Multiplexed imaging in live cells using pulsed interleaved excitation spectral FLIM.

Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety of excitation/detection optical designs and fluorescence unmixing schemes have been proposed to allow for multiplexed imaging, rapid and reliable differentiation and quantification of multiple fluorescent species at each imaging pixel is still challenging. Here we present a pulsed interleaved excitation spectral fluorescence lifetime microscopic (PIE-sFLIM) system that can simultaneously image six fluorescent tags in live cells in a single hyperspectral snapshot. Using an alternating pulsed laser excitation scheme at two different wavelengths and a synchronized 16-channel time-resolved spectral detector, our PIE-sFLIM system can effectively excite multiple fluorophores and collect their emission over a broad spectrum for analysis. Combining our system with the advanced live-cell labeling techniques and the lifetime/spectral phasor analysis, our PIE-sFLIM approach can well unmix the fluorescence of six fluorophores acquired in a single measurement, thus improving the imaging speed in live-specimen investigation.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina.

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Path-length-multiplexed scattering-angle-diverse optical coherence tomography for retinal imaging.

A low-resolution path-length-multiplexed scattering angle diverse optical coherence tomography (PM-SAD-OCT) is constructed to investigate the scattering properties of the retinal nerve fiber layer (RNFL). Low-resolution PM-SAD-OCT retinal images acquired from a healthy human subject show the variation of RNFL scattering properties at retinal locations around the optic nerve head. The results are consistent with known retinal ganglion cell neural anatomy and principles of light scattering. Application of PM-SAD-OCT may provide potentially valuable diagnostic information for clinical retinal imaging.

Patients' needs in proton therapy: A survey among ten European facilities.

Aims: The number of Proton Therapy (PT) facilities is still limited worldwide, and the access to treatment could be characterized by patients' logistic and economic challenges. Aim of the present survey is to assess the support provided to patients undergoing PT across Europe. Methods: Through a personnel contact, an online questionnaire (62 multiple-choice and open-ended questions) via Microsoft Forms was administered to 10 European PT centers. The questionnaire consisted of 62 questions divided into 6 sections: i) personal data; ii) general information on clinical activity; iii) fractionation, concurrent systemic treatments and technical aspects of PT facility; iv) indication to PT and reimbursement policies; v) economic and/ or logistic support to patients vi) participants agreement on statements related to the possible limitation of access to PT. A qualitative analysis was performed and reported. Results: From March to May 2022 all ten involved centers filled the survey. Nine centers treat from 100 to 500 patients per year. Paediatric patients accounted for 10-30%, 30-50% and 50-70% of the entire cohort for 7, 2 and 1 center, respectively. The most frequent tumours treated in adult population were brain tumours, sarcomas and head and neck carcinomas; in all centers, the mean duration of PT is longer than 3 weeks. In 80% of cases, the treatment reimbursement for PT is supplied by the respective country's Health National System (HNS). HNS also provides economic support to patients in 70% of centers, while logistic and meal support is provided in 20% and 40% of centers, respectively. PT facilities offer economic and/or logistic support in 90% of the cases. Logistic support for parents of pediatric patients is provided by HNS only in one-third of centers. Overall, 70% of respondents agree that geographic challenges could limit a patient's access to proton facilities and 60% believe that additional support should be given to patients referred for PT care. Conclusions: Relevant differences exist among European countries in supporting patients referred to PT in their logistic and economic challenges. Further efforts should be made by HNSs and PT facilities to reduce the risk of inequities in access to cancer care with protons.

Combined stent insertion and single high-dose brachytherapy in patients with advanced esophageal cancer--results of a prospective safety study.

