WO2019197726A1 - Dispositif et procédé pour commander le chauffage de l'épithélium pigmentaire rétinien - Google Patents

Dispositif et procédé pour commander le chauffage de l'épithélium pigmentaire rétinien Download PDF

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WO2019197726A1
WO2019197726A1 PCT/FI2019/050293 FI2019050293W WO2019197726A1 WO 2019197726 A1 WO2019197726 A1 WO 2019197726A1 FI 2019050293 W FI2019050293 W FI 2019050293W WO 2019197726 A1 WO2019197726 A1 WO 2019197726A1
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heating
beams
laser
electromagnetic radiation
rpe
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PCT/FI2019/050293
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Ossi KAIKKONEN
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Aalto University Foundation Sr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0625—Warming the body, e.g. hyperthermia treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A61F9/008—Methods or devices for eye surgery using laser
    • A61F2009/00861—Methods or devices for eye surgery using laser adapted for treatment at a particular location
    • A61F2009/00863—Retina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A61F9/008—Methods or devices for eye surgery using laser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0659—Radiation therapy using light characterised by the wavelength of light used infrared

Definitions

  • the invention relates to a method and device for controlling heating of retinal pigment epithelium (RPE).
  • RPE retinal pigment epithelium
  • the retinal pigment epithelium is a thin monolayer of cells underneath the neural retina.
  • the reduced ability of RPE cells to clear misfolded proteins and aggregates is a key factor in the pathogenesis of age-related macular degeneration (AMD).
  • Heat shock proteins enhance the ability of cells to refold misfolded proteins and to remove harmful aggregates. Increasing the amount of Hsps in RPE cells may therefore offer novel therapeutic approaches for AMD treatment.
  • RPE cells upregulate their Hsp production in elevated temperatures, which can be achieved through laser heating.
  • the temperature range where RPE cells significantly upregulate their Hsp production without suffering from thermal damage is very narrow.
  • a previously developed method for mouse RPE temperature determination aims to enable adaptive laser power control in order to reach the therapeutic window reliably.
  • the main difficulty of non-damaging RPE heating therapy has been a lack of effective dosimetry and retinal temperature control during therapy sessions. Due to the differences in the physiology of the eye between individuals and in one individual at different times, the same power setting for the heating laser may lead to sub therapeutic treatments in some individuals while causing retinal damage in others.
  • Previous long-pulse studies have relied on adjusting the optical power of the heating device to a suitable level a priori, while short-pulse studies have been carried out by first finding a threshold power that causes a visible lesion in the peripheral retina and using a certain fraction of that power.
  • Hsps heat shock proteins
  • Non-damaging RPE heating therapy aim to trigger Hsp production in the RPE through transpupillary delivery of near-infrared light.
  • the magnitude of temperature elevation in response to a standard laser power varies between individuals.
  • RPE laser irradiation without additional controls may therefore produce either a treatment without therapeutic effects or retinal damage.
  • An object of the invention is to overcome at least some problems relating to the known prior art. Especially the object of the invention is to provide a device and method for allowing retinal pigment epithelium (RPE) temperature determination and adaptive heating laser power control to enable safe and effective non-damaging RPE heating treatment.
  • RPE retinal pigment epithelium
  • a device that enables simultaneous RPE heating and temperature determination is described herein.
  • the aim of the variation of non-damaging RPE heating therapy described in this document is to upregulate heat shock protein production in RPE without causing thermal tissue damage.
  • the therapeutic temperature window, where the production of heat shock proteins is significantly upregulated but ocular tissues do not suffer from thermal damage, is very narrow. Therefore, the RPE must be brought to a temperature that is barely low enough not to cause apoptosis.
  • the object of the invention can be achieved by the features of independent claims.
  • the invention relates to a device for controlling heating of retinal pigment epithelium (RPE) according to claim 1.
  • the invention relates to a method for controlling retinal pigment epithelium (RPE) heating according to claim 22, as well as to a computer program product according to claim 23.
  • the invention relates to a device and computer program product to perform one or several of the temperature determination, heating power control and related functions described herein.
  • the device directs one heating laser beam and one or two stimulus laser beams on the retina.
  • the number of stimulus beams is determined by the temperature determination protocol.
  • laser beams are combined with beam splitters.
  • Each laser channel has its own adjustable optics before the beam splitter. These optics determine the direction of the beams, and whether the beams diverge, converges or propagates in collimation after passing the beam splitter. This adjusts the locations and sizes of the laser spots independently once the beams reach the retina.
  • a kinematic mirror may be used to redirect all of the beams at once, and either a lens system or an adjustable iris can be used to control the spot of all of the beams simultaneously.
  • the device has advantageously an ERG electrode required by the RPE temperature determination method, and a camera for fundus imaging.
  • the location of the heating laser spot on the RPE determines the heated area, while the location and size of the stimulus spots on the retina determine the area of the RPE whose temperature is determined.
  • the size and location of the stimulus spots and the heating spot may be controlled individually and as a group.
  • the stimulus and heating beams are produced advantageously by laser sources, e.g. laser diodes.
  • the power of each of the individual beams is modulated freely with a power control system, e.g. a photodiode feedback system.
  • the convergence/divergence of each of the individual beam is adjusted by an optical configuration, e.g. a moveable lens, and the beam is directed with a beam directing system, e.g. a kinematic mirror.
  • the individual beams are overlapped with optical components, e.g. beam splitters.
  • the beams may be cropped with an optical system, e.g.
  • an adjustable aperture and directed by a beam directing system, e.g. a kinematic mirror.
  • the beams are conveyed to the retina through the pupil with an optical system, e.g. a fundus lens. Adjusting the divergence/convergence of each individual beam adjusts the size of the laser spots on the retina. Redirecting each individual beam moves them on the retina. Adjusting the size of the aperture after the beams are combined may adjust the size of all of the laser spots on the retina while maintaining their relative sizes. Redirecting the combined beams may move the beams on the retina simultaneously while maintaining their positions relative to each other.
  • the retina is stimulated with light, e.g.
  • RPE temperature is determined from the ERG signal and heating laser power is adjusted based on an estimated power requirement for reaching optimal RPE temperature.
  • the size of a stimulus spot is smaller than the size of a heating spot, i.e. , the area illuminated by at least one of the stimulus beams is smaller than the area illuminated by at least one of the heating beams.
  • the areas illuminated by a heating spot and a stimulus spot may be concentric or nearly concentric. If a stimulus spot is smaller than a heating spot, temperature may be measured at the center of the area that is heated, where the temperature is highest. Towards the edges of the stimulus spot, the temperature may be reduced as shown in Figure 3. In this way, the temperature estimated from the stimulus may correspond more closely to the higher temperature closer to the center of the heating beam that can be important for safety reasons..
  • ERG is a technique for recording electrical responses that originate in the retina.
  • a nerve cell responds to light, as is the case with photoreceptor cells, or to a signal from a presynaptic nerve cell, as is the case with bipolar cells, the ion channels near the synapse open or close, causing a change in an extracellular current loop.
  • This current loop creates a gradient of electrical potential due to the electrical resistance of the extracellular matrix.
  • the source and sink of the extracellular currents produced by photoreceptor and bipolar cells are separated radially, meaning that one of them is closer to the back of the eye than the other. Therefore, a fraction of the extracellular current produced by photoreceptor and bipolar cells flows through the front of the eye and loop back from behind the retina. Electrodes placed in different locations along this current loop will detect a voltage that depends on the extracellular currents and thus reflects the signaling of neurons of the retina. For example, in mice, ERG is routinely recorded as a potential difference between two electrodes placed
  • the electrical responses to from different cell types have distinctly different waveforms, which are superimposed in the ERG trace.
  • flash stimuli are used to elicit these electrical responses.
  • the components of the flash-stimulated ERG response that are relevant for this text are the a-wave, which originates in the photoreceptor cells, and b- wave, which originates in bipolar cells.
  • a-wave which originates in the photoreceptor cells
  • b- wave which originates in bipolar cells.
