DE102011053775A1 - Methods and apparatus for detecting tau protein deposits in the eye - Google Patents

Abstract

Methods and apparatus are provided for detecting paired helical δ-filaments τ-PHF in the eye (100) in which, for example, an overview image is created by means of a camera device (103) and scanned areas of interest identified by a laser scanner based on the overview image to detect fluorescence from probes (108) bound to paired helical δ-filaments (107).

Description

The present invention relates to methods and devices for the detection of deposits of τ protein in the eye for the diagnosis of diseases from the group of so-called tauopathies, for example on the retina of a human eye.

So-called tauopathies belong to a group of neurodegenerative diseases that share a pathological deposit of τ-proteins in the brain. The group of tauopathies include, for example, Alzheimer's disease, Pick's disease, corticobasal degeneration, frontotemporal dementia or progressive supranuclear palsy. Alzheimer's disease, d. H. Alzheimer's disease, is by far the most common and also the most intensively researched disease of this group and therefore serves as an application example below.

Alzheimer's disease, the most common form of progressive neurodegenerative disease, affects approximately 40% -50% of the age group 85-90 years old in the elderly. 60% of people with dementia worldwide suffer from Alzheimer's disease. Currently, Alzheimer's can only be diagnosed with certainty after the death of the patient by autopsy of the brain.

Brains of people suffering from Alzheimer's disease contain neuronal tissue in addition to characteristic extracellular deposits, the so-called amyloid plaques, dystrophized neurites and neurofibrillary tangles (NFT). These neurofibrillary tangles consist of pathological deposits of the τ protein, which plays an important role in stabilizing microtubules, especially in axons. Abnormal hyperphosphorylation is considered to be the cause of the formation of paired helical filaments (π), which can no longer interact with the microtubules. As a result of these pathological changes, extracellular τ-PHF deposits can be observed as neurofibrillary lesions in brains of Alzheimer's patients.

At the cellular level, the formation of neurofibrillary tangles of τ-proteins proceeds approximately as follows:
In the course of their formation and accumulation, paired helical filaments of the τ protein first assemble within the cytoplasm, likely from τ oligomers, which are truncated during or before PHF formation. This results in classic intracellular neurofibrillary tangles. In this state, the τ PHFs consist of a core of truncated τ proteins and a frayed outer region with complete τ proteins. It is believed that these aggregates result in functional impairment of the affected neurons.

Currently, the extent of Alzheimer's disease in the living patient is determined substantially indirectly from cognitive tests, cerebrospinal fluid examinations, or morphological changes, for example, in brain volume or metabolic disorders of affected brain areas by magnetic resonance imaging (MRI) or FDG-PET (positron emission tomography using ^18F fluorodeoxyglucose ) diagnosed. The specificity of such indirect methods, which are often associated with high statistical variability of results, are considered to be low compared to direct detection of τ-PHF in the brain.

Therefore, it would be desirable to be able to detect τ-PHF deposits at an early stage or to be able to quantitatively evaluate the course of τ-pathy. This could facilitate therapies and enable them early.

For some time now, for purposes of study, positron emission tomograms (PT) of the brain have been performed using contrast agents (e.g., [ ¹¹ C] PIB, [ ¹⁸ F] FDDNP) that are selectively linked to amyloid plaques and / or τ-PHF give a direct indication of pathological changes of the nerve tissue. However, due to the very high costs for such examinations and a high expenditure on equipment, such examinations are hardly suitable for large-scale preventive examinations or for continuous monitoring of a therapy. In addition, such examinations can not be repeated as often as desired due to the radiation exposure to a patient.

In the WO 2011/038892 A1 It is proposed to detect τ-PHF in a retina of an eye, which is attributable to the brain, using fluorescent markers for this purpose. For this purpose, the use of various optical devices such as so-called fundus cameras, ie cameras for recording the fundus, or high-resolution laser scanning ophthalmoscopes (LSO) is proposed.

With this in mind, it is an object of the present invention to provide efficient methods and apparatus for detecting paired helical τ filaments in the eye.

This object is achieved by a method according to claim 1 and a device according to Claim 15. The subclaims define further embodiments.

According to the invention, a method is proposed for the detection of paired helical τ-filaments (τ-PHF) in the eye, comprising:
Taking an overview picture of a retina of the eye,
Identifying at least one area of interest of the retina on the basis of the overview image,
Scanning the at least one region of interest with a laser beam, and
Detecting light emission of paired helical τ-filament sensitive probes in response to scanning with the laser beam.

By first taking an overview image and then identifying at least one area of interest of the retina on the basis of the overview image, an efficient examination can be carried out. In particular, the areas of interest can be accurately examined by means of laser beam scanning, without a total duration of the investigation becoming unnecessarily long.

