Abstract:
An apparatus and a method for operating an endothelium reflection microscope. The apparatus includes an optical head, which comprises: (i) an illuminating system, (ii) a frontal eye observation optical system along a central channel in which an alignment-use light spot is received and imaged by a camera having a digital optical sensor, and an enlarged-imaging optical system for enlarged observation or photographing of the subject part by the digital camera. The apparatus further comprises a motor for operating the optical head, and a CPU controller for automatically controlling the motor, the illuminating system and the frontal eye observation optical system. The method comprises an endothelium image acquisition procedure in which the grey level inside a check area of the camera sensor is checked constantly during advancement along the Z-axis; when the grey level reaches a predetermined threshold value, a delay time (Δt) is triggered; and when the delay time (Δt) lapses, acquisition by the digital camera of one or more images of the endothelium is enabled.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates generally to microscopes and, more particularly, to non-contact endothelium microscopes and the like. 
       BACKGROUND OF THE INVENTION 
       [0002]    The endothelium is the innermost layer of tissues forming the cornea, consisting of a single layer of flat polygonal cells. One purpose of the endothelium is to control water content and, thus, permit suitable hydration of the cornea. Accordingly, the shape and number of cells in the endothelium influence the quality of one&#39;s vision. As the transparency of the cornea depends on a rather delicate balance of factors, there are a number of diseases that can readily disrupt this balance, cause a loss of transparency, and, thereby, hinder the quality of vision. 
         [0003]    The endothelium cells are of hexagonal in the children and in the young people. They do not reproduce themselves and. At birth, the density is about 4000 cells per square millimeter but as years pass the number decreases and the cells change their shape. The average density in an adult becomes of 2700 cells per square millimeter, in a range from 1600 to 3200 cells for square millimeter. The loss of cells brings about two main morphologic changes: the presence of cells with different surface area, and the increase in the amount of cells shaped differently from the basic hexagonal shape. 
         [0004]    The evaluation of the cornea endothelium is useful to have a first clinic indication regarding the risks of a surgical step, and for checking a diagnostic assumption or a therapy effectiveness. In this kind of evaluations, it is very important to observe heterogeneous parts, such as intracellular and intercellular areas of no reflectance (dark spots), hyper reflective areas (bright spots), empty areas in the cells layer (guttae), bubbles, Descemet&#39;s membrane rupture lines. 
         [0005]    Said parts can be checked in relationship with the evolution of different endothelium diseases of inflammatory or dystrophic nature. The quantity evaluation permits to assign to a determined photographic field a numeric parameter useful for the study of the endothelium variations in time, or for the comparison among different patients. 
         [0006]    The most easily accessible parameter is the average cellular density, obtained for comparison or and by counting the cellular elements. The first method is carried out by comparing the cellular dimensions with the dimensions of the hexagonal reticules that correspond to determined densities. The counting of the cellular elements, instead, is carried out by using fixed or variable reticules. 
         [0007]    The two methods give no information on the evolution of the cellular dimensions. This can be obtained by identifying, above and beyond the dimension of the average cellular area and its variability, also the perimeters of the cellus. The endothelium reflection microscope observation was first introduced in the ophthalmologic practice around 1960 by David Maurice who, by modifying a metallography microscope, was able to obtain photographic images of a rabbit corneal endothelium. Exploiting the same theoretic principles, a microscope was subsequently proposed capable of taking photographs of the endothelium without contacting the eye. 
         [0008]    The non-contact reflection microscope apparatus are generally derived from normal slit lamps with a high magnification microscopes. The technical principle on which they are based is the visualization of a determined structure in relation to its capability of reflecting an incident ray of light used for the illumination. In the commonly used technique (triangulation), the observation angle is of about 45°, the microscope being placed such that the bisector axis of the angle of view is perpendicular to the plane tangent to the corneal surface. 
