The dirty and clean dose concept: Towards creating proton therapy treatment plans with a photon-like dose response.

BACKGROUND: Applying tolerance doses for organs at risk (OAR) from photon therapy introduces uncertainties in proton therapy when assuming a constant relative biological effectiveness (RBE) of 1.1. PURPOSE: This work introduces the novel dirty and clean dose concept, which allows for creating treatment plans with a more photon-like dose response for OAR and, thus, less uncertainties when applying photon-based tolerance doses. METHODS: The concept divides the 1.1-weighted dose distribution into two parts: the clean and the dirty dose. The clean and dirty dose are deposited by protons with a linear energy transfer (LET) below and above a set LET threshold, respectively. For the former, a photon-like dose response is assumed, while for the latter, the RBE might exceed 1.1. To reduce the dirty dose in OAR, a MaxDirtyDose objective was added in treatment plan optimization. It requires setting two parameters: LET threshold and max dirty dose level. A simple geometry consisting of one target volume and one OAR in water was used to study the reduction in dirty dose in the OAR depending on the choice of the two MaxDirtyDose objective parameters during plan optimization. The best performing parameter combinations were used to create multiple dirty dose optimized (DDopt) treatment plans for two cranial patient cases. For each DDopt plan, 1.1-weighted dose, variable RBE-weighted dose using the Wedenberg RBE model and dose-average LETd distributions as well as resulting normal tissue complication probability (NTCP) values were calculated and compared to the reference plan (RefPlan) without MaxDirtyDose objectives. RESULTS: In the water phantom studies, LET thresholds between 1.5 and 2.5 keV/µm yielded the best plans and were subsequently used. For the patient cases, nearly all DDopt plans led to a reduced Wedenberg dose in critical OAR. This reduction resulted from an LET reduction and translated into an NTCP reduction of up to 19 percentage points compared to the RefPlan. The 1.1-weighted dose in the OARs was slightly increased (patient 1: 0.45 Gy(RBE), patient 2: 0.08 Gy(RBE)), but never exceeded clinical tolerance doses. Additionally, slightly increased 1.1-weighted dose in healthy brain tissue was observed (patient 1: 0.81 Gy(RBE), patient 2: 0.53 Gy(RBE)). The variation of NTCP values due to variation of α/ß from 2 to 3 Gy was much smaller for DDopt (2 percentage points (pp)) than for RefPlans (5 pp). CONCLUSIONS: The novel dirty and clean dose concept allows for creating biologically more robust proton treatment plans with a more photon-like dose response. The reduced uncertainties in RBE can, therefore, mitigate uncertainties introduced by using photon-based tolerance doses for OAR.

Increased relative biological effectiveness and periventricular radiosensitivity in proton therapy of glioma patients.

PURPOSE: Currently, there is an intense debate on variations in intra-cerebral radiosensitivity and relative biological effectiveness (RBE) in proton therapy of primary brain tumours. Here, both effects were retrospectively investigated using late radiation-induced brain injuries (RIBI) observed in follow-up after proton therapy of patients with diagnosed glioma. METHODS: In total, 42 WHO grade 2-3 glioma patients out of a consecutive patient cohort having received (adjuvant) proton radio(chemo)therapy between 2014 and 2017 were eligible for analysis. RIBI lesions (symptomatic or clinically asymptomatic) were diagnosed and delineated on contrast-enhanced T1-weighted magnetic resonance imaging scans obtained in the first two years of follow-up. Correlation of RIBI location and occurrence with dose (D), proton dose-averaged linear energy transfer (LET) and variable RBE dose parameters were tested in voxel- and in patient-wise logistic regression analyses. Additionally, anatomical and clinical parameters were considered. Model performance was estimated through cross-validated area-under-the-curve (AUC) values. RESULTS: In total, 64 RIBI lesions were diagnosed in 21 patients. The median time between start of proton radio(chemo)therapy and RIBI appearance was 10.2 months. Median distances of the RIBI volume centres to the cerebral ventricles and to the clinical target volume border were 2.1 mm and 1.3 mm, respectively. In voxel-wise regression, the multivariable model with D, D × LET and periventricular region (PVR) revealed the highest AUC of 0.90 (95 % confidence interval: 0.89-0.91) while the corresponding model without D × LET revealed a value of 0.84 (0.83-0.86). In patient-level analysis, the equivalent uniform dose (EUD11, a = 11) in the PVR using a variable RBE was the most prominent predictor for RIBI with an AUC of 0.63 (0.32-0.90). CONCLUSIONS: In this glioma cohort, an increased radiosensitivity within the PVR was observed as well as a spatial correlation of RIBI with an increased RBE. Both need to be considered when delivering radio(chemo)therapy using proton beams.