The regions of interest may in particular be regions in which light emission from the probes can be identified on the overview image.

The light emission may in particular be a fluorescence emission or an emission of inelastically scattered scattered light, for. By Raman scattering.

The method, in one embodiment, further comprises performing supplemental diagnostics, such as polarization-based or optical coherence tomography-based morphological characterization of the neural tissue of the retina, comprising e.g. B. a layer thickness measurement of the retinal nerve fiber cell layer or the entire retina.

On the basis of such measurements, various tauopathies, i. H. the presence of τ-PHF, associated diseases, such as eye diseases such as glaucoma of neurodegenerative diseases.

Preferably, the detection comprises a lifetime measurement of the fluorescence, whereby probes bound to paired helical τ filaments can be distinguished from unbound probes.

Morphological information can also be obtained via optical coherence tomography, which can be coupled with information from the detection of the fluorescence in order to enable the fluorescence of cellular structures in different layers of the eye.

The resolution of the scanning is preferably 20 μm or below, more preferably 10 μm or below.

As probes, one or more substances are preferably selected from the group comprising arylaminothiazoles, 4,6-divinylpyrimidines, 2,5- and 3,6-divinylpyrazines, [4- (1,3-benzothiazol-2-yl) phenyl] hydrazones , Diaryl ureas, tetracycline, 4,4 '- (1E, 1'E) -2,2' - (1-aryl-1H-pyrazole-3,5-diyl) bis (ethene-2,1-diyl) bis- 2-methoxyphenols, 2,5-bis (4-hydroxystyryl) benzonitrile and 2,5-bis (4-hydroxystyryl) -N-benzamide. However, other probes sensitive to paired helical τ filaments may also be used.

The invention will be explained in more detail with reference to the accompanying drawings with reference to embodiments. Show it:

1 a block diagram of an embodiment of a device according to the invention,

2 a diagram of another embodiment of a device according to the invention,

3 (a) to (j) various probes usable in embodiments of the invention,

4 a flowchart illustrating an embodiment of a method according to the invention,

5 histological sections of a retina of patients with tauopathies, and

6 In vivo images of τ-PHF in retinal ganglion cells of a mouse model of Alzheimer's disease.

In the following, various embodiments will be explained in more detail. It should be noted that features of various embodiments may be combined with each other unless otherwise specified. On the other hand, a description of an embodiment having a plurality of features is not to be construed as requiring all of these features for practicing the invention, as other embodiments may have fewer features or alternative features.

In 1 FIG. 4 is a block diagram of a paired helical τ filament (τ-PHT) detection apparatus according to the present invention. 107 in one eye 100 , for example, a human eye. For the detection of the paired helical τ-filaments probes are in the illustrated embodiment 108 . 109 which have characteristic fluorescence properties and which are sensitive to, ie bind to, paired helical τ filaments. At the in 1 illustrated embodiment, the probes 108 already paired helical τ filaments 107 tied while a probe 109 is unbound.

The device of the embodiment of 1 includes for examination of the eye 100 a laser scanner device 102 and a camera device 103 which in the embodiment of the 1 via a common ophthalmoscope lens 101 visually with the eye 100 coupled, in particular, a retina of the eye 100 in which τ-PHT 107 is embedded, to be able to illuminate and to be able to absorb optical radiation originating from the retina. The laser scanner device 102 and the camera device 103 be via a controller 104 controlled. The control 104 can be implemented, for example, by means of a computer and has a screen in the illustrated embodiment 106 and an input device 105 on, the input device 105 For example, a keyboard and / or a mouse may include.

For examination of the eye 100 is the controller 104 set up the camera setup 103 for taking an overview picture of the retina of the eye 100 head for. For this purpose, in particular the camera device 103 have illumination for illuminating the retina, which at an excitation wavelength of the probes 108 . 109 adapted to the probes 108 . 109 to stimulate fluorescence. In addition, a color filter can be provided for recording the overview image, which is essentially only light with wavelengths corresponding to the fluorescent light of the probes 108 . 109 after excitation by the illumination, so that an overview of the fluorescence of the retina is taken. In other embodiments, also or alternatively, other types of images, for example, multicolor images for identifying structures or infrared images may be included.