  • the embodiment of the present invention offers advantages over the known prior art. For example, continuous retinal pigment epithelium (RPE) temperature determination and adaptive heating laser power control enables safe and effective non-damaging RPE heating treatment. In addition a method for non-invasive RPE temperature determination during RPE heating therapy would enable adaptive heating power during the therapy session to reach a desired temperature. Additionally, retinal temperature estimation provides a foundation for thermal dosimetry, which would be beneficial in designing optimal treatment protocols.
  • RPE retinal pigment epithelium
  • a device which enables heating of the RPE while estimating its temperature.
  • the device is designed specifically for long-pulse non-damaging RPE heating therapy in vertebrate species, including humans.
  • the initial object and purpose of the device according to embodiments is to enable the development of optimal RPE heating treatment protocols for heat shock protein (Hsp) expression in the RPE.
  • the device heats up the RPE with a near-infrared laser beam while simultaneously stimulating the retina with flashes of light and registering the electroretinogram (ERG).
  • the ERG signal is used to estimate the temperature of the RPE, which serves as a feedback for heating power control.
  • the device also features a camera, which is used to correctly position the device on the eye and to adjust the size and location of the heated area on the RPE.
  • Embodiments of the invention may also provide temperature determination at higher temperatures than those which may be reliably determined by ERG response by using the stimulus response to estimate the temperature up to a certain temperature and beyond that, the temperature increase may be estimated based on the increase of laser power.
  • Figure 1 illustrates the layered structure of the retina.
  • Figure 2 illustrates the attenuation coefficients of water, blood and melanin in an RPE melanosome.
  • the absorption coefficient of blood os calcuated with 150g/l hemoglobin content and 98% oxygen saturation.
  • the melanosome absorption coefficient is an exponential model fitted to optical measurement data from RPE melanosomes.
  • Figure 3 shows a computer modeling of the lateral heat distribution in the RPE when heated with a 1.3 mm radius and 810 nm wavelength laser spot with 270 mW optical power with various choroidal prefusion rates.
  • the uneven heat distribution within the laser spot creates a need for a smaller stimulus spot for temperature determination.
  • Figure 4 shows the effect of temperature change on the kinetics of the ERG signal, which is one of the temperature dependent features used for RPE temperature determination. In higher temperatures, the onset and recovery of the signal become faster.
  • Figure 5 shows the effect of temperature on the spectral sensitivity of rhodopsin, which is one of the temperature dependent features used for RPE temperature determination.
  • rhodopsin becomes more sensitive to long-wavelength photons (e.g. 785 nm) relative to photons conventionally known as visible light (e.g. 515 nm). This is seen as a growing amplitude for dim-flash ERG responses elicited with long-wavelength light versus conventional visible light in high temperatures.
  • Figure 6 shows an example of an ERG signal preprocessing pipeline used for noise reduction.
  • Figure 7 shows the threshold retinal irradiance for mouse rod photoreceptor light adaptation
  • Figure 8 shows an examplary optical configuration of the RPE heating device.
  • Figure 9 shows and exemplary configuration for laser beam shaping used in the RPE heating device.
  • Figure 10 shows an exemplary configuration for achieving different sized heating and stimulus spots on the retina.
  • adjusting the size of the aperture adjusts the size of all heating and stimulus spots while maintaining their relative proportions.
  • Figure 1 1 shows an exemplary electronic circuit for an optical power feedback control of the RPE heating laser.
  • Figure 12 shows beam camera images from the laser diodes of the device at the location of the fundus lens without the fundus lens being present.
  • Figure 13 shows, how a fundus lens turns a collimated beam into a macroscopic spot on the retina.
  • Figure 14 shows the main sources of backreflection from collimated beams in the imaging system. The figure also shows, how the backreflections are mitigated with an obstruction placed in front of the imaging objective.
  • Figure 15 shows a photo of the exemplary RPE heating device designed for mice.
  • Figure 16 shows an exemplary user interface for controlling the heating laser.
  • Figure 17 shows and exemplary ERG electrode placement system for the treated eye.
  • Figure 18 shows an image from the camera system of the exemplary RPE heating device while placing the device on the eye of the mouse.
  • Figure 19 shows an image from the camera system of the exemplary RPE heating device during RPE heating therapy.
  • Figure 20 shows an image of RPE temperature monitoring during RPE laser heating. The simplified method used here for temperature determination relies on a single temperature dependent signal feature that relates to ERG signal kinetics.
  • Figure 21 shows, how the kinetics of the ERG signal may decelerate and the amplitude may drop when the retina is exposed to very high temperatures.
  • Figure 22 shows a treatment where the retina is exposed to very high temperatures.
  • the spectral sensitivity based RPE temperature determination method remains stable even at high temperatures.
  • Figure 23 shows the depth-wise Hsp70 staining in sagittal slices obtained from an untreated mouse eye and a mouse eye treated with the exemplary RPE heating therapy device. The figure shows that Hsp70 staining is strong in the RPE and weak in the choroid.
  • Figure 24 illustrates an exemplary device for RPE heating according to an advantageous embodiment of the invention.
  • Figure 25 shows one other exemplary device for RPE heating according to an advantageous embodiment of the invention.
  • the retina shown in Figure 1 , is a layered structure that transduces light into neural signals.
  • the neural signals originate in the light sensitive photoreceptor cells, which form the outermost layer of the neural retina.
  • the signal is relayed from photoreceptor cells to the brain through separate retinal layers, which process the signal into separate parallel streams of information, which include information about movement, shape and color.
  • the neurochemical signal that ultimately leads to visual perception begins when a rhodopsin pigment in a photoreceptor cell is activated through photon absorption. Rhodopsin activation triggers a signaling cascade, which causes a change in the release of neurotransmitters in the synaptic terminal of the photoreceptor cell.
  • Bipolar cells make connections with the synaptic terminals of photoreceptor cells and respond to the released neurotransmitters, transmitting the signals onward in the retinal neural network to the inner synaptic layer of the retina.
  • the synapses in the inner synaptic layer connect bipolar cells to amacrine cells and ganglion cells.
  • Amacrine cells receive neural signals from bipolar cells and other amacrine cells, and relay signals to ganglion cells, other amacrine cells and back to bipolar cells.
  • the network of bipolar cells and amacrine cells process the neural signals produced by photoreceptor cells in graded potentials.
  • Ganglion cells convert the graded potentials in their synapses to action potentials, which are transmitted to the brain through the optic nerve.
  • the retina In addition to cells that are directly involved in neural signaling, the retina also has cell populations, such as RPE cells and Miiller glial cells, which serve secondary functions in visual transduction.
  • RPE cells and Miiller glial cells, which serve secondary functions in visual transduction.
  • the following chapters outline aspects of the retinal function that are relevant for RPE heating therapy and ERG based retinal temperature determination.
  • Figure 2 shows absorption coefficients of water, blood and melanin in an RPE melanosome.
  • the absorption coefficient of blood was calculated with 150g/l hemoglobin content and 98% oxygen saturation with data from.
  • the melanosome absorption coefficient is an exponential model fitted to optical measurement data from RPE melanosomes.
  • photoreceptor cells in the outer retina are the first cells in the optical path that have significant amounts of light absorbing pigment.
  • Opsins in photoreceptor cells have a very high absorbance to visible light near their wavelength of peak absorption. Visible light is not suitable for non-damaging RPE heating therapy, because intensities high enough to cause significant heating in the RPE would produce a very strong visible stimulus.
  • the light transmitted by the RPE passes to the choroid, which contains both melanosomes and high amounts of blood. Therefore, the pigments in the choroid with significant absorption in the near-infrared region are melanin and hemoglobin. Unlike the absorption spectrum of melanin and rhodopsin, the absorption spectrum of hemoglobin is not monotonous in the near-infrared region. The absorbance increases when increasing the wavelength of light in the near-infrared region until the local maximum at approximately 900 nm, after which it slowly decreases.
  • Figure 3 shows the lateral thermal distribution in the RPE with 1.3 mm spot radius, 810 nm wavelength, 270 mW optical power and with various choroidal perfusion rates.
  • Photoreceptor cells lie directly adjacent and partially embedded in the RPE, and they can be approximated to be at the same temperature as the RPE during RPE heating therapy. Higher temperatures lead to a faster signal transduction in photoreceptor cells and bipolar cells, which is seen in ERG responses as a faster onset and recovery.