Comparing biological effectiveness guided plan optimization strategies for cranial proton therapy: potential and challenges.

BACKGROUND: To introduce and compare multiple biological effectiveness guided (BG) proton plan optimization strategies minimizing variable relative biological effectiveness (RBE) induced dose burden in organs at risk (OAR) while maintaining plan quality with a constant RBE. METHODS: Dose-optimized (DOSEopt) proton pencil beam scanning reference treatment plans were generated for ten cranial patients with prescription doses ≥ 54 Gy(RBE) and ≥ 1 OAR close to the clinical target volume (CTV). For each patient, four additional BG plans were created. BG objectives minimized either proton track-ends, dose-averaged linear energy transfer (LETd), energy depositions from high-LET protons or variable RBE-weighted dose (DRBE) in adjacent serially structured OARs. Plan quality (RBE = 1.1) was assessed by CTV dose coverage and robustness (2 mm setup, 3.5% density), dose homogeneity and conformity in the planning target volumes and adherence to OAR tolerance doses. LETd, DRBE (Wedenberg model, α/ßCTV = 10 Gy, α/ßOAR = 2 Gy) and resulting normal tissue complication probabilities (NTCPs) for blindness and brainstem necrosis were derived. Differences between DOSEopt and BG optimized plans were assessed and statistically tested (Wilcoxon signed rank, α = 0.05). RESULTS: All plans were clinically acceptable. DOSEopt and BG optimized plans were comparable in target volume coverage, homogeneity and conformity. For recalculated DRBE in all patients, all BG plans significantly reduced near-maximum DRBE to critical OARs with differences up to 8.2 Gy(RBE) (p < 0.05). Direct DRBE optimization primarily reduced absorbed dose in OARs (average ΔDmean = 2.0 Gy; average ΔLETd,mean = 0.1 keV/µm), while the other strategies reduced LETd (average ΔDmean < 0.3 Gy; average ΔLETd,mean = 0.5 keV/µm). LET-optimizing strategies were more robust against range and setup uncertaintes for high-dose CTVs than DRBE optimization. All BG strategies reduced NTCP for brainstem necrosis and blindness on average by 47% with average and maximum reductions of 5.4 and 18.4 percentage points, respectively. CONCLUSIONS: All BG strategies reduced variable RBE-induced NTCPs to OARs. Reducing LETd in high-dose voxels may be favourable due to its adherence to current dose reporting and maintenance of clinical plan quality and the availability of reported LETd and dose levels from clinical toxicity reports after cranial proton therapy. These optimization strategies beyond dose may be a first step towards safely translating variable RBE optimization in the clinics.

An ion-independent phenomenological relative biological effectiveness (RBE) model for proton therapy.