The control 104 is further set up to evaluate the recorded overview image and to define one or more regions of interest (ROI, from the English "region of interest"). The regions of interest may in particular be regions in which fluorescence from the probes is displayed on the overview image 108 . 109 is available. Such an evaluation can be carried out fully automatically by means of corresponding automatic image processing. In other embodiments, the selection of areas of interest can also be semi-automatic. For example, the overview picture on the screen 106 are displayed, with areas of fluorescence from the probes 108 . 109 can be highlighted or marked, and a user can then use the input device 105 select the areas of interest. The control 104 then controls the laser scanner device 102 scanning the areas of interest with a laser beam and detecting from the retina the light emitted in response to the laser beam, in particular fluorescent light. The spatial resolution in such scanning of the regions of interest is preferably less than 20 μm, more preferably less than 10 μm. The detection can be carried out in particular time-resolved, for example by means of FLI (Fluorescence Lifetime Imaging), and / or carried out spectrally resolved to probes bound in this way 108 from unbound probes 109 can differ, since typically changes by the binding to paired helical τ-filaments a lifetime and / or a spectrum of fluorescence. The laser scanner device can work in particular as a confocal laser scanning ophthalmoscope (cSLO, Confocal Scanning Laser Ophthalmoscope).

In addition to such fluorescence measurements, in some embodiments the laser scanner device may 102 Optical coherence tomography measurements or polarization-dependent measurements may also be carried out in order to obtain morphological information about the retina in addition to a preferably depth-resolved fluorescence distribution. The morphological information and the information obtained from the fluorescence can then be used by the controller 104 be linked and, for example, on the screen 106 for review, for example, be presented by a doctor.

In particular, fluorescence signals from the probes can be assigned in this way to cellular structures in the inner nuclear layer (INL) or the ganglion cell layer (GCL, or "ganglion cell layer") or other layers.

In 2 a more detailed diagram of a further embodiment of the present invention is shown, wherein in particular a laser scanner device and a camera device are shown in detail. In the embodiment of 2 is a controller like the controller 104 of the 1 not explicitly shown, but can according to the description of 1 be provided.

As a laser scanner device, the embodiment of the 2 a Confocal Laser Scanning Ophthalmoscope (cSLO), which contains one or more laser light sources 1 , a housing head 41 and a detection device 15 includes. In addition, the embodiment includes the 2 a camera device 40 ,

Number and type of laser light sources 1 can depend on probes used 50 which in one eye 10 are selected, so that the one or more laser light sources emit corresponding excitation wavelengths to the probe 50 to stimulate fluorescence. The one or more laser light sources 1 are over optical fibers 2 , preferably single-mode fibers, to the housing head 41 coupled. The light guides 2 emitted laser light is using collimator lenses 3 collimated. For multiple laser light sources 1 is the laser light of different laser light sources, in the example of 2 two laser light sources, via a deflection mirror 4 and a dichroic beam splitter 29 or another optical device combined into a single illumination beam, which via another deflection mirror 4 to another dichroic beam splitter 30 is steered.

The dichroic beam splitter 30 serves to separate an illumination beam path over which the laser light sources 1 Finally, laser light emitted into the eye 10 is directed, from a detection beam path, with which of the eye 10 outgoing light to the detector device 15 is steered. From the dichroic beam splitter 30 the laser light of the illumination beam reaches a scanner mirror 5 , which is preferably arranged in a conjugate pupil plane of the optical arrangement. The scanner mirror 5 For example, it may include a biaxial scanner mirror or two monochrome scanner mirrors connected in series to move the scanner mirror 5 an area in the eye 10 , especially on a retina of the eye 10 to be able to feel. The scanning motion of the scanner mirror 5 can, for example, by the controller 104 of the 1 to be controlled.

By means of a lens 6 , a lens 7 , a beam splitter 8th and an ophthalmoscope lens 9 The laser light is then on a desired layer, in particular the retina or a layer of the retina, in the eye 10 focused. The objective 6 can also be referred to as a scan lens, the lens 7 can also be referred to as a field objective. The beam splitter 8th For example, it can be swiveled in and out to either the laser scanner device or the camera device 40 to be able to use. In other embodiments, the laser scanner and camera device use different wavelengths, and the beam splitter 8th can be configured as a wavelength-selective, for example dichroic, beam splitter.

That in response to the illumination with laser light from the retina of the eye 10 emitted or scattered light, in particular fluorescent light, which by excitation of the probe 50 produced with laser light, is in the opposite direction as the illumination light via ophthalmoscope lens 9 , Beam splitter 8th , Objective 7 , Objective 6 and scanner mirror 5 to the dichroic beam splitter 30 steered, passes through this at least partially and is then on a Pinholelinse 11 on a pinhole 12 focused. The pinhole 12 preferably has a variable size, so that confocality and signal strength at the detection device 15 are adaptable, with a further open pinhole causes a poorer confocality at the same time higher signal strength. About a lens 13 then will that be the pinhole 12 passing light into a light guide 14 , preferably a multimode fiber coupled, which couples the light to the detection device 15 passes. The detection device 15 is hereby set up for spectrally resolved detection and / or for time-resolved detection or fluorescence lifetime imaging (FLI) in order to be able to distinguish bound from unbound probes as already explained.