  • the effect of the temperature dependence of signal transduction kinetics to dim-flash ERG responses is shown in Figure 4. Additionally, the sensitivity of rhodopsin to long- wavelength photons is temperature dependent. When the energy of an incident photon alone is insufficient to activate the rhodopsin, the activation requires contribution from thermal energy.
  • the amount of thermal energy stored in the rhodopsin must be more than the difference between the Arrhenius energy of rhodopsin activation and the energy of the incident photon.
  • the probability that a rhodopsin molecule has sufficient thermal energy depends on the temperature of the rhodopsin and the wavelength of the incident photon.
  • a stimulus in the visible spectrum e.g. green, 515 nm
  • the near-infrared spectrum e.g. 785 nm
  • TTM time-to-maximum
  • the TTM feature is calculated by fitting a second degree polynomial to the peak of the b-wave and determining the time point of the peak of the polynomial as Figure 4 shows.
  • the flash intensities are calibrated separately at the beginning of each experiment.
  • the first step in determining the appropriate flash intensities is finding the lowest intensity that induces a visible a-wave in the response in addition to the dominant b-wave.
  • the peak-to-peak amplitude between the bottom of the a-wave and the peak of the b-wave is then calculated, and it serves as a measure of photoreceptor sensitivity.
  • the green and NIR stimuli used for temperature determination are determined by finding the stimulus intensities that induce a b-wave whose amplitude is a certain percentage, typically 30% to 40%, of the previously mentioned peak-to-peak b-wave amplitude.
  • the green and NIR stimuli are fired in sequence every 2.5 seconds.
  • the sequence can naturally be different for different applications.
  • pre heating reference responses are registered.
  • the heating laser is turned on to a relatively low setting.
  • the temperature of the retina is determined by comparing the kinetics of the reference responses to the responses registered during heating. If the temperature determination implies that the retinal temperature is lower than the desired temperature, the power of the heating laser is increased. After the heating session is terminated, the temperature of the retina recovers back to normal body temperature. After the retina has returned back to normal body temperature, post-heating reference responses are registered.
  • the post-heating references are compared to the pre-heating references to determine, whether the baseline of the kinetics of the ERG signal changed due to heating. If the kinetics did not recover, an interpolated feature value between pre- and post-heating reference responses can be used as a reference instead of using only the pre-heating feature valued, which may lead in a higher accuracy in temperature determination.
  • the interpolation can only be performed after a heating session, and even though it may improve the accuracy of the temperature estimates during a heating session, changing signal kinetics imply a lower accuracy.
  • Temperature determination is advantageously determined from the ERG signal.
  • the first step in ERG based temperature estimation is pre-processing the signal. Pre-processing involves filtering out components other than the physiological signal. First, possible power line artefacts are removed with a 50 Hz notch filter. Second, the signal is low-pass filtered with a 60 Hz cut off FIR filter. Finally, an autoregressive model is used to forecast the baseline after the stimulus, and the forecasted baseline is subtracted from the signal. This removes periodic artefacts from the signal.
  • the next step after signal pre-processing is feature extraction.
  • the relative spectral sensitivity feature is calculated as a ratio of the peak value of responses elicited with NIR and green stimuli.
  • a relative feature values are used instead of the raw values.
  • To calculate the relative TTM feature value the peak time point of an ERG response recorded during heating is divided with the peak time point extracted from the reference ERG responses at normal body temperature.
  • To calculate the relative spectral sensitivity feature value The ratio between responses recorded with NIR and green stimuli during heating is divided with the same ratio calculated from responses at normal body temperature.
  • the light adaptation threshold for a wild type mouse rod is approximately 10 RY 1 (activated rhodopsins per rod cell per second). Brighter illumination levels affect the kinetics of the ERG response and confound the temperature determination. Since the retina must be dark adapted for the temperature estimator to work properly, the combined number of activated rhodopsins from the light stimulation and heating laser must not exceed this number.
  • the intensity of the incident light I is first converted to photon irradiance E q .
  • E p is the energy of a photon of the incident light.
  • a r0 d is the cross sectional area of a rod perpendicular to the propagation of light, which is approximately 1.54 pm 2 for mouse rods.
  • the optical density of the rod outer segment may further be separated to the maximal optical density D max and wavelength dependent normalized optical density of rhodopsin D norm .
  • the absorbance spectrum of mouse rhodopsin is calculated using a standard visual pigment template by Govardovskii et. al.. The absorbance in the near-infrared region is temperature dependent, but also template providing a crude estimate can also be used.
  • An absorbed photon does not always lead to rhodopsin activation.
  • the ratio between the number of absorbed photons and activated rhodopsins is termed quantum catch, F, and regardless of the wavelength, it is approximately 0.67.
  • the probability that an incident photon activates a rhodopsin is:
  • Equation 9 can be used to estimate the light adaptation threshold irradiance at a given wavelength.
  • the maximal heating irradiance may be calculated for each wavelength.
  • the threshold irradiance which is shown in Figure 7, shows the maximum heating irradiance that does not cause light adaptation in mice. This figure is useful for selecting an optimal heating laser wavelength, which is able to cause sufficient heating without causing significant light adaptation.
  • One object of this invention is to provide a device that enables simultaneous heating and temperature determination of a desired portion of the RPE.
  • the device directs three laser beams on the retina, one for heating and two for stimulation required for ERG based retinal temperature determination.
  • the device also includes a monochrome camera and an electrode for ERG recording.
  • the laser beams are conveyed to the retina through a fundus lens, which prevents the laser beams from focusing on the retina and enables the retina to be viewed with a camera.
  • the size and location of all of the laser spots on the retina need can be adjusted simultaneously with the same controls.
  • the entire optical configuration is mounted on a micromanipulator stage, which is used to precisely place the device on the eye of the mouse.
  • the fundus lens must contact the cornea by fluid without pressing on the eye. Since the device is placed on the eye under dim red lights, a visual aid is needed to place the device securely.
  • the monochrome camera which also detects infrared light, was used to look through the fundus lens while positioning the device on the cornea.
  • the eye of the mouse is illuminated using external LED lights and the heating laser with a low power setting. After the device is properly positioned, the same camera is also used to adjust the size and location the heating and stimulus spots on the retina.
  • Figure 8 shows the overall optical configuration of an exemplary device according to an advantageous embodiment of the invention.
  • the components will henceforth be referenced with numbers 1 - 29 as they appear in Figure 8.
  • the heating and stimulus laser beams are produced by laser diodes #6-8 and collimated by lenses #9, #1 1 and #22. Cylindrical lenses #10-1 1 and #20- 21 shape the heating and infrared stimulus beams and correct their astigmatism.
  • Beam splitters #4 and #5 combine the laser beams to a single optical path. The combined beams then go through an adjustable aperture #12, which crops the beams to an identical size.
  • Kinematic mirrors #27-29 are used to accurately overlap the beams.
  • An additional kinematic mirror #26 is used to direct all of the beams simultaneously.
  • the size and location of the heating and stimulus spots are adjusted by adjusting the size of the aperture #12 and the orientation of the common kinematic mirror #26.
  • the beams reflected from mirror #26 are reflected to the fundus lens #2 by beam splitter #3.
  • the light that scatters back from the eye passes through beam splitter #3 to camera #16 through imaging objective #17.
  • a central obstruction #18 prevents back-reflected laser beams from entering the imaging optics.
  • One photodiode system #13-15 provides feedback for operating current control of the heating laser and another photodiode system #23-25 measures the optical power that reaches the eye.
  • the ERG based retinal temperature determination protocol was calibrated ex-vivo using 532 and 780 nm stimulus lights.
  • This exemplary device uses laser diodes with similar wavelengths: 515 and 785 nm.
  • the 785 nm stimulus laser has high power requirements, as photoreceptors are very insensitive to it.
  • the power requirement is approximately 20 mW for the light that reaches the retina based on former ex-vivo experiments with a 2 mm diameter beam.
  • the power requirement for the 515 nm stimulus light is only a few microwatts.
  • a 785 nm laser diode with 1 W maximal power (Roithner Lasertechnik RLT780- 1000G) was used as the NIR stimulus laser and a 515 nm laser diode with 10 mW maximal power (Roithner Lasertechnik LD-515-10MG) can be used as the green stimulus laser, for example.