BACKGROUND: A relative biological effectiveness (RBE) of 1.1 is used for proton therapy though clinical evidence of varying RBE was raised. Clinical studies on RBE variability have been conducted for decades for carbon radiation, which could advance the understanding of the clinical proton RBE given an ion-independent RBE model. In this work, such a model, linear and simple, using the beam quantity Q = Z2/E (Z = ion charge, E = kinetic energy per nucleon) was tested and compared to the commonly used, proton-specific and linear energy transfer (LET) based Wedenberg RBE model. MATERIAL AND METHODS: The Wedenberg and Q models, both predicting RBEmax and RBEmin (i.e., RBE at vanishing and very high dose, respectively), are compared in terms of ion-dependence and prediction power. An experimental in-vitro data ensemble covering 115 publications for various ions was used as dataset. RESULTS: The model parameter of the Q model was observed to be similar for different ions (in contrast to LET). The Q model was trained without any prior knowledge of proton data. For proton RBE, the differences between experimental data and corresponding predictions of the Wedenberg or the Q model were highly comparable. CONCLUSIONS: A simple linear RBE model using Q instead of LET was proposed and tested to be able to predict proton RBE using model parameter trained based on only RBE data of other particles in a clinical proton energy range for a large in-vitro dataset. Adding (pre)clinical knowledge from carbon ion therapy may, therefore, reduce the dominating biological uncertainty in proton RBE modelling. This would translate in reduced RBE related uncertainty in proton therapy treatment planning.

Clinical use and future requirements of relative biological effectiveness: Survey among all European proton therapy centres.

BACKGROUND AND PURPOSE: The relative biological effectiveness (RBE) varies along the treatment field. However, in clinical practice, a constant RBE of 1.1 is assumed, which can result in undesirable side effects. This study provides an accurate overview of current clinical practice for considering proton RBE in Europe. MATERIALS AND METHODS: A survey was devised and sent to all proton therapy centres in Europe that treat patients. The online questionnaire consisted of 39 questions addressing various aspects of RBE consideration in clinical practice, including treatment planning, patient follow-up and future demands. RESULTS: All 25 proton therapy centres responded. All centres prescribed a constant RBE of 1.1, but also applied measures (except for one eye treatment centre) to counteract variable RBE effects such as avoiding beams stopping inside or in front of an organ at risk and putting restrictions on the minimum number and opening angle of incident beams for certain treatment sites. For the future, most centres (16) asked for more retrospective or prospective outcome studies investigating the potential effect of the effect of a variable RBE. To perform such studies, 18 centres asked for LET and RBE calculation and visualisation tools developed by treatment planning system vendors. CONCLUSION: All European proton centres are aware of RBE variability but comply with current guidelines of prescribing a constant RBE. However, they actively mitigate uncertainty and risk of side effects resulting from increased RBE by applying measures and restrictions during treatment planning. To change RBE-related clinical guidelines in the future more clinical data on RBE are explicitly demanded.

Towards harmonizing clinical linear energy transfer (LET) reporting in proton radiotherapy: a European multi-centric study.

BACKGROUND: Clinical data suggest that the relative biological effectiveness (RBE) in proton therapy (PT) varies with linear energy transfer (LET). However, LET calculations are neither standardized nor available in clinical routine. Here, the status of LET calculations among European PT institutions and their comparability are assessed. MATERIALS AND METHODS: Eight European PT institutions used suitable treatment planning systems with their center-specific beam model to create treatment plans in a water phantom covering different field arrangements and fulfilling commonly agreed dose objectives. They employed their locally established LET simulation environments and procedures to determine the corresponding LET distributions. Dose distributions D1.1 and DRBE assuming constant and variable RBE, respectively, and LET were compared among the institutions. Inter-center variability was assessed based on dose- and LET-volume-histogram parameters. RESULTS: Treatment plans from six institutions fulfilled all clinical goals and were eligible for common analysis. D1.1 distributions in the target volume were comparable among PT institutions. However, corresponding LET values varied substantially between institutions for all field arrangements, primarily due to differences in LET averaging technique and considered secondary particle spectra. Consequently, DRBE using non-harmonized LET calculations increased inter-center dose variations substantially compared to D1.1 and significantly in mean dose to the target volume of perpendicular and opposing field arrangements (p < 0.05). Harmonizing LET reporting (dose-averaging, all protons, LET to water or to unit density tissue) reduced the inter-center variability in LET to the order of 10-15% within and outside the target volume for all beam arrangements. Consequentially, inter-institutional variability in DRBE decreased to that observed for D1.1. CONCLUSION: Harmonizing the reported LET among PT centers is feasible and allows for consistent multi-centric analysis and reporting of tumor control and toxicity in view of a variable RBE. It may serve as basis for harmonized variable RBE dose prescription in PT.