         [0009]    Non-contact endothelium microscopy is particular indicated in all cases where the contact with the cornea can be dangerous, and therefore immediately after surgery or when there is an extreme structural fragility of the cornea. With the integration of the microscope with techniques of image analysis, the apparatus is able to give also a quantitative description of the endothelium tissue, expressed by the average cellular density and specific morphometric parameters. 
         [0010]    A non-contact endothelium microscope according to the prior art is shown for example in European Patent Application n. EP628281. The optical unit in this apparatus comprising an illuminating system, for obliquely illuminating through a slit an eyeball surface of a subject eye, and an eye-front observation optical system in which alignment-use indicator light for positional adjustment of the imaging optical axis is projected towards the eye and the resulting reflected light is received and imaged by a TV camera. An enlarged-imaging optical system is also provided for enlarged observation or enlarged photographing of the subject part of the TV camera based on slit illuminating light with which the eyeball surface has been illuminated. 
         [0011]    A photo-detector is arranged so as to detect a position at which the enlarged-imaging optical system has been focused on the subject part, via a reflected optical path other than that via which the enlarged image has been formed by the enlarged-imaging optical system. The whole optical unit is automatically moved both in a transversal direction and in a direction toward the eye, in response to the location of the above mentioned indicator light as displayed on a screen of a video monitor, so that the location chases a specified position on the screen. The enlarged visual image of the subject portion of the cornea is thus photographed via the TV camera when the photo-detector detects the focusing. 
         [0012]    The above described system, with the use of a focusing detection photo-detector placed along a supplementary reflected optical path, renders the apparatus sophisticated, and thus costly to be produced and maintain in order to have reliable results. 
       OBJECTS AND SUMMARY OF THE INVENTION 
       [0013]    Accordingly, it is an object of the present invention to provide testing of the endothelium without the use of sensors, photosensors or placement of other devices in a reflected optical path. 
         [0014]    Another object of the present invention is to provide an apparatus that achieves a higher quality endothelium image than that of conventional arrangements while reducing or eliminating the need for electronic components and, thereby, providing greater reliability, completeness and flexibility of use. 
         [0015]    The essential characteristics of the microscope apparatus for the morphometric analysis of the cornea endothelium with direct image acquisition according to the invention are defined by the first of the annexed claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    A specific, illustrative apparatus for morphometric analysis of the cornea endothelium by direct image acquisition, according to the present invention, is described below with reference to the accompanying drawings, in which: 
           [0017]      FIG. 1  shows schematically an optical pathway according to a first embodiment of the present invention; 
           [0018]      FIG. 2  shows schematically an optical pathway according to a second embodiment of the present invention; 
           [0019]      FIG. 3  illustrates schematically a hardware configuration of an apparatus according to one aspect of the present invention; 
           [0020]      FIG. 4  shows a first image displayed on a monitor screen during image acquisition procedures, according to one aspect of the present invention; 
           [0021]      FIG. 5  shows a second image displayed on a monitor screen during image acquisition procedures according to  FIG. 4 ; 
           [0022]      FIG. 6  represents schematically selected reflections obtained using an apparatus according to the present invention; 
           [0023]      FIG. 7  is a flowchart showing a first procedure for image acquisition using an apparatus, according to one aspect of the present invention; 
           [0024]      FIG. 8  is a flowchart showing a second procedure for image acquisition using an apparatus according to the present invention; and 
           [0025]      FIG. 9  is a flowchart showing a third procedure for image acquisition using an apparatus according to the present invention. 
       
    
    
       [0026]    The same numerals are used throughout the drawing figures to designate similar elements. Still other objects and advantages of the present invention will become apparent from the following description of the preferred embodiments. 
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0027]    Referring now to the drawings and, more particularly, to  FIGS. 1-9 , there is shown generally a specific, illustrative apparatus for examination of the corneal endothelium and a method of operating the same, according to various aspects of the present invention. According to one embodiment, the apparatus comprises a movable optical head or microscope  1  having a CCD high speed camera  2 , e.g., a monochrome digital camera with shooting capacity of at least one hundred frames per second with FireWire high speed data output, such as an IEEE 1394 port or equivalent. 