  • 810 nm laser diodes have been used successfully in RPE heating studies. However, 810 nm light causes strong light adaptation at intensities required by RPE heating therapy, and a longer wavelength must therefore be used to enable ERG based retinal temperature determination. Since longer wavelengths penetrate deeper into the ocular tissues, the heating becomes less specific, and the choroid and the sclera are exposed to higher temperatures. Higher wavelength heating lasers may also require a higher overall intensity to achieve the same temperature elevation in the RPE.
  • the optical power required for Hsp70 expression in rabbits with a 60 second heating duration and 1.3 mm spot diameter was approximately 120 mW. Even though the test animal and wavelength were different, this power can be used as a crude estimate when estimating the required heating power.
  • 120 mW converts to 93 mW of total power if the total power is increased in proportion to spot diameter rather than area. This converts to an irradiance of 12 Wcmr 2 .
  • an irradiance of 12 Wcmr 2 with a 950 nm light source would be at the threshold of light adaptation.
  • the power requirements will be higher for light, whose absorption is weaker in the RPE.
  • the power requirement grows linearly with spot diameter, smaller spot sizes require higher irradiances.
  • the ERG based temperature estimation method requires light stimulation, which also contributes to light adaptation.
  • the model used to estimate the sensitivity of rhodopsin in the near-infrared region is not highly accurate, and some room must be left for error.
  • the attenuation coefficient of water has a local maximum of 0.43cm - 1 at 980 nm. With this wavelength, approximately 12% of light is absorbed in the vitreous. As the vitreous does not have blood perfusion to cool it down, there would be a risk of damage.
  • the attenuation coefficient of water has a local minimum of 0.43cm - 1 at 1060 nm. With this wavelength, only 3.5% is absorbed in the vitreous reducing the risk of damage.
  • 1064 nm was chosen as the heating laser wavelength.
  • 93 mW which corresponds to what has been used with an 810 nm heating laser, may be indicative as a power requirement for a 1 mm heating spot.
  • higher powers are required for a 1064 nm laser due to lower absorbance in the RPE.
  • much of the light intensity of a laser is lost in the optical shaping of the beam.
  • a 1064 nm laser diode with 2W maximal power (Roithner Lasertechnik RLCO-1064-2000-T03) can be chosen as the heating laser source.
  • Suitable wavelengths for different purposes can be selected for the stimulus beams as well as the heating beam.
  • the light emitted by a laser diode diverges in a large angle.
  • a collimating lens is placed in front of the laser emitter at a distance equal to the focal length of the lens. If the lens is placed closer than its focal length, the beam diverges, and if the lens is placed further from the laser diode than the focal length of the lens, the beam converges after the lens.
  • the beam emitted by a laser diode has an elliptical shape.
  • the axis in which the beam diverges more is termed the fast axis
  • the slow axis is termed.
  • Cylindrical lenses which focus light only on one axis, can be used both to shape an elliptical beam into a circular shape and to correct astigmatism.
  • Both the 1064 nm heating laser and the 785 nm NIR stimulus laser can be multi-mode lasers and have a wider laser cavity, and therefore exhibit significant astigmatism. Additionally, both of the lasers is advantageous to be collimated with a short focal length optics to create intense beams. Both the heating laser and the NIR stimulus were collimated with similar optical schemes, but with different lenses.
  • a spherical collimation lens was first placed accordingly for the light to be collimated along the fast axis.
  • the spherical lens causes an astigmatic beam to converge along the slow axis.
  • the light starts to diverge again along the slow axis.
  • Two cylindrical lenses were used both to correct the astigmatism along the slow axis and to transform the elliptical beam into spherical.
  • Figure 9 shows the collimation scheme for the heating laser diode, but the NIR stimulus laser was shaped using the same principle.
  • the heating laser was collimated using a 3.1 mm focal length aspheric lens.
  • the NIR stimulus laser was collimated using a 20.5 mm focal length microscope objective. Both the heating and the NIR stimulus lasers were collimated by a pair of cylindrical lenses, the first one having a focal length of 15 mm and the second one 40 mm.
  • the heating laser beam is cropped by the aperture (#12 in Figure 8), it remains the same size until it arrives to the fundus lens.
  • the ERG based method described in chapter 4.2 determines the temperature of the RPE from the area illuminated by the stimulus laser beams. In order to determine the temperature from the center of the heating spot, which may be warmer than the periphery, the stimulus laser spots were adjusted to be smaller than the heating spot. This was achieved by placing the collimation lenses of the stimulus lasers slightly further from the laser emitters than the focal length of the lenses to create slightly converging beams. As the stimulus beams are cropped by the aperture, they converge slightly between the aperture and the fundus lens. When the stimulus beams reach the fundus lens, they are slightly smaller than the heating laser beam.
  • Figure 10 shows how the stimulus beams converge between the iris and the retina while the heating beam maintains its size.
  • the magnified image shows the formation of the heating spot (H) and stimulus spots (S).
  • H heating spot
  • S stimulus spots
  • Most of the components are removed from the image for clarity, and only a single beam splitter, the aperture and the fundus lens are shown.
  • the collimation optics of the green stimulus source are very different from the collimation optics of the NIR stimulus, both of the beams are shaped to converge the same way.
  • the light was collimated very far from the source using a 25 mm diameter and 80 mm focal length lens (manufacturer and model unknown). This configuration creates a very wide beam, and since only the center of the beam passes through the aperture, there was no need to reshape the elliptical beam into a spherical one.
  • the optical power of a laser diode depends on its temperature.
  • the stimulus sources might not have any feedback control, whereupon their intensities are temperature dependent. If the stimulus sources are used with very low duty cycles, they do not heat up as they are used. Naturally any feedback control can be used, even if it is not shown here. However, the temperature of the air may change during experimentation leading to a slight variation in intensity.
  • the heating laser diode is feedback-controlled ensuring that its optical power remains constant during use. However, the diode heats up significantly when used, and cooling is necessary to ensure high optical power.
  • all of the laser diodes are cooled with a water circulation that runs through custom made heat transfer elements attached to each photodiode mount.
  • the temperature of the cooling water is set to 5°C, and it runs through the heat transfer elements of green stimulus, infrared stimulus and heating laser diodes in that order.
  • the water circulation can be maintained by a temperature controlled water circulation system.
  • the relationship between the operating current and optical power of a laser diode depends greatly on the temperature of the laser diode. Typically, the threshold current of a laser diode is higher and the slope efficiency is lower in higher temperatures. If a laser diode is used with high power and high duty cycle, it heats up and its optical power reduces. This makes optical power feedback necessary to for reliable optical power control of a high power laser diode when operated at high duty cycles.
  • the heating laser diode is operated with high power levels and duty cycles, and its temperature does not remain constant even if good thermal stabilization is used. According to an embodiment, the optical power of the heating laser diode is advantageously continuously measured and used as feedback for adjusting the operating current to ensure reliable optical power.
  • the optical power of the heating laser is continuously measured by a photodiode (#15 in Figure 8, D1 in Figure 1 1 ). Approximately 2% of the light produced by the infrared laser diode is transmitted by the shortpass dichroid beam splitter (#4). The transmitted portion is passed to the photodiode through a diffuser film (#13), which makes the illumination of the photodiode less sensitive to the orientation of the incident beam. Additionally, a neutral density filter was used to attenuate the transmitted portion of the infrared beam to fall within the dynamic range of the photodiode.
  • the photodiode In order for feedback system of the heating laser to be stable, the photodiode must have a low response time. In this configuration, the photodiode was reverse-biased with a 10V voltage in a configuration known as photoconductive mode. Reverse biasing the photodiode thickens its depletion layer, reducing its capacitance and response time. Light causes electrons to leak in the reverse direction of the photodiode. When the leakage current flows through a resistor, the optical power can be measured by measuring the voltage across the resistor.
  • the speed at which a photodiode in photoconducive mode responds to light depends on the reverse bias voltage and the load resistance (R3 in Figure 1 1 ), and at 10 V reverse bias and 10 kQ load resistance the photoconductive mode is considerably faster than standard photovoltaic mode. These values lead to a sufficiently fast response, but the response time could be improved significantly by decreasing the load resistance, however at the cost of inducing noise in the feedback signal.
