. The treatment parameters have not yet been optimized as there is a need for a better understanding of the process. The skin is an important target and serves as a good model for monitoring and evaluating the interaction of light with biological tissue. OBJECTIVES: The tissue perfusion and the temperature of basal cell carcinomas were measured in connection with PDT in order to investigate the biological mechanisms involved. METHODS: An infrared camera was used during the treatment to measure skin temperature and a laser Doppler perfusion imaging device was used to image the superficial perfusion before and after treatment. Six hours after topical application of 5-aminolaevulinic acid (ALA) or methyl esterified ALA (ALA-ME), 38 basal cell carcinomas were treated using light from a diode laser at 633 nm. RESULTS: In the lesions, the perfusion immediately after PDT was similar to that before PDT. One hour after the treatment the perfusion in the lesion was increased 50% compared with before PDT. However, in the skin surrounding the lesions the perfusion was doubled immediately after PDT and was still increasing 1 h after treatment. A temperature increase in the lesions of about 1-3 degrees C was observed for light fluence rates of 100-150 mW cm-2. In all patients treated, a diffuse temperature increase was visible outside the lesions. In some of the patients, the outlines of the blood vessels surrounding the treated lesions became visible in the thermal images. Measurements of temperature on healthy volunteers not administered photosensitizer, but illuminated with light of the same fluence rate, showed a similar increase in temperature in the illuminated spots. However, no temperature increase was observed outside the illuminated area. No statistically significant differences were found between the measurements on patients treated with ALA and ALA-ME. CONCLUSIONS: The increased perfusion in the area surrounding the lesions after PDT, as seen by perfusion and temperature measurements, is the result of an inflammatory reaction to the PDT process. However, directly after PDT the perfusion in the lesions was the same as before irradiation. The combination of these observations suggests the presence of local blood stasis during and immediately after the treatment. The temperature measurements showed that the increased temperature was well below the temperature limit of hyperthermal damage. Furthermore, the measurements indicate that the increase in temperature was primarily a consequence of the heat absorbed in the tissue.

Photodynamic therapy vs. cryosurgery of basal cell carcinomas: results of a phase III clinical trial.

BACKGROUND: A previously reported randomized clinical trial showed treatment of Bowen's disease using photodynamic therapy (PDT) with topically applied delta-aminolaevulinic acid (ALA) to be at least as effective as cryosurgery and to be associated with fewer adverse effects. OBJECTIVES: To compare ALA-PDT and cryotherapy in the treatment of histopathologically verified basal cell carcinomas (BCCs) in a non-blinded, prospective phase III clinical trial. METHODS: One lesion from each of 88 patients was included. The BCCs were divided into superficial and nodular lesions. The follow-up period was restricted to 1 year with close follow-up for the first 3 months. Efficacy was assessed as the recurrence rate 12 months after the first treatment session, verified by histopathology. Tolerability was evaluated as the time of healing, pain and discomfort during and after the treatment, and final cosmetic outcome. RESULTS: Histopathologically verified recurrence rates in the two groups were statistically comparable and were 25% (11 of 44) for ALA-PDT and 15% (six of 39) for cryosurgery. However, clinical recurrence rates were only 5% (two of 44) for PDT and 13% (five of 39) for cryosurgery. Additional treatments, usually one, had to be performed in 30% of the lesions in the PDT group. The healing time was considerably shorter and the cosmetic outcome significantly better with PDT. Pain and discomfort during the treatment session and in the following week were low, and were equivalent with the two treatment modalities. CONCLUSIONS: In terms of efficacy, ALA-PDT is comparable with cryosurgery as a treatment modality for BCCs. Retreatments are more often required with PDT than with cryosurgery. This can easily be performed due to the shorter healing time, less scarring and better cosmetic outcome that follows ALA-PDT.

Multivariate analysis of laryngeal fluorescence spectra recorded in vivo.

BACKGROUND AND OBJECTIVE: The potential of using various multivariate analysis methods for classification of fluorescence spectra acquired in vivo from laryngeal tissues in Patients was investigated. STUDY DESIGN/MATERIALS AND METHODS: Autofluorescence spectra were measured on 29 normal tissue sites and 25 laryngeal lesions using 337-nm excitation. Four different multivariate analysis schemes were applied. Laryngeal fluorescence spectra from patients who had been administered delta-aminolevulinic acid (ALA) were obtained using 405-nm excitation and were classified using partial least squares discriminant analysis (PLS-DA). RESULTS: For autofluorescence spectra, logistic regression based on principal component analysis (PCA) or PLS, or PLS-DA all resulted in sensitivities and specificities around 90% for lesion vs. normal. Using ALA and 405-nm excitation gave a sensitivity of 100% and a specificity of 69%. CONCLUSION: Multivariate analysis of fluorescence spectra could allow classification of laryngeal lesions in vivo with high sensitivity and specificity. PLS performs at least as well as PCA, and PLS-DA performs as well as logistic regression techniques on these data.