         [0028]    The high speed camera  2  is directly connected to a CPU unit  3 . The unit  3  comprises a controller  4 , e.g. a 65XX type controller produced by the company National Instruments (United States, Texas) or equivalent. The controller  4  controls a power driver board  5 , so that the signal coming from the CPU unit  3  is suitable for driving electric DC motors  6  as described hereinafter. 
         [0029]    The function of the motors  6  is to set in position the microscope  1  with the camera  2 , following to automatic control by the CPU unit  3  so that the eye center  7  to be examined is found. Such a finding is obtained via a reflection into the cornea surface of the light emitted by an infrared LED  8  mounted onto the mobile head of the apparatus, consisting of the microscope  1  with the camera  2 . 
         [0030]    The cited electronic components are connected each other according to a known configurations. Considering instead more in detail the optical scheme of  FIG. 1 , a second LED  9  with associated optics  10  is arranged nearby the infrared LED  8  for providing the fixation point in association with a semireflecting mirror  11  and a semireflecting mirror  12 , necessary to arrange the microscope in a way to center the patient eye and to obtain the triangulation necessary for the test. These components, like the other that follow and that form the optical scheme, are triangulation elements for the endothelium test, known and already in use for this kind of application. 
         [0031]    The optical scheme comprises then a side projection axis  13 , a side reflection axis  14  and a central channel  15 . In the embodiment of  FIG. 1 , transversally to the side projection axis  13 , a halogen lamp  16  is arranged with a lamp condenser  17  and a slit  18 . Along the side projection axis  13  there is also placed a semireflecting mirror  19  receiving the light beam generated by the halogen lamp  16  and the beam that can be generated by a halogen lamp  16  and the beam that can be generated by a photoflash  20  located at the start of the side projection axis  13 . On the same axis, the photoflash  20  is followed by a photoflash condenser  21 , a slit  22  and, beyond the mirror  19 , by a an optical unit  23  that concentrates the beam onto the patient eye  7 . In the embodiment of  FIG. 2  the lamp  16 , the condenser  17 , the slit  18 , the semireflecting mirror  19  and the photoflash  20  are replaced by a stroboscopic lamp  36  activated analogously and with the same function the previous elements. 
         [0032]    Along the side reflection axis  14  there is arranged a side reflection optical unit  24  the reflected beam and the endothelium image to a mirror  25 , from which the beam and the image signal are reflected to the central channel  15  passing through the a filter  26  and a magnifying optical unit  27 . The beam, and the endothelium image conveyed therewith, joins the central channel  15  in a point where a dichroic mirror  28  is arranged. 
         [0033]    The channel  15  also provides for, starting from the examined eye  7 , the above mentioned semireflecting mirror  12  and a central optical unit  29  that concentrates the image of the eye  7  and of the LED  8  to the high speed camera  2 , passing through the dichroic mirror  28 . 
         [0034]    The system is controlled by two pulses  30  and  31  coming from the controller  4 . The first pulse  30  transmits the on/off signal to the LEDs  8  and  9 , to the photoflash  20  and to the halogen lamp  16 . The pulse  31  transmits the signal for the operation of the motors  6 . 
         [0035]    The optical head is driven by the motors along three Cartesian directions where the low-high direction corresponds to a Y-direction, the direction of horizontally approaching to and mowing away from the eye corresponds to a Z-direction, and the transversal sideways direction corresponds to a X-direction. 
         [0036]    With reference also to  FIGS. 4 to 6  and to the self-explanatory flowcharts of  FIGS. 7 to 9 , the microscope according to the invention works in the following way. After arranging the optical head at the desired position, the test starts with the turning on of LED  9  giving the fixation point for the patient. At the same time, the infrared LED  8  is switched on, projecting via the reflecting mirror  12  a spot of light onto the cornea surface. This spot is detected by the camera  2  along the central channel  15 . Camera  2  starts then acquiring images, with a resolution of at least 656×400 pixels, taken continuously with a frequency of about 100 Hz. 