The camera device 40 of the embodiment of 2 has a lighting 16 which can have one or more light sources, in particular broadband emitting light sources, for example a flash lamp, one or more white light LEDs or combinations of light emitting diodes which emit in different spectral ranges. About a collimator 17 , a lens 18 (Field objective), a lens 20 (Ring aperture lens) and a lens 22 , which may be configured in particular as an anti-reflection lens and may have corresponding coatings, and a beam splitter 23 the light passes through the beam splitter already described 8th to the ophthalmoscope lens 9 and illuminate the eye 10 , In addition, there is a retina 19 and a field stop 21 provided. About a preferably changeable color filter 31 can be a spectral range to illuminate the eye 10 be selected, for example, a spectral region in which a fluorescence of the probe 50 can be stimulated.

The beam splitter 23 is designed as a hole mirror in the illustrated embodiment, so that in the illustrated example, the detection beam path of the camera device as shown "inside" the illumination beam path runs.

That of the eye 10 , especially the retina of the eye 10 , in response to that from the lighting 16 emitted light emitted light is transmitted through the ophthalmoscope lens 9 through the beam splitter 8th and 23 through to a lens 24 steered from where it has a beam splitter 28 and a camera lens 25 to a camera 27 is directed, with which a picture can be taken. In addition, through the beam splitter 28 passing light through a camera lens 25 to another camera 26 directed. The other camera 26 For example, an infrared camera may be while the camera is on 27 For example, a color camera can be. However, it can also be provided two color cameras or two infrared cameras or a black and white camera. In front of the camera 27 is in the illustrated embodiment, a preferably changeable color filter 32 arranged, which may be designed, for example, only light in the wavelength range of fluorescence of the probe 50 place it, so with the camera 27 an overview image of the fluorescence in the eye 10 can be made on the basis of then then as already with reference to 1 explains the laser scanner device, in particular the scanner mirror 5 can be driven to scan areas of interest. The other camera 26 For example, to track movements of the eye 10 or for other settings.

In the illustrated embodiment, the lenses 7 and 24 a variable focus on, so that by means of these lenses on a region of interest of the eye 10 , especially the retina of the eye 10 , can be focused.

As probes for the detection of paired helical τ-filaments, in particular aggregates thereof, for example, in 3 used, for example, arylaminothiazoles ( 3 (a) ), 4,6-divinylpyrimidines ( 3 (b) ), 2,5-divinylpyrazines ( 3 (c) ), Benzothiazolylphenylhydrazones (e.g., the in 3 (d) shown substance), 3,6-divinylpyridanzine ( 3 (e) ), Diaryl ureas ( 3 (f) ), Tetracyclines ( 3 (g) ), Stilbene derivatives, 3,5-divinylpyrazoles or the in 3 (h) to 3 (j) structures shown, namely 4,4 '- (1E, 1'E) -2,2' - (1-aryl-1H-pyrazole-3,5-diyl) bis (ethene-2,1-diyl) bis-2 methoxyphenols ( 3 (h) ), 2,5-bis (4-hydroxystyryl) benzonitrile ( 3 (i) ), or 2,5-bis (4-hydroxystyryl) -N-benzamide ( 3 (j) ).

The radicals shown can be, for example, C ₁ -C ₆ -alkyl radicals.

However, the choice of probes is not limited to the illustrated examples.

The compounds mentioned preferably act as fluorescent probes. They have a (preferably high) affinity for the Aβ protein, α-synuclein and / or tau-PHF aggregates and bind to them - preferably specifically. The binding of the compounds of the invention to one or more of the above target proteins is optically detectable.

Preferably, probes used have sufficient solubility, low lipophilicity (logP <5) and selective binding to τ-PHF even in the presence of other β-sheet aggregates such as β-amyloid. Preferably, a dissociation constant of probes used is in an in vitro displacement assay against thioflavin-S or thioflavin-T or auto-displacement assay against radiolabelled analogs at IC / EC ₅₀ <200 nM. A one-photon fluorescence excitation of the probes bound to τ-PHF is preferably between 390 and 550 nm, although other wavelengths are possible, in which case the corresponding excitation wavelength, for example by the laser light sources 1 or the lighting 6 to be adjusted accordingly.