Fiber-optic probe for noninvasive real-time determination of tissue optical properties at multiple wavelengths.

We present a compact, fast, and versatile fiber-optic probe system for real-time determination of tissue optical properties from spatially resolved continuous-wave diffuse reflectance measurements. The system collects one set of reflectance data from six source-detector distances at four arbitrary wavelengths with a maximum overall sampling rate of 100 Hz. Multivariate calibration techniques based on two-dimensional polynomial fitting are employed to extract and display the absorption and reduced scattering coefficients in real-time mode. The four wavelengths of the current configuration are 660, 785, 805, and 974 nm, respectively. Cross-validation tests on a 6 x 7 calibration matrix of Intralipid-dye phantoms showed that the mean prediction error at, e.g., 785 nm was 2.8% for the absorption coefficient and 1.3% for the reduced scattering coefficient. The errors are relative to the range of the optical properties of the phantoms at 785 nm, which were 0-0.3/cm for the absorption coefficient and 6-16/cm for the reduced scattering coefficient. Finally, we also present and discuss results from preliminary skin tissue measurements.

Analysis of gas dispersed in scattering media.

Monitoring of free gas embedded in scattering media, such as wood, fruits, and synthetic materials, is demonstrated by use of diode laser spectroscopy combined with sensitive modulation techniques. Gas detection is made possible by the contrast of the narrow absorptive feature of the free-gas molecules with the slow wavelength dependence of the absorption and scattering cross sections in solids and liquids. An absorption sensitivity of 2.5 x 10(-4), corresponding to a 1.25-mm air column, is demonstrated by measurements of dispersed molecular oxygen. These techniques open up new possibilities for characterization and diagnostics, including internal gas pressure and gas-exchange assessment, in organic and synthetic materials.

Blood perfusion studies on basal cell carcinomas in conjunction with photodynamic therapy and cryotherapy employing laser-Doppler perfusion imaging.

Superficial blood perfusion was monitored using laser-Doppler perfusion imaging in connection with a phase III clinical trial comparing photodynamic therapy, utilizing topically applied delta-aminolevulinic acid, with cryotherapy of basal cell carcinomas. A total of 526 images were recorded before and immediately after the treatment and during the follow-up period. Before treatment, the lesions exhibited a blood perfusion 3+/-2 times that in normal tissue. Both treatment modalities induced an increased blood perfusion inside the lesions, which slowly approached normal values in conjunction with successful treatments. The blood perfusion in successfully treated lesions approached normal values 2 months after photodynamic therapy, and about 1 year after cryotherapy. The tissue perfusion in recurrent lesions did not decrease to normal values after the treatment, suggesting that the laser-Doppler perfusion imaging technique can be used to follow the healing process and discover possible persistent tumour growth.

Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin.

BACKGROUND AND OBJECTIVE: Fluorescence techniques can provide powerful noninvasive means for medical diagnosis, based on the detection of either endogenous or exogenous fluorophores. The fluorescence of delta-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) has already shown promise for the diagnosis of tumors. The aim of the study was to investigate the localization of skin tumors after the topical application of ALA, by detecting the PpIX fluorescence either in the spectral or in the time domain. STUDY DESIGN/MATERIALS N AND METHODS: Two fluorescence imaging systems were used to identify basal cell carcinomas of the skin in humans, after topical application of 20% ALA ointment. Both systems rely on the comparison between the exogenous and the endogenous fluorescence, performed either in the spectral domain or in the time domain. The first system works by using three images acquired through different spectral filters, whereas the second one measures the spatial map of the average fluorescence lifetime of the sample. RESULTS: A clear demarcation of skin malignancies was successfully performed in vivo noninvasively with both fluorescence imaging systems. CONCLUSION: The two complementary approaches considered in the present study show promise for skin tumor detection and delineation based on specific fluorescence features.

Multiple polynomial regression method for determination of biomedical optical properties from integrating sphere measurements.

We present a new, to our knowledge, method for extracting optical properties from integrating sphere measurements on thin biological samples. The method is based on multivariate calibration techniques involving Monte Carlo simulations, multiple polynomial regression, and a Newton-Raphson algorithm for solving nonlinear equation systems. Prediction tests with simulated data showed that the mean relative prediction error of the absorption and the reduced scattering coefficients within typical biological ranges were less than 0.3%. Similar tests with data from integrating sphere measurements on 20 dye-polystyrene microsphere phantoms led to mean errors less than 1.7% between predicted and theoretically calculated values. Comparisons showed that our method was more robust and typically 5-10 times as fast and accurate as two other established methods, i.e., the inverse adding-doubling method and the Monte Carlo spline interpolation method.

Quantifying the absorption and reduced scattering coefficients of tissuelike turbid media over a broad spectral range with noncontact Fourier-transform hyperspectral imaging.