         [0037]    On each acquired frame, data acquisition procedures are carried out for identifying the points (pixels) in which the grey level is inside a certain predetermined range, so as to eliminate the darker and the clearer points of the prefixed range, and to identify all the points that belong to the light spot reflected by the cornea, and thus to precisely outline the same spot. 
         [0038]    Of all the pixels that form the image of the reflected spot the X- and Y-coordinates are calculated, with reference to the upper left angle of the image that coincides with the same position on the sensor of the camera  2  (point Φ in  FIG. 4 ). 
         [0039]    Subsequently, average, variance and standard deviation of the X-, Y-coordinates are calculated so as to define the center of the reflected spot and to identify the interference of possible remote luminous signals that could be mistakenly associated with the spot. 
         [0040]    The driver board  5  is continuously operated to make the luminous spot given by the LED  8  coincide with the center of the sensor of the camera  2 , as a result of the action of the electronic motors  6 . In practice, the apparatus according to the invention makes the center position of the eye  7  coincide with the center of the CCD sensor of the camera and of the video signal processed by the FireWire IEEE 1394 port and the controller  4 , with a feedback control loop to automatically drive the electric motors  6 . 
         [0041]    In greater detail, the CPU unit  3  determines two concentric areas  32  and  33  (see  FIGS. 4 and 5 ). A bigger area  32 , is the area of the image useful to the test, the borders of the image being discarded due to the fact that they are often affected by undesired external reflections. When the center of the above mentioned light spot is outside the area  32 , the continuation of the test is not permitted. The area  2  can be circular, as in the example, or shaped differently (oval, squared etc.) 
         [0042]    The radius of the area  32  may be defined by the medical operator, or established as a design parameter, the center coinciding with the CCD camera sensor center. A smaller area  33  is instead the optimal area for the centering, i.e. the target area to be reached by the center of the spot in order to deem the eye  7  and camera sensor centered with respect to each other. 
         [0043]    After that the center of the reflected spot has been calculated as mentioned, the distance of this from the center of the small area  33  (which can even be a single pixel), and the motors are continuously operated to drive the optical head  1  along the X- and Y-directions until such distance is minimized, that is to say the center of the reflected spot is brought (and kept) inside the area  33 . In practice, the system automation is therefore to calculate the center position of the reflected spot with respect to the area center  33  so as to instruct the motors accordingly. In this way, through the driver board  5  and the motors  6  placed on two X Y-directions, the movement of the optical head is driven with a frequency equal to that with which the frames are taken, i.e. every ten milliseconds. 
         [0044]    When the reflected image (spot) is deemed centered to the sensor (step A in  FIGS. 7 and 8 ), through a suitable TTL signal that activates the driver board  5 , the lamp  16  is switched on. Said lamp  16  illuminates the slit  18  through the lamp condenser  17 . The luminous slit that is formed is projected on the eye along the axis  13  through the mirror  19  and the lens  23 . The optical head is now moved along the Z-direction, until the triangulation takes place, i.e. until the luminous slit, to the geometric conditions that regulate the optical reflection, can be reflected by the corneal surface via the reflection axis  14 . When this reflection occurs, the image of the slit becomes superimposed to the image acquired by the camera  2  coming from the central channel  15 . The same geometric conditions just mentioned are such that the advancement of the optical head along the Z-direction corresponds to a shifting, from the left towards the right (considering the camera sensor as seen in  FIGS. 4 and 5 ) of the image of the slit reflected by the cornea. 