In some of the described materials, especially the 2,5-divinylpyramizines and 4,6-divinylpyrimidines, different geometries and quatrupole moments with similar affinities to τ-PHF, especially tau aggregates, and consistent selectivity towards other β-sheet aggregates result in different Photon capture cross sections for single photon excitation and two photon excitation.

• a) eine größer fünffache, bevorzugt größer zehnfache Extinktionszunahme bei einer Detektionswellenlänge nach Bindung an τ-PHF-Aggregate (oder auch an β-Amyloid) gegenüber einem ungebundenen Zustand,
• b) einen Stokes-Shift von > 10 nm, bevorzugt > 20 nm,
• c) einen Extinktionskoeffizienten E > 5000 L·mol^–1·cm^–1, bevorzugt größer 10000 L·mol^–1·cm^–1
• d) EC50 < 400 nM, bevorzugt < 200 nM in einem Verdrängungsassay gegen fluoreszente Referenzsonde oder im Autoverdrängungsassay gegen die radioisotopenmarkierte Verbindung
• e) eine Löslichkeit/Lipophilie mit logP < 5, bevorzugt zwischen 1 und 2,8, und
• f) eine topologische polare Oberflächenfläche (TPSA, vom Englischen „Topological Polar Surface Area”) < 200 Ų, bevorzugter < 100 Ų, noch bevorzugter < 80 Ų.

In summary, substances used as probes preferably have 3 or more of the following properties a) -f):

• a) a greater than five-fold, preferably greater than ten-fold, increase in absorbance at a detection wavelength after binding to τ-PHF aggregates (or also to β-amyloid) compared to an unbound state,
• b) a Stokes shift of> 10 nm, preferably> 20 nm,
• c) an extinction coefficient E> 5000 L · mol ^-1 · cm ^-1 , preferably greater than 10000 L · mol ^-1 · cm ^-1
• d) EC50 <400 nM, preferably <200 nM in a displacement assay against fluorescent reference probe or in the auto-displacement assay against the radiolabeled compound
• e) a solubility / lipophilicity with log P <5, preferably between 1 and 2.8, and
• f) Topological Polar Surface Area Topographic Polar Surface Area (TPSA) <200 Å ² , more preferably <100 Å ² , even more preferably <80 Å ² .

The abovementioned increase in absorbance when binding to, for example, τ-PHF In particular, an improvement of the signal-to-noise ratio and can be determined experimentally, for example by changing, for example reducing, a background noise. The determination of the extinction coefficient E can be measured for example at 25 ° C, a pH of 7, at a respective absorption maximum of the respective substance with DMSO (dimethyl sulfoxide) as a solvent.

The Stokes shift denotes a difference between excitation (excitation) maximum and emission maximum. A larger Stokes shift facilitates separation of excitation light and fluorescence, for example, by the dichroic beam splitters or color filters mentioned above.

The above-mentioned EC50 value characterizes the affinity of the respective probe for the substance to be detected, for example τ-PHF, and can be indirectly determined, for example, by displacing fluorescent or radioactive reference ligands, for example displacement of thioflaviri S, thioflavin T or 11C-FIB , A suitable measuring method is, for example, in Lockhart, et al., The Journal of Biological Chemistry, 280, 7677-7684, March 4, 2005, under Materials and Methods described.

In embodiments, probes, for example the above-mentioned arylaminotyazoles, include 4,6-divinylpyrimidines, 3,6-divinylpyridazines, 2,5-divinylpyrazines, [4- (1,3-benzothiazol-2-yl) phenyl] hydrazones and / or diaryl ureas 2, preferably at least 3 aromatic rings which are bonded together directly via a vinyl bridge or a urea bridge. In particular, materials used may have 3 aromatic rings which are linked together via vinyl bridges, resulting in extended p-electron systems.

In embodiments, compounds of the abovementioned classes with delocalized electrons, for example p-electron systems, are used in particular via at least 15 atoms involved, for example at least 20 atoms, in particular 22 or more atoms.

Preferably, probes whose conformation in the excited state are stabilized by binding to the target protein. This stabilization may be by hydrogen bonding or van der Waals interactions of the aryl moieties with the target protein. This leads to stabilization of the non-planar, excited state in preferred probes. Such twisting can be induced by 1,3-allyl stress or by 1,4-butadiene voltage alkylated vinyl aromatic compounds.

Particular preference is furthermore given to compounds which have a half-life in vivo of> 60 min. For this purpose, in certain embodiments, residence time and excretion rate of correspondingly labeled probes (eg 3H, 11C, 18F) are determined.

Preference is given to compounds which have an increased potential brain activity with reduced binding to white brain mass and have reduced plasma protein binding. The diffusivity of a compound through the endothelium of the blood-brain barrier is largely determined by its lipid solubility (lipophilicity) and size. In a preferred embodiment, the compounds of the invention have a molecular weight <500 g / mol.