Visualizing minute details in light-sheet and confocal microscopy data by combining 3D rolling ball filtering and deconvolution.

We developed an open-source deconvolution software that stunningly increases the visibility of minute details, as for example, neurons or nerve fibers in light-sheet microscopy or confocal microscopy data by combining rolling ball background subtraction in three directions with deconvolution using a synthetic or measured point spread function. Via automatic block-wise processing image stacks of virtually unlimited size can be deconvolved even on small computers with 8 or 16 GB RAM. By parallelization and optional GPU-acceleration, the software works with high speed: On a PC equipped with a state-of-the-art NVidia graphic board a three dimensional (3D)-stack of about 1 billion voxels can be deconvolved within 5 to 10 minutes. The implemented variation of the Richardson-Lucy deconvolution algorithm preserves the photogrammetry of the image data by using flux-preserving regularization, an approach that to our knowledge has not been applied for deconvolving microscopy data before.

Impact of range uncertainty on clinical distributions of linear energy transfer and biological effectiveness in proton therapy.

PURPOSE: Increased radiation response after proton irradiation, such as late radiation-induced toxicity, is determined by high dose and elevated linear energy transfer (LET). Steep dose-averaged LET (LETd ) gradients and elevated LETd occur at the end of proton range and might be particularly sensitive to uncertainties in range prediction. Therefore, this study quantified LETd distributions and the impact of range uncertainty in robust dose-optimized proton treatment plans and assessed the biological effect in normal tissues and tumors of patients. METHODS: For each of six cancer patients (two brain, head-and-neck, and prostate), two nominal treatment plans were robustly dose optimized using single- and multi-field optimization, respectively. For each plan, two additional scenarios with ±3.5% range deviation relative to the nominal plan were derived by global rescaling of stopping-power ratios. Dose and LETd distributions were calculated for each scenario using the beam parameters of the corresponding nominal plan. The variability in relative biological effectiveness (RBE) and probability of late radiation-induced brain toxicity (PIC ) was assessed. RESULTS: The optimization technique (single- vs multi-field) had a negligible impact on the LETd distributions in the clinical target volume (CTV) and in most organs at risk (OARs). LETd distributions in the CTV were rather homogeneous with arithmetic mean of LETd below 3.2 keV/µm and robust against range deviations. The RBE variability within the CTV induced by range uncertainty was small (≤0.05, 95% confidence interval). In OARs, LETd hotspots (>7 keV/µm) occurred and LETd distributions were inhomogeneous and sensitive to range deviations. LETd hotspots and the impact of range deviations were most prominent in OARs of brain tumor patients which translated in RBE values exceeding 1.1 in all brain OARs. The near-maximum predicted PIC in healthy brain tissue of brain tumor patients was smaller than 5% and occurred adjacent to the CTV. Range deviations induced absolute differences in PIC up to 1.2%. CONCLUSIONS: Robust dose optimization generates LETd distributions in the target volume robust against range deviations. The current findings support using a constant RBE within the CTV. The impact of range deviations on the considered probability of late radiation-induced toxicity in brain tissue was limited for robust dose-optimized treatment plans. Incorporation of LETd in robust optimization frameworks may further reduce uncertainty related to the RBE-weighted dose estimation in normal tissues.

Total Synthesis of (±)-Thebainone A by Intramolecular Nitrone Cycloaddition.