Previous randomized studies comparing the two commonly used palliative treatments for incurable esophageal cancer, i.e. stent insertion and intraluminal brachytherapy, have revealed the pros and cons of each therapy. While stent treatment offers a more prompt effect, brachytherapy results in more long-lasting relief of dysphagia and a better health-related quality of life (HRQL) in those living longer. This prospective pilot study aimed to explore the feasibility and safety of combining these two regimes and incorporating a single high dose of internal radiation. Patients with newly diagnosed, incurable cancer of the esophagus and dysphagia were eligible for inclusion, and stent insertion followed by a single dose (12 Gy) of brachytherapy was performed as a two-stage procedure. Clinical parameters including HRQL and adverse events were registered at inclusion, and 1, 2, 3, 6, and 12 months later. Twelve patients (nine males) with a median age of 73 years (range 54-85) were included. Stent insertion followed by a single dose of brachytherapy was successfully performed in all but one patient who was treated with stent only. Relief of dysphagia was achieved in the majority of cases (10/11, P < 0.05), but HRQL did not improve except for dysphagia-related items. Only minor adverse events, including chest pain, reflux, and restenosis, were reported. The median survival time after inclusion was 6.6 months. Our conclusion is that the combination of stent insertion and single high-dose brachytherapy seems to be a feasible and safe palliative regime in patients with advanced esophageal cancer. Randomized trials comparing the efficacy of this strategy to stent insertion or brachytherapy alone are warranted.

BMEntored: Enhancing the First-Year Experience in a BME Doctoral Program.

We describe our experiences with the first offering of a new program, BMEntored, for supporting first-year doctoral students in Biomedical Engineering (BME) during their first semester. The goal of BMEntored was to enhance the first-semester experience of first-year doctoral students in BME with an emphasis on guiding students in selecting a research supervisor and promoting cross-cohort, cross-lab social connections.

Generative adversarial network enables rapid and robust fluorescence lifetime image analysis in live cells.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is up to 2,800 times faster than the gold standard time-domain maximum likelihood estimation (TD_MLE) and that flimGANE provides a more accurate analysis of low-photon-count histograms in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis in live cells. With its advantages in speed and reliability, flimGANE is particularly useful in fundamental biological research and clinical applications, where high-speed analysis is critical.

Complex polarization ratio to determine polarization properties of anisotropic tissue using polarization-sensitive optical coherence tomography.

Complex polarization ratio (CPR) in materials with birefringence and biattenuance is shown as a logarithmic spiral in the complex plane. A multi-state Levenberg-Marquardt nonlinear fitting algorithm using the CPR trajectory collected by polarization sensitive optical coherence tomography (PS-OCT) was developed to determine polarization properties of an anisotropic scattering medium. The Levenberg-Marquardt nonlinear fitting algorithm using the CPR trajectory is verified using simulated PS-OCT data with speckle noise. Birefringence and biattenuance of a birefringent film, ex-vivo rodent tail tendon and in-vivo primate retinal nerve fiber layer were determined using measured CPR trajectories and the Levenberg-Marquardt nonlinear fitting algorithm.

Ciclosporin use in multi-drug therapy for meningoencephalomyelitis of unknown aetiology in dogs.

OBJECTIVE: To evaluate the efficacy and safety of ciclosporin therapy alone or in combination with corticosteroids and/or ketoconazole in dogs with diagnosis of meningoencephalomyelitis of unknown aetiology. METHODS: Medical records of 10 dogs diagnosed with meningoencephalomyelitis of unknown aetiology and treated with ciclosporin therapy alone or in combination with corticosteroids and/or ketoconazole were reviewed at the Veterinary Medical Teaching Hospital, University of Wisconsin-Madison. Laboratory abnormalities, side effects, clinical and cerebrospinal fluid responses to treatment and association between blood ciclosporin level and response to treatment were evaluated. Histopathological diagnosis was available in three patients. RESULTS: No significant abnormalities were detected on serial complete blood count and serum chemistry panel in any of the dogs. Side effects of ciclosporin therapy included excessive shedding, gingival hyperplasia and hypertrichosis. Overall median survival time for all dogs in the study was 930 days (range, 60 to more than 1290 days). In all dogs, serial cerebrospinal fluid analysis showed a marked improvement in the inflammation. CLINICAL SIGNIFICANCE: Results suggest that ciclosporin either alone or in combination with ketoconazole may be a safe and effective treatment for meningoencephalomyelitis of unknown aetiology in dogs.

Fibre orientation contrast for depth-resolved identification of structural interfaces in birefringent tissue.

Incorporation of polarimetric sensitivity into optical coherence tomography can provide additional image contrast when structures of interest are optically anisotropic (e.g., fibrous tissue). We present a generalized technique based on polarization-sensitive optical coherence tomography to detect changes in depth-resolved fibre orientation and thus increase image contrast in multiple-layered birefringent tissues. A high contrast B-scan image of collagen fibre orientation is shown for a porcine intervertebral disc cartilage specimen that exhibited low backscattering intensity contrast. Interfaces in the annulus fibrosus identified using depth-resolved fibre orientation allowed quantification of lamellae thickness. Moreover, the technique detects changes in fibre orientation without intense processing needed to effectively quantify tissue retardation and diattenuation.