The log P value and the log D value are model measures of the relationship between lipophilicity (fat solubility) and hydrophilicity (water solubility) of a substance. The expectation is to be able to estimate the distribution coefficients of this substance in other systems with an aqueous and a lipophilic phase with the help of the octanol-water partition coefficient. The log P value is greater than one if a substance is more soluble in fat-like solvents such as n-octanol, less than one if it is better soluble in water. Accordingly, log P value is positive for lipophilic and negative for hydrophilic substances. Preference is given to compounds which have a log P value of from 1 to 2.8. In further embodiments, compounds with a log D <5 are preferred. The measurement of the log P value or of the log D value takes place via an octanol / water two-phase system and UV / VIS spectroscopy at 25 ° C. and pH 7. Since not all chemicals have the log P value and / or the log D value can be measured, there are other models for the prediction, eg. By Quantitative Structure Activity Relationships (QSAR) or by Linear Free Energy Relationships (LFER).

The potential brain penetration of the compounds can also be defined via the topological polar surface area (TPSA). This is defined as the sum of the surface contributions of the polar atoms (usually oxygen atoms, nitrogens and / or hydrogen atoms) in a molecule. The calculation was among others by Ertl, P. et al., Molecular Chemistry, 2000, 43, 3714-3717 described.

Freedom of charge or a weak basicity of the compounds promote good penetration through the blood-brain barrier. The compounds of the invention are further characterized by good photostability (low photobleaching) and by a short-lived singlet excitation is characterized by a long-lived triplet stimulus.

In 4 a flow chart illustrating an embodiment of a method according to the invention is shown. The procedure of 4 in particular using the devices of the 1 or 2 but can be implemented independently.

To prepare the actual procedure will be in one step 140 For example, to a human patient or an animal to be examined τ-PHF sensitive probes fed, for example one of with reference to 3 explained probes. These probes attach themselves after a certain time to τ-PHF in the eye.

In step 141 is then an overview image of an eye fundus with a corresponding camera device as with reference to 1 and 2 explained explained camera devices. Here, as with reference to 2 already explained a wavelength of illumination and detection by means of a camera, for example by means of color filters such as the color filter 31 and 32 of the 2 be spectrally adjusted to the excitation wavelength or emission wavelength of the corresponding probe.

When creating the overview image, the fundus of the eye is generally excited to fluorescence over a large area. In order to avoid the influence of scattered light or other extraneous light, for example scattered light on a layer located in front of the fundus, for example the eye lens, various measures can be taken. For example, the fundus may be illuminated in a structured manner, resulting in lighter and darker areas on the ocular fundus. From intensity measurements in the darker areas, a spatially resolved background signal can be determined as a false light distribution via the camera, which is subtracted from a picture of the fundus taken later and evenly illuminated. Thus, a picture of the fundus essentially free of false light can be generated. The spatial resolution corresponds to the resolution of the camera used as the camera 27 of the 2 and depends among other things on the number of pixels of the camera.

In this case, two different light patterns are preferably generated in such a way that the bright areas of the two light patterns complement each other to form a complete gapless image (light image) and the darker areas of the two light patterns also complement each other without gaps to form an image (dark image). Thus scattering centers in an entire cross section of the beam path of the camera device can be taken into account in the scattered light distribution. In addition, such a scattered light distribution can be determined with high accuracy. Details on corresponding methods of scattered light determination can be found, for example, in US Pat DE 103 30 716 , of the DE 103 47 389 , of the DE 10 2004 053 730 and the DE 10 2006 031 177 ,

In one step 142 are then defined area of interest, in particular areas in which fluorescence is visible on the overview image. This can be done fully automatically by image analysis or semi-automatically involving user input.

In some embodiments, when generating the overview image and defining the regions of interest, no distinction is made between fluorescence of probes bound to τ-PHF and unbound probes. In other embodiments, a fluorescence lifetime and / or a spectral fluorescence distribution, for example by using a color camera, determined in order to distinguish bound from unbound probes already in the preparation of the overview image, since a binding of the probes to τ-PHF in particular a slow component of a exponential decay of fluorescence decay behavior of fluorescence. Such a lifetime measurement can be accomplished, in particular, by combining the illumination at at least two frequencies, preferably in the megahertz range, and using a runtime camera as the camera. By using spectral analysis, also referred to as "spectral unmixing", fluorescence from the probes of interest can also be distinguished from other sources of fluorescence, such as autofluorescence of the ocular fundus.

Using such methods, the regions of interest can then be used in step 142 only based on the fluorescence bound to τ-PHF.