Using an intramolecular nitrone cycloaddition and a Heck cyclization as the crucial transformations, a total synthesis of the racemic morphine alkaloid thebainone A was accomplished in 22 steps commencing with isovanillin.

Deconvolution of light sheet microscopy recordings.

We developed a deconvolution software for light sheet microscopy that uses a theoretical point spread function, which we derived from a model of image formation in a light sheet microscope. We show that this approach provides excellent blur reduction and enhancement of fine image details for image stacks recorded with low magnification objectives of relatively high NA and high field numbers as e.g. 2x NA 0.14 FN 22, or 4x NA 0.28 FN 22. For these objectives, which are widely used in light sheet microscopy, sufficiently resolved point spread functions that are suitable for deconvolution are difficult to measure and the results obtained by common deconvolution software developed for confocal microscopy are usually poor. We demonstrate that the deconvolutions computed using our point spread function model are equivalent to those obtained using a measured point spread function for a 10x objective with NA 0.3 and for a 20x objective with NA 0.45.

High-resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO).

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep-tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)-expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP-expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity-dependent expression pattern of tdTomato.

Simultaneous study of molecular and micelle diffusion in a technical microemulsion system by dynamic light scattering.

HYPOTHESIS: The application of dynamic light scattering (DLS) is well-established for measuring diffusion coefficients related to either molecular or translational micelle diffusion. The simultaneous determination of both transport properties should be feasible, but has not been reported in the literature yet. EXPERIMENTS: Different diffusion modes present in a microemulsion and selected subsystems consisting of a polyol mixture, a binary surfactant mixture, and carbon dioxide (CO2) were investigated systematically by DLS at temperatures of (314, 333, and 353) K and corresponding pressures of (10, 13, and 16) MPa. FINDINGS: Diffusion coefficients related to molecular and translational micelle diffusion could be measured simultaneously and increase with increasing temperature. From the translational diffusion coefficients, an increase in the hydrodynamic diameter of the micelles from their non-swollen to the CO2-swollen state being in agreement with literature data for the same and similar microemulsions was found. The effective diffusion coefficients related to the faster molecular diffusion process only observable in the presence of CO2 are not affected significantly by the surfactant. The time-dependent parts of the recorded intensity correlation functions related to molecular diffusion processes are heterodyne because the scattered light modulated by molecular concentration fluctuations is superimposed with light scattered by the micelles.

High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster.

The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique we developed, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows us to visualise long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applying image fusion from orthogonal directions. This methodological integration of novel chemical, optical, and computational techniques allows a major advance in the analysis of global neural circuit organisation.

Generation and annihilation time of magnetic droplet solitons.

Magnetic droplet solitons were first predicted to occur in materials with uniaxial magnetic anisotropy due to a long-range attractive interaction between elementary magnetic excitations, magnons. A non-equilibrium magnon population provided by a spin-polarized current in nanocontacts enables their creation and there is now clear experimental evidence for their formation, including direct images obtained with scanning x-ray transmission microscopy. Interest in magnetic droplets is associated with their unique magnetic dynamics that can lead to new types of high frequency nanometer scale oscillators of interest for information processing, including in neuromorphic computing. However, there are no direct measurements of the time required to nucleate droplet solitons or their lifetime-experiments to date only probe their steady-state characteristics, their response to dc spin-currents. Here we determine the timescales for droplet annihilation and generation using current pulses. Annihilation occurs in a few nanoseconds while generation can take several nanoseconds to a microsecond depending on the pulse amplitude. Micromagnetic simulations show that there is an incubation time for droplet generation that depends sensitively on the initial magnetic state of the nanocontact. An understanding of these processes is essential to utilizing the unique characteristics of magnetic droplet solitons oscillators, including their high frequency, tunable and hysteretic response.

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet.