Reflectance feedback control of photocoagulation in vivo.

OBJECTIVE: The objective of this research was to build a real-time feedback system that controlled lesion size. Two-dimensional reflectance images were acquired with a charge-coupled device camera during irradiation, and argon laser exposure was ended when parameters of the image reached prespecified values. METHODS: The real-time feedback control system was tested by creating lesions at different power levels in pigmented rabbits. Laser exposure time was controlled by monitoring the central reflectance. RESULTS: Results of feedback-controlled lesions formed in vivo in pigmented rabbits are presented. An ability to produce uniform lesions despite variation in tissue absorption or changes in laser power is demonstrated. CONCLUSIONS: Reflectance control of photocoagulation is possible; incorporation of feedback during photocoagulation has numerous therapeutic and safety benefits over current ballistic delivery.

Maintaining the cornea and the general physiological environment in visual neurophysiology experiments.

Neurophysiologists have been investigating the responses of neurons in the visual system for the past half-century using monkeys and cats that are anesthetized and paralyzed, with the non-blinking eyelids open for prolonged periods of time. Impermeable plastic contact lenses have been used to prevent dehydration of the corneal epithelium, which would otherwise occur in minutes. Unfortunately, such lenses rapidly introduce a variety of abnormal states that lead to clouding of the cornea, degradation of the retinal image, and premature termination of the experiment. To extend the viability of such preparations, a new protocol for maintenance of corneal health has been developed. The protocol uses rigid gas permeable contact lenses designed to maximize gas transmission, rigorous sterile methods, and a variety of methods for sustaining and monitoring the overall physiology of the animal. The effectiveness of the protocol was evaluated clinically by ophthalmoscopy before, during, and after the experiments, which lasted 8-10 days. Histopathology and quantitative histology were performed on the corneas following the experiment. Our observations showed that this protocol permits continuous contact lens wear without adversely affecting the corneas. Thus, it is possible to collect data 24 h each day, for the entire duration of the experiment.

Initial in vivo results of a hybrid retinal photocoagulation system.

We describe initial in vivo experimental results of a new hybrid digital and analog design for retinal tracking and laser beam control. An overview of the design is given. The results show in vivo tracking rates which exceed the equivalent of 38 degrees/s in the eye. A robotically assisted lesion pattern is created for laser surgery to treat conditions such as diabetic retinopathy and retinal breaks.

Chronic measurement of epidural pressure with an induction-powered oscillator transducer.

An induction-powered oscillator transducer (IPOT) was designed for the chronic measurement of epidural pressure. The transducer was completely implantable so all pressure measurements were made through the intact skin. The IPOT had a linear pressure range from -50 to +200 cm H2O, was sensitive to 1 mm H2O and had a zero drift of less than 1 mm H2O/day under full load. Zero drift was minimized by using a hermetically-sealed metal bellows transducing element which was chemically treated to prevent corrosion and creep. The correlation between epidural pressure and intraventricular pressure was determined during the first 24 hours after implantation in six dogs. Epidural pressure was found to be a linear function of intraventricular fluid pressure. Epidural pressure and intraventricular pressure were essential equal provided the epidural wedge pressure was minimized by proper insertion of the transducer. The correlation between epidural pressure and intraventricular pressure was determined after chronic implantation in five dogs. Epidural pressure was a linear function of intraventricular pressure in the chronically implanted dogs, but epidural pressure was not equal to intraventricular pressure. After chronic implantation, the epidural pressure transducer was not responsive to changes in intraventricular pressure because of mechanical changes in the dura. The dura became stiff and non-compliant. Maximum correlation between epidural pressure and intraventricular fluid pressure in chronic implantations will depend on judicious material selection and mechanical design at the transducer-dura interface.

Corneal photocoagulation with continuous wave and pulsed holmium: YAG radiation.

