Chronic persistent middle ear infections (otitis media) can lead to cholesteatoma, an ingrowth of multi-layered keratinizing squamous epithelium, which requires complete removal in a surgical procedure. However, intraoperative detection is difficult and may result in remaining inflammatory tissue, leading to recurrence. Fluorescence lifetime imaging microscopy (FLIM) is a technique to measure and image the fluorescence lifetime (FLT) of fluorescent molecules. To date, there is no commercial system available to monitor FLT of intrinsic fluorophores during middle ear surgery. In this work, we performed FLIM on cryopreserved human middle ear tissue samples: two non-inflammatory biopsy specimens (external auditory canal and middle ear mucosa), otitis media and cholesteatoma. The thin-sliced tissue samples were examined with FLIM using excitation wavelengths of 375 nm and 473 nm. Results showed different FLTs among the various tissue types. Differences in the FLTs were observed in the 500-575 nm emission range with the 473 nm excitation. This could either be related to the presence and amount of enzymes in the cells or also refer to the structural diversity in the human middle ear tissues and the respective content of, for instance, collagen, elastin and keratin. Additional series of experiments are needed for more detailed analyses on the possible sources of the emission signals. These initial measurements should provide an overview of the occurring endogenous autofluorescence and the FLTs of different human middle ear tissue, in order to distinguish tissue types.
Phase-sensitive optical coherence tomography (OCT) is emerging as an imaging modality that detects functional changes in the retina. Besides imaging photoreceptor function, recently, functional changes in the inner plexiform layer (IPL) have been detected using full-field swept-source OCT. The IPL connects neuronal cells which are dedicated for processing different aspects of the visual information, such as edges in the image or temporal changes. A characteristic of signal processing in the IPL is that different aspects of the visual impression are only processed in very specific depths. Here, we present an investigation of these functional signals for different depths in the IPL with the aim to separate different properties of the visual signal processing. Therefore, we investigate the phase changes of three different sub-layers. Whereas the first two depths, closest to the ganglion cell layer, exhibit an increase in the optical path length, the third depth, closest to the bipolar cell layer, exhibits a decrease in the optical path length. Additionally, we found that the second or middle depth is sensitive to temporal changes, showing a maximum increase of the optical path length at a stimulation frequency of around 10 Hz. The results suggest that the responses from different cell types, which are sensitive to different features of the stimulation signal, can be distinguished by phase-sensitive OCT.
We demonstrate functional in vivo imaging of photoreceptor and neuronal layers within the living human retina by looking at the expansion of their optical path length. To this end, we use a special full-field swept-source optical coherence tomography system that acquires all lateral points in parallel, achieving a high-speed data acquisition with up to 200 volumes per second. A combination of computational motion and aberration correction with a suitable phase evaluation scheme yields minuscule changes after exposing the photoreceptors to a white light stimulus.
Non-invasive functional retinal imaging in humans is of tremendous interest. By using phase-sensitive full-field swept-source OCT (FF-SS-OCT) we demonstrated simultaneous quantitative imaging of the optical activation in the photoreceptor and ganglion/inner plexiform layer. Since the signals from the ganglion cells layer are ten-fold smaller than those from the photoreceptor cells a new algorithms for suppression of motion artifacts and pulsatile blood flow in the retinal vessels is important. With improved data evaluation we simultaneously measured the activation of photoreceptors and ganglion/inner plexiform with high quality and were able to analyze the spatial and temporal response of cells in the ganglion/inner plexiform over more than 10 seconds.
Using phase-sensitive full-field swept-source optical coherence tomography we already showed that morphological changes in the photoreceptor outer segments are detectable. Those signals manifest themselves in an elongation of the optical path length. Using improved post.processing we report on progress in detecting signals in the neuronal layers of the human retina. The spatially resolved signals show a characteristic time course and by combining these with simultaneous measurements of the photoreceptors we were able to generate a wiring map of the neuronal retina.
This paper describes the further investigation into the capabilities of the already established noncontact optoacoustic method to measure temperature profiles in cell cultures during controlled heating. The technic is scalable in spatial and temporal resolution. The intra and extracellular medium is heated by a thulium laser (wavelength 1.94 μm; power up to 25W). With a second Q-switched thulium laser (2.01 μm; up to 3 mJ) the sample medium temperature is simultaneously probed in the dish (20 mm diameter) via the photoacoustic effect. The pressure waves emitted due to the thermoelastic expansion of water are measured with an ultrasonic hydrophone at the side of the dish. The amplitudes of the waves are temperature dependent and are used to calculate the temperature/time course at 10 locations. Temperatures of up to 70°C with a heating power of up to 25 W after 5 s were measured, as well as lateral temperature profiles over time. Measurements in water show temperature fluctuations likely due to thermal convection and water circulation. Since measurements in agar do not show similar temperature fluctuations, this theory seems to be confirmed. In conclusion optoacoustics can serve as a real-time non-contact technique to determine temperature changes in cell and organ cultures as well as in vivo and during hyperthermia based therapies.
We implemented a real-time video-rate 4D-OCT system with virtual reality display. To achieve the required low latencies we optimized the dataflow path and the placement of the necessary synchronization points. Employing temporal reprojection enables to perform volume rendering at 1/3 of the display refresh rate, yet maintaining smooth updates to the HMD; thus we achieve display updates at 90Hz, volume rendering at 30Hz and C-scan acquisition at <15Hz. By mounting of a tracking accessory to the scanning head we can render the OCT volume in virtual space in the position of the actual imaging volume.