  • a circuit diagram for the control electronics of the heating laser is shown in Figure 1 1.
  • a photodiode feedback circuit (red area in Figure 1 1 ) provides optical power feedback to the heating laser current control circuit (green area Figure 1 1 ).
  • the photodiode feedback circuit converts light absorbed by the photodiode D1 into a voltage, which is passed to the heating laser current control circuit via switch SW2.
  • the leakage current of the photodiode passes through resistor R1 , and the voltage over the resistor serves as an optical power measurement.
  • the voltage from the photodiode feedback system is passed to the inverting input of operational amplifier U1 where it is compared to a voltage produced either by the local control circuit (red area in Figure 1 1 ) or by an external source via jack J2.
  • the operational amplifier adjusts the gate current of Darlington transistor Q1 , which in turn controls the operating current of the heating laser diode, to match the output voltages of the photodiode feedback circuit and the local control circuit.
  • a slow operational amplifier (slew rate 1 was used to ensure a stable feedback.
  • the heating laser may be operated by adjusting the operating current of the laser diode instead of the optical power.
  • the switch from photodiode feedback to operating current feedback is managed by switch SW2.
  • the operating current is obtained from the voltage over resistor R6.
  • Instrumentation amplifier U2 converts the voltage over the resistor R6 into a potential relative to ground, enabling its use as a feedback potential at U1.
  • Both the current and the optical power measurements are modified so that at zero current or optical power, the voltage measurement does not drop near zero. This is done to ensure that the device is completely off, even if the control voltage at the non-inverting input of U1 is slightly higher than zero.
  • Resistor R2 causes an additional current to leak through the R3, causing the voltage reading to be approximately 0.3V without contributions from light.
  • the stimulus laser diodes are operated with short pulses and low duty cycles.
  • the diodes do not heat up significantly when used and optical power feedback is not required.
  • the laser diodes are controlled by a laser diode switch (iC-HG, IC Haus), which is capable of producing constant current pulses down to five nanoseconds.
  • the shape of the ERG response to a flash stimulus lasting less than a millisecond does not significantly depend on the duration of the flash if the number of photons delivered in the flash is constant.
  • the output current of the laser diode switch is set to a constant value and the flash intensity is adjusted by adjusting the length of the control pulse given to the laser diode switch.
  • the dynamic range of the flash intensity is set at the short end by the shortest flash that the laser control electronics are capable of reproducing reliably, and on the long end, the maximal flash duration that appears instantaneous to the retina. In this case, the dynamic range is approximately 50 decibels.
  • Beam splitters are optical devices that, in addition to splitting one beam into two, can overlap two separate beams. Different beam splitters separate incident light based on wavelength and polarization. In the device described here, two beam splitters were used to combine three laser beams of different wavelengths to propagate on the same path. An additional beam splitter was used to enable imaging.
  • the green and NIR stimulus beams are combined using a 50-50 beam splitter (#5 in Figure 8).
  • the beam splitter reflects 53% of the NIR stimulus beam and transmits 37% of the green beam.
  • the transmitted green stimulus beam and the reflected NIR stimulus beam pass to a shortpass dichroic beam splitter (#4 in Figure 8, which transmits wavelengths between 420 and 900 nm and reflects wavelengths between 990 and 1600 nm near perfectly. More specifically, the beam splitter reflects 98% of 1064 nm light and transmits 98% of both the 515 nm and 785 nm laser beams.
  • An additional polarizing beam splitter (#3 in Figure 8) was used to enable retinal imaging. The beams that arrive at the beam splitter are almost entirely S-polarized, and the beam splitter reflects nearly all of S-polarized light. The beam splitter does not therefore attenuate the beams significantly.
  • the intensity of the small fraction of the heating beam that pass through beam splitters #3 and #4 are continuously measured using photodiodes #25 and #15.
  • the signal from photodiode #15 is proportional to the total optical output power of the heating laser, and the signal is used as a feedback for controlling the operating current of the laser diode.
  • the signal from photodiode #25 is proportional to the optical power that passes through the iris and it is used to measure the optical power that reaches the retina.
  • Figure 12 shows the intensity profiles of the heating beam and the stimulus beam without the fundus lens.
  • the images were taken with 2 mm aperture diameter, which is the maximal spot size that can enter through the dilated mouse pupil, using a beam profile camera (for example Spiricon SP503U).
  • the images were taken without the fundus lens with the beam camera sensor placed where the fundus lens would be (A: color mapping for relative intensity, B: Heating beam, C: Infra-red stimulus beam, D: Green stimulus beam).
  • the stimulus beam is produced by a single mode laser that has a relatively long coherence length, and is therefore prone to diffraction patterns such as the diffraction rings seen in the beam.
  • the heating beam is produced by a multi- mode laser with a wider spectrum and is less prone to form diffraction patterns when passing through an aperture.
  • the maximal output power of the heating laser that reaches the fundus lens was measured with an optometer (Thorlabs PM121 D) and determined to be approximately 300 mW with a 2 mm aperture, which results in a 1 mm diameter beam on the retina.
  • the focal length of the lens of the lens of the eye is approximately the same as its distance from the retina. This means that a collimated laser beam that is pointed to the eye will focus a very small area on the retina (see Figure 13A).
  • the device described in this text is designed to heat up a larger area of the retina, and additional optics must therefore be added close to the eye to prevent the laser beam from focusing. A standard way of achieving this is to utilize a fundus lens.
  • a fundus laser lens (Ocular 2mm Fundus Laser Lens, Ocular instruments) was used to achieve a large spot size.
  • the size of a laser spot on the retina depends on the diameter of the collimated beam and on the optical properties of the eye and the fundus lens.
  • Beam magnification is the ratio between the diameter of the spot on the retina and the diameter of the collimated beam.
  • the manufacturer of the lens does not provide information about beam magnification, but a crude estimate may be calculated with certain assumptions.
  • the refracting surfaces of the eye are at the surface of the cornea. In reality, both the cornea and the lens refract light separately, but the assumption that there is only one refracting surface simplifies optic calculations.
  • the fundus lens is in perfect contact with the cornea, i.e. that the refracting surfaces overlap.
  • the diameter of the mouse eye, as well as its focal length are 3 mm.
  • the focal length of the fundus lens is -6 mm in air.
  • the focal length f of the combination of the fundus lens and the optics of the eye can now be calculated:
  • a 6 mm focal effective focal length at the surface of the eye results in a beam magnification of 0.5. This means, that the diameter of a collimated beam will reduce to one half when it passes to the retina.
  • Figure 13 shows the effect of the fundus lens on a collimated beam (A: An eye without a fundus lens will focus collimated light to a point. B: The fundus lens without the eye has a focal length of -6 mm. The combination of the fundus lens and the eye has a focal length of 6 mm, leading to a 0.5 beam magnification.)
  • one of the principal challenges is to illuminate the retina while avoiding the illumination beam to be reflected directly to the camera sensor.
  • the problem arises as the illumination beam entering the eye and the light scattered from the retina share the same optical path.
  • Each surface on the optical path such as the surface of a lens or a beam splitter, reflects a portion of the illumination beam towards the camera.
  • Anti-reflective coatings can reduce the reflectance significantly.
  • the light scattered back from the retina to form the image is very faint compared to the light used to illuminate the retina. Even very faint back-reflections from the beam splitters are bright compared to the image of the retina, reducing the quality of the image significantly. Additional techniques are therefore needed to mitigate back-reflection.
  • the advantageous optical components which are both on the imaging and illumination path, are the fundus lens and the beam splitter (#2 and #3 in Figure 8). More specifically, the surfaces that cause back-reflection are the two surfaces of the fundus lens and the internal surfaces of the beam splitter. The main sources of back-reflection are shown in Figure 14.
  • All of the surfaces that cause back-reflection, other than the fundus lens - eye interface, are flat. As a collimated beam is reflected by a flat surface, the beam stays collimated. These collimated beams stay in the center of the optical path, and they are blocked by an opaque circular obstruction placed directly in front of the imaging optics. The obstruction reduces the luminous intensity of the image but since it is very close to the imaging optics, it does not create a visible dark spot in the image.