Absorption (mu(a)) and reduced scattering (mu(s)') spectra of turbid media were quantified with a noncontact imaging approach based on a Fourier-transform interferometric imaging system (FTIIS). The FTIIS was used to collect hyperspectral images of the steady-state diffuse reflectance from turbid media. Spatially resolved reflectance data from Monte Carlo simulations were fitted to the recorded hyperspectral images to quantify mu(a) and mu(s)' spectra in the 550-850-nm region. A simple and effective calibration approach was introduced to account for the instrument response. With reflectance data that were close to and far from the source (0.5-6.5 mm), mu(a) and mu(s)' of homogeneous, semi-infinite turbid phantoms with optical property ranges comparable with those of tissues were determined with an accuracy of +/-7% and +/-3%, respectively. Prediction accuracy for mu(a) and mu(s)' degraded to +/-12% and +/-4%, respectively, when only reflectance data close to the source (0.5-2.5 mm) were used. Results indicate that reflectance data close to and far from the source are necessary for optimal quantification of mu(a) and mu(s)'. The spectral properties of mu(a) and mu(s)' values were used to determine the concentrations of absorbers and scatterers, respectively. Absorber and scatterer concentrations of two-chromophore turbid media were determined with an accuracy of +/-5% and +/-3%, respectively.

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.

Photodynamic therapy utilising topical delta-aminolevulinic acid in non-melanoma skin malignancies of the eyelid and the periocular skin.

PURPOSE: Photodynamic therapy utilising delta-aminolevulinic acid-induced protoporphyrin IX photosensitisation, was evaluated as a treatment modality for nonmelanoma skin malignancies of the eyelids and the periocular skin. METHODS: Photodynamic therapy with laser light at 635 nm was performed in 13 patients with 19 basal cell carcinomas and one patient with 3 cutaneous T-cell lymphoma lesions. A topical application regimen was used and in median 3 treatment sessions were given to each lesion. RESULTS: A visually judged complete response was achieved in 42% (8/19) of the basal cell carcinomas and in 100% of the T-cell lymphoma lesions (3/3). Partial tumour response was seen in 42% (8/19) and no response in 16% (3/19) of the basal cell carcinomas. The median follow-up time was 12 months. No, or very little scarring and loss of cilia were induced by the treatment. CONCLUSION: Photodynamic therapy with delta-aminolevulinic acid can eradicate eye-close non-melanoma skin malignancies without compromising the function of the eyelids. Further development of the method is needed to reach the same cure rates as those of the conventional treatment modalities.

Clinical spectral characterisation of colonic mucosal lesions using autofluorescence and delta aminolevulinic acid sensitisation.

BACKGROUND AND AIMS: Laser induced fluorescence (LIF) from colonic mucosa was measured in vivo with and without delta aminolevulinic acid (ALA) in an attempt to differentiate between neoplasia and non-neoplasia in real time during colonoscopy. METHODS: Spectra from 32 adenomas, 68 normal sites, and 14 hyperplastic polyps in 41 patients were obtained with a point monitoring system. Twenty one of the patients had been given a low dose of ALA as a photosensitiser before the examination. Light of 337, 405, or 436 nm wavelength was used as excitation. Stepwise multivariate linear regression analysis was performed. RESULTS: With 337 nm excitation, 100% sensitivity and 96% specificity was obtained between normal mucosa and adenomas. Seventy seven per cent of the hyperplastic polyps were classified as non-neoplastic. When exciting with 405 and 436 nm, the possibility of distinguishing different types of tissue was considerably better in the ALA patients than in the non-ALA patients. CONCLUSIONS: The in vivo point measurements imply that a good discrimination between normal tissue and adenomatous polyps can be obtained using the LIF technique. Excitation at 337 nm and at 405 nm or 436 nm using ALA gives good results. LIF also shows potential for distinguishing adenomatous from hyperplastic polyps. The number of detection wavelengths could be reduced if chosen properly.

Changes in local hepatic blood perfusion during interstitial laser-induced thermotherapy of normal rat liver measured by interstitial laser Doppler flowmetry.

Interstitial laser Doppler flowmetry was used to measure the effect of interstitial laser-induced thermotherapy on local blood perfusion in normal rat liver in the peripheral treatment region elevated to hyperthermic temperatures. The Nd:YAG laser emitting at 1064 nm was utilised as heat generation source. The plane-cut tip of an optical fibre was placed in the middle of the exteriorised left liver lobe. Blood perfusion and temperature were measured in the liver parenchyma 4 mm from the laser fibre. The temperature at the location of the liver temperature sensor was maintained at 41 or 44°C during 30 min by regulating the power of the heating laser. The laser Doppler signal was recorded during and after heat treatment, for a total time of 60 min. At 41°C, a significant increase in perfusion up to 1.3 times the initial value was observed 2-16 min after start of treatment. At 44°C, perfusion decreased continuously during and after treatment, and was significantly different from control 40 min after start of treatment. The results may be valuable in assessing the thermal response of tissues surrounding the target in interstitial laser-induced thermotherapy of liver tumours during conditions of normal blood flow.