MHz-OCT systems based on FDML swept laser sources combined with the massive parallel processing capabilities of modern computer hardware enable volumetric imaging, processing and stereoscopic display at video rates. The increasing image quality and speed might enable new fields of application where the volumetric OCT completely replaces stereoscopic microscopes instead of being a mere supplement. Aside from the depth resolving capability, a particular advantage is the ability to display a whole image volume from arbitrary points of view without the need to move the actual microscope or to rotate the patient’s eye. Purely digital microscopy is already offered as alternative to traditional through-an-eyepiece surgical microscope. We explore the use of virtual reality to present digital OCT microscopy images to a trained surgeon, carrying out a series of surgical procedures ex-vivo on a porcine eye model.
Selective retina therapy (SRT) targets the retinal pigment epithelium (RPE) with pulsed laser irradiation by inducing microbubble formation (MBF) at the intracellular melanin granula, which leads to selective cell disruption. The following wound healing process rejuvenates the chorio-retinal junction. Pulse energy thresholds for selective RPE effects vary intra- and interindividually. We present the evaluation of an algorithm that processes backscattered treatment light to detect MBF as an indicator of RPE cell damage since these RPE lesions are invisible during treatment. Eleven patients with central serous chorioretinopathy and four with diabetic macula edema were treated with a SRT system, which uses a wavelength of 527 nm, a repetition rate of 100 Hz, and a pulse duration of 1.7 μs. Fifteen laser pulses with stepwise increasing pulse energy were applied per treatment spot. Overall, 4626 pulses were used for algorithm parameter optimization and testing. Sensitivity and specificity were the metrics maximized through an automatic optimization process. Data were verified by fluorescein angiography. A sensitivity of 1 and a specificity of 0.93 were achieved. The method introduced in this paper can be used for guidance or automatization of microbubble-related treatments like SRT or selective laser trabeculoplasty.
Laser photocoagulation has been a treatment method for retinal diseases for decades. Recently, studies have demonstrated therapeutic benefits for subvisible effects. A treatment mode based on an automatic feedback algorithm to reliably generate subvisible and visible irradiations within a constant irradiation time is introduced. The method uses a site-individual adaptation of the laser power by monitoring the retinal temperature rise during the treatment using optoacoustics. This provides feedback to adjust the therapy laser power during the irradiation. The technique was demonstrated on rabbits in vivo using a 532-nm continuous wave Nd:YAG laser. The temperature measurement was performed with 523-nm Q-switched Nd:YLF laser pulses with 75-ns pulse duration at 1-kHz repetition rate. The beam diameter on the fundus was 200 μm for both lasers, respectively. The aim temperatures ranged from 50°C to 75°C in 11 eyes of 7 rabbits. The results showed ophthalmoscopically invisible effects below 55°C with therapy laser powers over a wide range. The standard deviation for the measured temperatures ranged from 2.1°C for an aim temperature of 50°C to 4.7°C for 75°C. The ED50 temperature value for ophthalmoscopically visible lesions in rabbits was determined as 65.3°C. The introduced method can be used for retinal irradiations with adjustable temperature elevations.
The induced thermal damage in retinal photocoagulation depends on the temperature increase and the time of irradiation. The temperature rise is unknown due to intraocular variations in light transmission, scattering and grade of absorption in the retinal pigment epithelium (RPE) and the choroid. Thus, in clinical practice, often stronger and deeper coagulations are applied than therapeutically needed, which can lead to extended neuroretinal damage and strong pain perception. This work focuses on an optoacoustic (OA) method to determine the temperature rise in real-time during photocoagulation by repetitively exciting thermoelastic pressure transients with nanosecond probe laser pulses, which are simultaneously applied to the treatment radiation. The temperature-dependent pressure amplitudes are non-invasively detected at the cornea with an ultrasonic transducer embedded in the contact lens. During clinical treatment, temperature courses as predicted by heat diffusion theory are observed in most cases. For laser spot diameters of 100 and 300 μm, and irradiation times of 100 and 200 ms, respectively, peak temperatures range between 70°C and 85°C for mild coagulations. The obtained data look very promising for the realization of a feedback-controlled treatment, which automatically generates preselected and reproducible coagulation strengths, unburdens the ophthalmologist from manual laser dosage, and minimizes adverse effects and pain for the patient.
Retinal photocoagulation is a long time established treatment for a variety of retinal diseases, most commonly applied for
diabetic macular edema and diabetic retinopathy. The damage extent of the induced thermal coagulations depend on the
temperature increase and the time of irradiation. So far, the induced temperature rise is unknown due to intraocular
variations in light transmission and scattering and RPE/choroidal pigmentation, which can vary inter- and intraindividually
by more than a factor of four. Thus in clinical practice, often stronger and deeper coagulations are applied than
therapeutically needed, which lead to extended retinal damage and strong pain perception. The final goal of this project
focuses on a dosimetry control, which automatically generates a desired temperature profile and thus coagulation
strength for every individual coagulation spot, ideally unburden the ophthalmologist from any laser settings. In this paper
we present the first realtime temperature measurements achieved on patients during retinal photocoagulation by means of
an optoacoustic method, making use of the temperature dependence of the thermal expansion coefficient of retinal tissue.
Therefore, nanosecond probe laser pulses are repetitively and simultaneously applied with the treatment radiation in
order to excite acoustic waves, which are detected at the cornea with an ultrasonic transducer embedded in the contact
lens and then are processed by PC.
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