  • the eye - fundus lens interface is curved, and light reflected from it does not maintain its collimation. Instead, the reflected beam diverges and passes the circular obstruction (see Figure 14). However, the light reflected from this surface maintains its polarization, and is therefore mostly reflected away from the camera by the polarizing beam splitter #3. Additionally, the divergence of the reflected beam causes only a fraction of it to reach the camera sensor. Even though beam splitter #3 reflects most of S-polarized light away from the camera, it does not reduce the luminous intensity of the light scattered by the retina as significantly. Scattering reduces the polarization of the light that forms the image, leading to a significant portion of the light to pass through the beam splitter as P-polarized light.
  • a 108 mm focal length macro lens can be used as the imaging objective connected to allied vision U503B monochrome camera.
  • the front surface of the imaging objective was placed at a distance of 13cm from the fundus lens.
  • the magnification of the objective at 13 cm working distance is 1.1 , and the 2 mm diameter fundus lens window will form an image that covers a fairly large portion of the 5.76x4.29 mm sensor.
  • the device Before experimentation, the device is set to a desired angle, as is shown in Figure 15.
  • the angle of the device determines the location of the accessed area in the retina in the vertical axis.
  • the mouse is placed on a thermoregulatory device for the duration of the experiment.
  • the orientation of the thermoregulatory device with respect to the heating device determines the accessed area on the retina in the horizontal axis.
  • the device can be mounted on a custom-built mount that includes a three- axis manual micromanipulator stages, a rotation mount and a rail system.
  • the micromanipulators enable the device to be placed accurately on the cornea of the mouse, the rotation mount enables the vertical angle of the device to be adjusted, and the rail system enables the device to be moved close to the eye quickly at the beginning of an experiment.
  • the control station is the physical interface between the data acquisition computer and the heating device.
  • the control station case shown in Figure 16, has numerous cable connections and controls, and it houses electronics. The following list details the contents of the control station and relates them to the letters in Figure 16 (a-o in sublists).
  • Heating laser lock button The user locks the current optical power setting from dial B. The device adjusts heating power accordingly.
  • Heating laser power adjust dial The user rotates the dial to select a desired optical power. Device does not lock the optical power until button A is pushed.
  • External LED switch Possibility to add an external LED to the device.
  • Heating laser feedback mode switch The user chooses between optical feedback and direct current adjustment. SW2 in Figure 1 1.
  • Heating laser control source switch The user selects the source that adjusts the heating laser power between the external data acquisition computer and the local controls in the control station. The third position of the switch keeps the device on, but turns the heating laser off. SW1 in Figure 11.
  • the LCD screen displays the locked value of the optical power, the unlocked value from dial B, an optical power measurement from the heating laser photodiode feedback circuit, the operating current of the heating laser, feedback mode from switch E, the heating laser control source and state of the external LED (on/off).
  • Green stimulus control voltage jack Connects to the TTL port of the IC-HG laser driver described in 5.1.3.
  • Heating laser external control voltage jack Analog voltage input for controlling the power of the heating laser from the data acquisition computer. J2 in Figure 11.
  • Heating laser feedback circuit output voltage Used for recording the optical power of the heating laser. J1 in Figure 11.
  • NIR stimulus control voltage jack Input for the green stimulus laser. Connects to the TTL port of the IC-HG laser driver described in 5.1.3.
  • L Cable bundle that connects the control station to the optical device. Contains cables for the stimulus and heating lasers and the photodiode circuit.
  • the LDC display (G), the pushbutton (A) and dial (B) are connected to an electrician DUE board housed within the control station casing.
  • the electrician sets the local control voltage in the heating laser photodiode feedback circuit.
  • the electrician also gets the optical power measurement from the photodiode feedback circuit, the operating current measurement of the heating laser, as well as position of each pushbutton and shows them on the display.
  • the cable bundle (I) connects to an external housing that contains the stimulus laser driver circuit.
  • Figure 17 shows the designed instrument according to an exemplary embodiment, that enables electrical contact between the eye and the electrode.
  • an Ag/AgCI electrode can be connected to the eye by a saline gel filled custom-made glass capillary (outer diameter 1.5mm, inner diameter 1.17mm).
  • a gel is used instead of a fluid to prevent dripping.
  • the glass capillary is curved and its tip lies directly next to the fundus lens.
  • a small amount of saline gel is pushed out of the capillary both to create an electrical contact and to hydrate the eye.
  • the saline fluid comes from a syringe that is operated using a micrometer screw. Very small amounts of saline fluid can be pushed out of the capillary by turning the micrometer screw slightly.
  • Figure 18 shows an image from the camera while approaching the eye.
  • the device is brought closer to the eye until the space between the cornea of the eye and the fundus lens is in contact via liquid.
  • the focus of the camera is adjusted to see the location of the optic nerve, and the size and location of the heating spot are adjusted accordingly.
  • Figure 19 shows the location of the optic nerve during heating therapy.
  • the impedance between the two ocular electrodes was typically 20 - 50 kQ. There was significant variation in the electrode placement between different experiments. At best, the ERG signal was stable and had no significant power line or respiratory artefacts. Power spectral analysis revealed that the amplitude of the power line artefact in experiments with successful electrode placement was typically between 1 and 3 microvolts. With suboptimal electrode placement, a 50 Hz power line and respiratory artefact was present in the signal.
  • FIG. 20 shows the RPE temperature estimate from two successive heating sessions. Two successive heating sessions with 41.5°C target temperature. The experiment was conducted before a proper optical power measurement system was implemented, and therefore the heating laser power is only indicative.
  • FIG. 21 shows averages of 5 dim flash ERG responses before, during and after a heating session. The figure shows how the amplitude reduces dramatically during the heating session and recovers partly thereafter. The responses were significantly slower after the heating session than before.
  • Response A is the dim flash ERG response before heating.
  • B is the response after 4 minutes of intense heating therapy.
  • C is the response 14 minutes after turning off the heating laser.
  • the vertical lines show the time-to-maximum of each response.
  • the preliminary data from intense treatments indicates that high temperatures indicate that the b-wave suppresses significantly more than the a-wave in strong flash responses.
  • dim flash responses the delay before the b-wave appears to be the main decelerating factor, and the waveform of the response after the delay does not change as much. This indicates that the cause for the suppression of the b-wave may lie in the synaptic terminal of the photoreceptor cell.
  • Section A shows the deceleration of response kinetics as the temperature estimate falls below body temperature after laser heating is terminated.
  • the kinetics-based temperature estimate in section B is calculated by using a linearly interpolated reference value individually for each response during heating therapy. Even though the linear interpolation method is a crude estimate of the baseline kinetics, the two independent temperature determination methods are in good accordance.
  • Figure 23 shows a staining image from ocular slices from a treated and untreated eye.
  • the experiment that produced the samples was in the very early stages of experimentation, and the thermal dose is not well defined.
  • the examination of stained ocular slices revealed that Hsp70 staining was strong in the RPE and negligible in the choroid.
  • the thermal dose was not well defined in this experiment, the fact that Hsp70 expression was strongest in the choroid implies that RPE temperature is higher than choroidal temperature. This, in turn, indicates that non-damaging RPE heating therapy can be administered for example with 1064 nm light.
  • the intensity profile of the light emitted by the heating laser depends on its operating current.
  • the heating laser is used at low power settings, such as when it is used to illuminate the retina, the beam is very inhomogeneous.
  • the homogenous intensity profile of the beam could be preserved at all power levels by using pulse width modulation (PWM) with a constant operating current for optical power control. If PWM modulation was used, the device that controls the PWM duty cycle would manage the optical power feedback digitally instead of using an analog circuit.
  • PWM pulse width modulation
  • imaging of the retina is not the main feature of the device, the imaging capabilities of the device were not developed with rigor and possible improvements are numerous. First, imaging with laser light without wavefront correction produces an image with strong speckling. Broadband light sources would solve the speckling problem and could improve image quality significantly.
  • the back-reflections from the surfaces of the beam splitter cube could be completely eliminated using a plate or pellicle beam splitter instead of a beam splitter cube.
  • a flat beam splitter would not affect the back-reflections from the fundus lens, which could be removed by using polarizers with very high extinction ratios.