In this study, the effectiveness of pulsed and continuous wave (CW) holmium: YAG lasers in coagulating in vitro pig corneas was analyzed. With the CW laser, irradiance and exposure time were varied; irradiance, from 162 to 324 W/cm2 and exposure time, from 200 to 800 ms. With the pulsed laser, number of pulses and radiant exposure were varied; number of pulses per lesion, from 4 to 30 and radiant exposure, from 10 to 25 J/cm2. Laser-induced corneal damage was determined by analyzing histological cross sections of each lesion. Depth and diameter of the lesions were plotted against the varying laser parameters. Light and birefringent photomicrographs of typical lesion histology show that the pulsed laser significantly damaged superficial layers of the cornea and could not achieve the coagulation depths produced by the CW laser. Additional histology demonstrates that minimal surface damage (intrastromal coagulation) occurred when the CW laser beam was delivered with a sapphire-tipped contact probe. The results provide empirical data on the sensitivity of each parameter in producing a range of coagulation end points. In addition, the experimental results describe trends between the parameters of either laser and the extent of coagulation.

Real-time algorithm for retinal tracking.

Conventional retinal laser photocoagulation is presently performed by an ophthalmologist manually aiming a low-power laser beam at a desired site and firing a high-power laser for a preselected interval of time. To automate this process a retinal tracker must acquire a target, track small saccades, and identify loss of track during a large saccade. The authors successfully implemented a real-time algorithm that used a simple computer, video digitizing card, low light video camera, and fundus camera to perform rudimentary tracking on a photograph of a retina undergoing smooth circular motion. The algorithm tracked speeds up to 5 Hz, or 27 degrees/s, which equated to the retina moving in a 525 microns diameter circle.

Preliminary results on reflectance feedback control of photocoagulation in vivo.

The size of therapeutic laser-induced retinal lesions is critical for effective treatment and minimal complications. Due to tissue variability, the size of a lesion that results from a given set of laser irradiation parameters cannot be predicted. Real time feedback control of lesion size is implemented based on two-dimensional reflectance images acquired during irradiation. Preliminary results of feedback controlled lesions formed in pigmented rabbits demonstrate an ability to produce uniform lesions despite variations in tissue absorption or changes in laser power.

Reflectance as an indirect measurement of the extent of laser-induced coagulation.

Two-dimensional reflectance images and surface thermal distributions were recorded during argon laser induced coagulation. During laser irradiation, coagulated egg yolk formed a white lesion. The whiteness, or reflectance caused by backscattering of light from the forming lesion, would be measured after a short delay from the onset of laser irradiation. In the experiments which covered exposure time from 4.5 to 17.0 s, we found that it started slowly, the reflectance increased rapidly once the surface temperature of the lesion reached approximately 90 degrees C. After this rapid rise, the reflectance began to taper off until no change in reflectance was recorded. There was a 0.98 correlation between lesion diameter measured from a two dimensional reflectance image and the lesion diameter that was measured microscopically. There was a 0.92 correlation between reflectance at the center of the lesion and microscopically measured depth of coagulation at the same point. However, the correlation between microscopically measured coagulation depth and width was only 0.88.

The influence of the design of two different bioresorbable barriers on the results of guided tissue regeneration therapy. An intra-individual comparative study in the monkey.

The aim of the present study was to compare two bioresorbable barriers to evaluate whether differences in design influence the result of guided tissue regeneration (GTR) therapy. Twenty-four (24) plaque exposed, recession type defects in 4 monkeys were treated. Contralateral defects were randomized for test or control treatment. During a healing period of 6 weeks, gingival recession resulting in device exposure occurred at 3 test and 10 control sites. One control barrier was exfoliated. Histologically, 9 of the 12 test barriers were completely integrated with the surrounding tissues. At 3 test sites, epithelium had migrated apically outside the barrier to a level not exceeding one-third of the height of the device. Seven of the 11 control barriers were enclosed by dentogingival epithelium. The adjacent connective tissue exhibited local inflammatory cell infiltrates (ICT). At the remaining 4 control sites, the epithelial downgrowth as well as the adjacent ICT areas were limited to the coronal 1/3 of the device. New attachment; i.e., new cementum with inserting collagen fibers, averaged 2.2 mm and 0.8 mm at the test and control sites respectively (P < 0.01). Based on the results of the present study, it was concluded that a bioresorbable GTR device, designed to prevent epithelial downgrowth along the barrier surface, has a higher potential to promote new attachment formation than a device which does not have this property.