         [0045]    In order to have high quality images of the endothelium, it is important that the images be captured, and also (preferably) the cornea be illuminated by the photoflash  20 , in the time in which the incident beam coming from the side projection axis  13  is in the optimal position to create the necessary reflection on the layer of the endothelium cells. To this purpose, the apparatus according to the invention proceeds in the following manner. 
         [0046]    A check area or band  34  ( FIG. 5 ) is established on the image taken by the CCD camera sensor, in the left part thereof. In the example the check area  34  is a five pixels wide band starting from the left border of the sensor, but it may be less displaced with respect to the center, and be less wide and long according to the circumstances. In the absence of a triangulation, the image in the check band  34  is generally composed by a grey background. 
         [0047]    The area  34  is constantly checked, during the advancement along the −Z direction, with the maximum frequency allowed by the characteristics of the camera (for example around 100 frames per second). With particular reference also to  FIG. 6 , there is represented a beam  14 B reflected by the cornea C, and more precisely by the superficial part thereof, the epithelium Cep. The reflected beam  14 B is captured by the camera as a luminous strip  35  (the above mentioned image of the illuminated slit) moving from left to right. 
         [0048]    When the luminous strip  35  enters the check area  34  the grey level intensity detected therein increases to a bigger value than a predetermined threshold value; this time t o  is fixed like a temporal reference. The grey level intensity detection in the check area is carried out by beverage calculations over all the pixels forming the area. 
         [0049]    From the time t o  a suitable delay At is set to control the acquisition. In fact, considering the advancement speed of the head along the Z-direction and above all the thickness of the cornea, it is only with a certain delay after the image  35  reflected by the epithelium Cep has been detected in the check area  34 , that an image reflected by the endothelium comes to an optimal position for being taken by the camera  2 . This situation is clearly represented in the same  FIG. 6 , where the beam  14 A reflected by the endothelium Cend produces a strip image  37  which is displaced rearward with respect to the image  35  reflected by the epithelium Cep. 
         [0050]    The period of time Δt that passes between t o  (reference) and the time in which the image of the endothelium is taken is then fundamental, and is evaluated on the basis of the advancement speed and the average thickness of the human cornea. The delay time Δt can in any case be adjusted manually or automatically. As the delay time At passes, the photoflash  20  is turned on, illuminating the cornea, and the image of the endothelium is taken through the camera  2 . A number of different images can also be taken, so that the one having the best quality can be chosen. The images are stored in a database for possible further processing or treatment. As the acquisition cycle is closed, the apparatus returns in the start configuration awaiting a new test to be done. 
         [0051]    As mentioned, both the At delay and the position of the check area  34  can be changed so as to give to the medical operator the possibility to obtain better images also in case of corneas with particular morphologies. The photoflash lamp  20 , thanks to its supplementary luminous impulse, permits to lower the gain of the camera  2  and so to have less noisy images. Said photoflash can be activated with a certain advance with respect to the lapse Δt, considering the intrinsical lag of the device. 
         [0052]    The advantageous characteristics of the apparatus according to the invention attain the object stated in the introductory part. The absence of a photosensor or of a linear sensor along an optical reflection path; the acquisition procedure, controlled and realized by means of simple software instructions given to the apparatus as described above, ensures a better reliability, lower costs and a better use flexibility. Furthermore, by the possibility of taking a number of frames, and then choosing the highest quality one. 
         [0053]    The patients, the tests and the captured images are stored in a database, permitting to work on the taken data even after the test. This permits to rely on useful clinical parameters and, subsequently, to process the same so as to define the number and the density of the cells, their shape, their surface, i.e. their maximum, minimum and average area, the deviation from the standard parameters, a variance coefficient, the ratio of cells of various forms, graphics of the distribution, of the dimension of cells areas and graphics of the perimeters distribution. The test can be carried out with a reduced assistance by the medical operator, thanks to the automatic control of the same test as described above. 
         [0054]    Various modifications and alterations may be appreciated based on a review of this disclosure. These changes and additions are intended to be within the scope and spirit of the invention as defined by the following claims.