In step 143 then the ones in step 142 scanned areas of interest with a laser and fluorescence detected. For this purpose, in particular a confocal laser scanning ophthalmoscope as well as in 2 shown used. In particular, pixel-by-pixel spectral detection can take place on at least two channels during this image recording. In the following, structures can then be segmented, ie identified, which have features which resemble or correspond to features of paired helical τ-filaments. In addition, one can For this purpose, a number of data points in some embodiments are limited to one or a few data points in the regions of interest and adjacent regions or in the structures mentioned above with the τ-PHF-like features and adjacent regions. Instead of the lifetime measurement can be analogous by a microspectroscopic examination, z. As Raman spectroscopy, molecule-specific, the presence and binding of the probe to helical τ-filaments are detected. Such measurements of scattered light inelastic scattering, z. B. Ramangestreutem light, can be used in some embodiments, alternatively or in addition to fluorescence measurements to detect the probes.

In addition or as an alternative to such a lifetime measurement, in some embodiments an optical coherence tomography image can be taken with a corresponding laser scanner device. By defining areas of interest, which may be relatively small, and by restricting the lifetime or spectroscopic measurements to only a few data points, a relatively short examination time can be achieved, which is more comfortable for a patient and more accurate at the same time the areas of interest.

Different possibilities and variants of the implementation of the step 143 , in particular the measures mentioned above, will now be explained in more detail.

In general, with laser scanner arrangements, a serial point-like excitation of the fluorescent objects takes place, wherein the point-shaped excited field is confocally imaged in a field stop, which is preferably arranged in front of a detector, preferably a single detector. From successively detected light from each point, a structure of a two-dimensional image of the fluorescent object takes place after passing through a notch filter. Due to the confocality, which in the embodiment of the 2 for example, by the size of the pinhole 12 can be influenced, the influence of fluorescence or scattered light from layers that are not in the focal plane of the excitation or imaging system, greatly reduced.

The laser wavelength is selected in particular matching the probe used, d. H. a laser wavelength is selected in which the probe has an increased absorption and / or scattering and / or excitation for fluorescence or bioluminescence. The laser wavelength can be in the visible or infrared spectral range.

A spectrally resolved detection, for example for the aforementioned "Spectral Unmixing" to distinguish bound probes from unbound probes or other fluorescence sources, for example, a spectral splitting of the light and simultaneous detection in multiple channels, for example in the detection device 15 of the 2 , To determine the fluorescence lifetime imaging (Fluorescence Lifetime Imaging), for example, a time-correlated single photon counting as in the DE 199 20 158 or the DE 101 45 823 be used described.

In a used detection device such as the detection device 15 can be separated from fluorescent light to the reflection light, ie light with the excitation wavelength, for example by appropriate color filter. Thus, a reflection image can be recorded simultaneously with fluorescence measurements, on the basis of which, for example, a compensation of eye movements between the individual measurements, for example measurements at different points or between spectrally resolved measurement and lifetime measurement, can take place.

Optionally, in step 144 still a complementary diagnostic done, and this can also be done in parallel to the previous steps. For example, an autofluorescence detection can take place, or other types of measurements can be carried out, such as optical coherence tomography measurements, for example in order to additionally detect eye diseases, especially eye diseases associated with neurodegenerative diseases.

The by the inventive method of 4 The information obtained can then be used for diagnosis. For this purpose, the data can be compared, for example, with data from previous examinations on the same patient or data from a database. For example, the size and structure of fluorescent regions, spectral signatures and fluorescence lifetime in the fluorescent regions compared to the environment, the assignment of fluorescent areas to layers of the retina, for example on the basis of lifetime data and / or compared to a morphology of the retina taken for example with optical coherence tomography, and take into account an overall distribution of the areas in the retina.

During the imaging, ie the steps 141 and 143 of the 4 , doing a move the patient and thus the eye to be examined are detected and taken into account, wherein such a movement, for example by the infrared camera 26 of the 2 can be detected.

In particular, an ante-mortem diagnosis of tauopathies can be carried out by the presented methods and devices according to the invention, a course of the disease can be monitored and / or a corresponding therapy can be controlled.

To further illustrate the invention are in 5 histological sections of the retina of patients who suffered from two different tauopathies. On the left is a retina of a patient with Alzheimer's disease (AD), and on the right is the retina of a patient suffering from progressive supranuclear palsy (PSP). Arrows indicate τ-PHF deposits in the inner granule layer (INL), which in this case were visualized by staining with a specific AT8 antibody.