  • One polarizer would be used to ensure that all beams that arrive at the fundus lens are near-perfectly polarized.
  • Another polarizer would be placed in front of the camera to block all light that has the same polarization as the heating and stimulus beams, allowing only scattered light from the retina to pass to the image sensor.
  • the sharpness of the image could be improved by using a shorter focal length objective closer to the eye.
  • the main difficulty with temperature determination during RPE heating was the suppression and deceleration of the ERG response in high retinal temperatures, reducing the accuracy of ERG based RPE temperature determination.
  • the problem requires a solution if high retinal temperatures are required for sufficient Hsp70 expression.
  • One possible solution is mixing the anesthetic gas into pure oxygen instead of regular air. High temperatures increase the metabolic rate and oxygen consumption of tissues.
  • the suppression of the ERG response may be a sign of local hypoxia, which could be reduced by higher blood oxygenation.
  • ERG based RPE temperature at high retinal temperatures could be heating the retina to a certain stable temperature and raising the temperature further simply by controlling the heating power. For example, the temperature could be raised to 42°C guided by the ERG based temperature determination. After reaching a stable 42°C, a 50% increase in laser power could cause a predictable increase in temperature beyond 42°C even though the temperature can no longer be reliably determined from the ERG signal.
  • the stimulus response may be used to estimate the temperature up to a certain temperature and beyond that the temperature increase may be estimated based on the increase of laser power.
  • Figure 24 illustrates an exemplary device for retinal pigment epithelium (RPE) temperature determination and adaptive heating laser power control to enable safe and effective non-damaging RPE heating treatment according to an advantageous embodiment of the invention.
  • the exemplary device comprises an optical configuration, where the optical configuration may include one or more laser sources, and one or more optical elements.
  • the device comprises optics for each laser beam.
  • the optics for each laser beam may include one or more of the following: means for beam power control, means for beam direction control, means for beam divergence / convergence control and means for beam diameter control.
  • the means for beam direction control may comprise a kinematic mirror 7, 8, 9.
  • the means for beam divergence/convergence system may comprise a lens system 12, 13, 14.
  • the means for beam diameter control may comprise an aperture (not shown).
  • the device may comprise means to combine the individual laser beams. Combining here means to arrange the beams in an orientation in which all the beams may be introduced to the same object, such as the eye to be subjected to the heating and temperature determination.
  • the means for combining the laser beams may comprise beam splitters 4 and 5.
  • the device may comprise optics for all the beams after the combination of the beams, hereinafter referred to as the joint beam optics.
  • the joint beam optics may comprise one or more of the following: means for joint beam direction control 6, means for joint beam divergence / convergence control (not shown) and means for joint beam diameter control 24.
  • the means for joint beam direction control may comprise a kinematic mirror.
  • the means for joint beam divergence/convergence system may comprise a lens system.
  • the means for joint beam diameter control may comprise an aperture.
  • the device may comprise a fundus lens 2.
  • the fundus lens may be used to convey the laser beams through the pupil so they may progress through the eye towards the retina.
  • two or more of the laser beams are arranged to have differences in their convergence/divergence.
  • the area illuminated by each beam may differ from each other even if the beams pass through the same aperture.
  • changing the aperture size will change the size of the areas illuminated by each.
  • the optics are arranged so that the area illuminated by at least one of the stimulus beams is within the area illuminated by the heating beam.
  • the signals elicited by the stimulus beam represent the temperature within the area where the heating is applied.
  • the area illuminated by at least one of the stimulus beams is smaller than the area illuminated by the heating beam. In this way the signal elicited by the stimulus beam may correspond more closely to the temperature near the center of the heated area, and exclude areas towards the edge where the temperature may be lower due to conduction of heat to the surrounding areas not heated by the heating beam.
  • the area illuminated by at least one of the stimulus beams is concentric or nearly concentric with the area illuminated by the heating beam.
  • the area illuminated by at least one stimulus beam is from 1 % to 99% of the area illuminated by the heating beam. In a preferred embodiment, the area illuminated by at least one stimulus beam is from 50% to 90% of the area illuminated by the heating beam. In a further embodiment, the area illuminated by at least one stimulus beam is from 70% to 90% of the area illuminated by the heating beam.
  • the direction of each beam may be adjusted prior to the joining of the beams so that the beams are not necessarily concentric. This may be useful for example to provide scanning of the temperature of different areas within the area that is heated and the area outside of the heated area.
  • the device may include a photometric measurement device 25, e.g. optical power meter or beam camera, measures the optical power that passes aperture 24 and is passed by beam splitter 3. This optical power is directly proportional to the optical power that reaches the retina. Additionally, the measured intensity distribution corresponds with the intensity distribution at the retina.
  • - 3 means to provide a camera view of the fundus function: Direct heating and stimulus beams towards the eye and direct image from the fundus to the camera example: Polarizing beam splitter that reflects the S-polarized heating and stimulus beams towards the eye, and transmits the P-polarized component of the light scattered from the retina to the camera system.
  • Combining means to arrange the beams in an orientation in which all the beams may be introduced to the same object, such as the eye to be subjected to the heating and temperature determination e.g. beam splitters
  • - 6 means for joint beam direction control function: adjust direction of all beams example: kinematic mirror
  • - 7,8,9 means for beam direction control function: adjust direction of each beam example: kinematic mirrors
  • - 10 means to mitigate back-reflection artefacts in the camera image function: block collimated back-reflections from reaching the camera sensor example: opaque obstruction placed at the center of the imaging path, wide enough diameter to catch all of the back-reflected imaging beams, e.g. 5 mm.
  • means for fundus imaging function image the fundus and the size and location of the heating and stimulus spots on the retina example: camera system with a microscope or macro imaging objective
  • means for shaping the heating and stimulus laser beams function controlling the size and divergence/convergence of the heating and stimulus laser beams independently example: collimation lenses with independent x-y-z translation
  • laser sources for RPE heating and retinal stimulation function produce laser beams for retinal heating and stimulation with visible and near infrared light example: 1064 nm laser diode with 2 Watt maximum optical power, for RPE heating, 785 nm laser diode with 1 W maximum optical power for near infrared stimulation, 515 nm laser diode with 10 mW maximal power for visible stimulation.
  • the RPE heating laser diode is operated by an optical power measuring feedback circuit.
  • the stimulus laser diodes are operated by a laser pulse driver circuit.
  • - 21 means for data acquisition and stimulation and RPE heating control function: Amplifies and acquires ERG signal from the ERG electrode, modulates the stimulus and RPE heating lasers, measures the optical power of the heating laser that reaches the RPE, acquires video image from the fundus.
  • the system has a user interface, and the system runs an RPE treatment protocol determined by the user.
  • ERG electrode function acquire the ERG signal from the heat treated eye.
  • - 24 means for controlling the size of the stimulus and heating beams function: control the size of the stimulus and heating beams simultaneously while maintaining their relative sizes on the retina example: adjustable aperture
  • - 25 means for measuring the optical power that reaches the retina function: to measure the optical power that reaches the retina example: optical power meter.
  • FIG. 25 shows one other exemplary device according to an embodiment of the invention.
  • the device comprises at 101 , the output fiber of an electromagnetic radiation source for RPE heating (e.g. fiber coupled 810 nm laser module).
  • a stimulus light source 102 e.g. white LED
  • a background light source 103 e.g. white LED is depicted, which is an optional feature of the device.
  • a collimation lens 104 for the RPE heating laser may be provided, which may create a uniform illumination profile on spatial light modulator 107 and make the beam travel in a narrow angle to fall within the boundaries of a projection lens 1 1 1.
  • a collimation lens 105 for the stimulus light may create a uniform illumination profile on spatial light modulator 108 and make the beam travel in a narrow angle to fall within the boundaries of projection lens 1 1 1.
  • a collimation lens 106 for the background light may create a uniform illumination profile at the distance from the projection lens 1 1 1 as spatial light modulators 107-108.
  • a spatial light modulator 107 for the RPE heating light may create a desired light profile, which is projected in front of the fundus lens, which may project it further onto the retina, e.g. an adjustable aperture.
  • a spatial light modulator 108 for the stimulus light may create a desired light profile, which is projected projected in front of the fundus lens, which may project it further onto the retina, e.g. an adjustable aperture.