Based on the modal analysis method, we developed a model that describes the output beam of a diode-pumped solid state (DPSS) laser emitting a multimode beam. Measuring the output beam profile in the near field and at the constructed far field the individual modes, their respective contributions, and their optical parameters are determined. Using this information, the beam is optically reshaped into a quasi-Gaussian beam by the interference and superposition of the various modes. This process is controlled by a mode modulator unit that includes different meso-aspheric elements and a soft-aperture. The converted beam is guided into a second optical unit comprising achromatic-aspheric elements to produce a thin light sheet for ultramicroscopy. We found that this light sheet is markedly thinner and exhibits less side shoulders compared with a light sheet directly generated from the output of a DPSS multimode laser.

Outlook on optimizing ultramicroscopy imaging technique through optical characterization.

Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented. Using the new light sheet in combination with our objective equipped with a modulator unit to compensate the refractive index mismatch between air and mediums with indices of 1.45-1.56, we present high resolution images of various biological samples that were chemically cleared using different methods. They demonstrate a marked improvement in quality, contrast and resolution.

Study on the applicability of dynamic light scattering (DLS) to microemulsions including supercritical carbon dioxide-swollen micelles.

The applicability of dynamic light scattering (DLS) for the characterization of the size of supercritical carbon dioxide (sc-CO2)-swollen micelles in a polyester polyol-based multicomponent microemulsion with nonionic surfactant has been thoroughly proved for the first time in this work. Systematic experiments confirming that a hydrodynamic mode is observable in either a homodyne or a heterodyne detection scheme as well as the evaluation of the influence of the laser power applied to the slightly colored microemulsion have ensured an accurate implementation of this technique for a technically relevant system. The correlation times associated with the translational diffusion coefficient of the swollen micelles in a continuous liquid phase were measured for temperatures from (298.15 to 338.15)K at pressures of (90 and 100)bar. While there was no significant effect of pressure, it was found that the translational diffusion coefficient increases with increasing temperature as expected. We postulate this is primarily related to the effect of decreasing viscosity of the continuous phase. An estimation of the hydrodynamic diameter of the sc-CO2-swollen micelles is in good agreement with values for similar systems reported in the literature. For the derivation of absolute sizes for corresponding systems, also dynamic viscosity and refractive index data will be determined simultaneously in a currently developed closed experimental loop.

Birds of a Feather Laugh Together: An Investigation of Humour Style Similarity in Married Couples.

The present research investigated the degree of similarity in humour styles between spouses as assessed with the Humour Styles Questionnaire (HSQ). Furthermore, self-esteem was investigated as a potential moderator of partner humour style similarity. A sample of 116 heterosexual, married couples independently completed questionnaires assessing self-reported humour styles across the 32 item HSQ, as well as global self-esteem. Results indicated that there is significant positive association between the humour styles of married partners. This association was moderated by individual self-esteem. Specifically, participants with high self-esteem were found to have greater humour style similarity with their partners. Similarity was also greater for positive compared to negative types of humour. Implications for the use of dyadic data in investigating the roles of humour within couples are discussed.

Ultramicroscopy: development and outlook.

We present an overview of the ultramicroscopy technique we developed. Starting from developments 100 years ago, we designed a light sheet microscope and a chemical clearing to image complete mouse brains. Fluorescence of green fluorescent protein (GFP)-labeled neurons in mouse brains could be preserved with our 3DISCO clearing and high-resolution three-dimensional (3-D) recordings were obtained. Ultramicroscopy was also used to image whole mouse embryos and flies. We improved the optical sectioning of our light sheet microscope by generating longer and thinner light sheets with aspheric optics. To obtain high-resolution images, we corrected available air microscope objectives for clearing solutions with high refractive index. We discuss how eventually super resolution could be realized in light sheet microscopy by applying stimulated emission depletion technology. Also the imaging of brain function by recording of mouse brains expressing cfos-GFP is discussed. Finally, we show the first 3-D recordings of human breast cancer with light sheet microscopy as application in medical diagnostics.

Reduction of photo bleaching and long term archiving of chemically cleared GFP-expressing mouse brains.

Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years.