The 6 shows in vivo recordings of τ-PHF in retinal ganglion cells of a mouse model of Alzheimer's disease. On the left is a picture taken during a preliminary examination, on the right is a picture 48 Hours after administration of a specific probe (FSB), the illustrated laser scanning ophthalmoscope image shows lightly marked τ-PHF deposits. From this it can be seen that the described devices and methods are suitable for the detection of τ-PHF in a retina.

It should be noted that in other embodiments, only one camera device or only one laser scanner device can be used.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2011/038892 A1 [0009]
• DE 10330716 [0068]
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Cited non-patent literature

• Lockhart, et al., The Journal of Biological Chemistry, 280, 7677-7684, March 4, 2005, for Material and Methods [0055]
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Claims (17)

Method for detecting paired helical τ filaments ( 107 ) in the eye ( 10 ; 100 ), comprising: taking an overview image of a retina of the eye ( 10 ; 100 ), Identifying at least one region of interest of the retina on the basis of the overview image, scanning the at least one region of interest with a laser beam, and detecting light emission from for paired helical τ-filaments ( 107 ) sensitive probes in response to scanning with the laser beam. The method of claim 1, wherein determining at least one region of interest comprises identifying light emission from the probes. 108 . 109 ) in the overview image and determining the at least one region of interest based on the identified fluorescence. The method of claim 2, wherein identifying the light emission distinguishes from paired helical τ filaments ( 107 ) bound probes ( 108 ) light emission from unbound probes ( 109 ) comprises light emission. The method of any one of claims 1-3, wherein detecting light emission in response to the laser beam distinguishing paired helical τ filaments ( 107 ) bound probes ( 108 ) light emission from unbound probes ( 109 ) comprises light emission. A method according to claim 3 or 4, wherein the discriminating is based on a spectrum of the light emission and / or on the basis of a duration of the light emission. The method of claim 5, wherein detecting light emission in response to the scanning comprises a time-resolved measurement of the light emission, wherein the time-resolved measurement is performed only in a part of the regions of interest. The method of any of claims 1-6, wherein taking an overview image comprises illuminating the retina of the eye in a first waveband in which an excitation wavelength of the probes ( 108 . 109 ), and filtering from the retina in response to illuminating outgoing light such that the overview image is based on light in a second wavelength range which is a light emission wavelength of the probes ( 108 . 109 ), wherein the first wavelength range is different from the second wavelength range. The method of any one of claims 1-7, wherein the scanning and the detecting are done with a laser scanning ophthalmoscope. The method of any of claims 1-8, further comprising taking a morphology of the retina of the eye, and correlating the detected light emission with the morphology of the eye. The method of any one of claims 1-9, further comprising performing an additional optical measurement for detection of features typical of eye diseases. Method according to one of claims 1-10, wherein the scanning is carried out with a spatial resolution <20 microns. The method of any one of claims 1-11, wherein the probe is selected from the group consisting of arylaminothiazoles, 4,6-divinylpyrimidines, 2,5- and 3,6-divinylpyrazines, [4- (1,3-benzothiazol-2-yl ) phenyl] hydrazones, diaryl ureas, tetracycline, 4,4 '- (1E, 1'E) -2,2' - (1-aryl-1H-pyrazole-3,5-diyl) bis (ethene-2,1- diyl) bis-2-methoxyphenols, 2,5-bis (4-hydroxystyryl) benzonitrile, 2,5-bis (4-hydroxystyryl) -N-benzamides. The method of any of claims 1-12, wherein the light emission comprises fluorescence. The method of any of claims 1-13, wherein the light emission comprises inelastically scattered scattered light. Device for detecting paired helical τ-filaments ( 107 ) in a retina of an eye, comprising: a camera device ( 103 ; 40 ) for taking an overview image of a retina of the eye ( 10 ; 100 ), a controller ( 104 ) for determining at least one region of interest of the eye on the basis of the overview image and for driving a laser scanner device ( 102 ; 41 . 1 . 2 . 14 . 15 ) such that the laser scanner device ( 102 ; 41 . 1 . 2 . 14 . 15 ) scans the at least one region of interest with a laser beam and light emission from probes sensitive to paired τ-filaments ( 108 . 109 ) is detected in response to the scanning with the laser beam. Apparatus according to claim 15, wherein the camera device ( 103 ; 40 ) a lighting ( 16 ), a color filter ( 31 ) for selecting a wavelength range comprising an excitation wavelength of the probe ( 50 ; 108 . 109 ) for illuminating the retina, a camera ( 27 ) and a second color filter ( 32 ) for selecting the light emission from the probes ( 108 . 109 ). Apparatus according to claim 15 or 16, wherein the apparatus is adapted to carry out the method according to any one of claims 1-14.

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