  • a beam splitter 109 may combine the stimulus and light beams with the RPE heating beam. Preferably beam splitter 109 may be a dichroic shortpass beam splitter with the pass band ending at around 700 nm.
  • a beam splitter 1 10 may combine the stimulus beam with the full field background beam (e.g. 90:10 reflectiomtransmission plate beam splitter).
  • a projection lens 1 1 1 may project images of spatial light modulators 107 and 108, as well as the full field background, into a virtual image (1 16) in front of the fundus lens (1 13). If the distance from spatial light modulators (107 and 108) to lens 1 1 1 is dobject, and the distance between lens 1 1 1 to the virtual image 1 16 is dimage, the focal length of the lens must be
  • a projection mirror 1 12 may turn all of the beams towards the eye, and may be coaxial with the imaging system 1 14.
  • a fundus lens 1 13 may project an image of the stimulus and heating spots onto the retina.
  • Imaging optics 1 14 of a slit lamp biomicroscope system may be utilized in the device
  • 1 15 depicts the eye of the patient, while 1 16 depicts a projection plane or virtual image. This is where the heating and stimulus spots are projected, and where the camera focuses.
  • the fundus lens may project an image of the retina here, and virtual images of the heating and stimulus spots onto the retina.
  • the system may be mounted on a slit lamp biomicroscope. Moving the slit lamp biomicroscope may enable translation of all of the beams as a group in the fundus. Additionally, a doctor may slightly tilt the fundus lens to slightly move the beams on the fundus. In addition to controlling the beams as a group, it is possible to move the beams independently. Beams may also be moved independently by moving the light source, lens, spatial light modulator -combination (1 ,4,7 for RPE heating and 2,5,8 for stimulation).
  • Devices disclosed herein may be utilized on humans or animals. Some embodiments of the invention have been designed for use with mice and some of the parameters disclosed are preferable for this use case.
  • a device for mouse treatments may be designed to enable retinal temperature determination from rod driven dark-adapted mouse ERG.
  • a device designed for humans may determine retinal temperature from light adapted cone driven ERG, and therefore the brightness of the stimulus light is advantageously much higher. Additionally, the light should stimulate both M and L (red and green) cones, and the wavelength should be chosen accordingly. In practice, this may mean either using a stimulation wavelength, which stimulates both M and L cones similarly, or using white light. White light may be advantageous here, because it enables better retinal imaging with the same light. When using white light, it is more convenient to consider illuminance/luminance rather than irradiance/power because illuminance/luminance describes the amount of stimulation more accurately.
  • the stimulation frequency may be between 10 and 40 Hz, the most likely range being between 10 and 20 Hz.
  • the heating laser should preferably have a wavelength long enough not to cause significant light adaptation. Longer wavelengths are absorbed less by the retinal pigment epithelium, and penetrate deeper into the ocular tissues, which is why longer wavelengths are not advantageous strictly from a heating perspective.
  • 810 nm is a wavelength often used with these types of treatments.
  • a retinal irradiance of up to 10 W/cm 2 may be utilized from the 810 nm laser into a spot diameter between 1 and 7 mm on the retina.
  • first, second and third electromagnetic radiation sources may be laser sources so that the first laser source is for RPE heating and the second and third laser sources for retinal stimulation and producing for example laser beams with visible and near infrared light, used wavelengths may be about 1064 nm laser diode producing a maximum retinal irradiance of 10W/cm 2 at the macula with 2 Watt maximum optical power, for RPE heating, 785 nm laser diode with 1 W maximum optical power for near infrared stimulation, 515 nm laser diode with 10 mW maximal power for visible stimulation.
  • At least one first electromagnetic radiation source may be used for RPE heating and at least one second electromagnetic radiation source may be used for retinal stimulation, where the first electromagnetic radiation source may be a laser source such as a 810 nm fiber coupled laser diode for heating, and white LED producing a retinal illuminance of up to 10 7 trolands for stimulation may be used as a stimulus light source.
  • the first electromagnetic radiation source may be a laser source such as a 810 nm fiber coupled laser diode for heating
  • white LED producing a retinal illuminance of up to 10 7 trolands for stimulation may be used as a stimulus light source.
  • the wavelength provided by the first electromagnetic radiation source may be in the range of 750-870 nm, more advantageously in the range of 800 - 820 nm, and most advantageously around 810 nm.
  • the second electromagnetic radiation source may for example provide white, broadband light.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Radiation-Therapy Devices (AREA)

Abstract

L'invention concerne un dispositif développé pour le traitement thermique de l'épithélium pigmentaire rétinien (EPR). Le but du dispositif est de provoquer un choc thermique transitoire au niveau de l'EPR, ce qui induit une régulation à la hausse de la production de protéine de choc thermique. Des faisceaux laser sont combinés à des diviseurs de faisceau (4, 5). Chaque canal laser a sa propre optique réglable située avant le diviseur de faisceau (4, 5). Ces optiques déterminent la direction des faisceaux et le fait que les faisceaux divergent, convergent ou se propagent de manière collimatée après le passage à travers le diviseur de faisceau (4, 5). Ceci permet d'ajuster indépendamment l'emplacement et la taille des points laser lorsque les faisceaux atteignent la rétine. Après le diviseur de faisceau (4, 5), un miroir cinématique (26) est utilisé pour rediriger tous les faisceaux à la fois et, soit un système de lentilles, soit un diaphragme réglable peut être utilisé pour commander le point de tous les faisceaux simultanément.
PCT/FI2019/050293 2018-04-10 2019-04-10 Dispositif et procédé pour commander le chauffage de l'épithélium pigmentaire rétinien WO2019197726A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022013483A1 (fr) * 2020-07-17 2022-01-20 Maculaser Oy Dispositif et procédé d'obtention de signaux d'erg
WO2022053745A1 (fr) * 2020-09-10 2022-03-17 Maculaser Oy Agencement permettant d'obtenir des signaux erg pendant le chauffage rétinien et de réguler le chauffage rétinien sur la base de ces derniers
EP4248924A1 (fr) * 2022-03-25 2023-09-27 OD-OS GmbH Procédé de détermination des paramètres de rayonnement, ainsi que dispositif de rayonnement

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011050056A2 (fr) * 2009-10-20 2011-04-28 Boston Biocom Llc Traitement au laser des maladies de l'œil
WO2016174310A1 (fr) * 2015-04-30 2016-11-03 Aalto University Foundation Sr Dispositif et procédé pour une surveillance non effractive de la température des tissus rétiniens
US20170203132A1 (en) * 2015-10-26 2017-07-20 Ojai Retinal Technology, Llc System and process utilizing pulsed energy to treat biological tissue
US20170319383A1 (en) * 2016-03-21 2017-11-09 Ojai Retinal Technology, Llc System and process for treatment of myopia

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011050056A2 (fr) * 2009-10-20 2011-04-28 Boston Biocom Llc Traitement au laser des maladies de l'œil
WO2016174310A1 (fr) * 2015-04-30 2016-11-03 Aalto University Foundation Sr Dispositif et procédé pour une surveillance non effractive de la température des tissus rétiniens
US20170203132A1 (en) * 2015-10-26 2017-07-20 Ojai Retinal Technology, Llc System and process utilizing pulsed energy to treat biological tissue
US20170319383A1 (en) * 2016-03-21 2017-11-09 Ojai Retinal Technology, Llc System and process for treatment of myopia

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022013483A1 (fr) * 2020-07-17 2022-01-20 Maculaser Oy Dispositif et procédé d'obtention de signaux d'erg
WO2022053745A1 (fr) * 2020-09-10 2022-03-17 Maculaser Oy Agencement permettant d'obtenir des signaux erg pendant le chauffage rétinien et de réguler le chauffage rétinien sur la base de ces derniers
EP4248924A1 (fr) * 2022-03-25 2023-09-27 OD-OS GmbH Procédé de détermination des paramètres de rayonnement, ainsi que dispositif de rayonnement
WO2023180561A1 (fr) * 2022-03-25 2023-09-28 Od-Os Gmbh Procédé de détermination de paramètres d'irradiaton et appareil d'irradiation

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