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 (iii) 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 
   The present invention relates generally to microscopes and, more particularly, to non-contact endothelium microscopes and the like. 
   BACKGROUND OF THE INVENTION 
   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. 
   Endothelium cells in children and young people are typically hexagonal in shape These cells, however, do not reproduce themselves. At birth, the density of endothelium cells is about 4000 per square millimeter but, as the years pass, the cells begin to change in shape, and the total number of cells decreases. In an adult, the average density is about 2700 cells per square millimeter, with a range of about 1600 to about 3200 cells per square millimeter. The loss of endothelium cells with age is accompanied by two main morphological changes: (i) the presence of cells with different surface areas, and (ii) an increase in the number of cells that are shaped differently from their original hexagonal shape. 
   Evaluation of the corneal endothelium has been found useful for providing a first clinical indication as to the potential risks of surgery, and for verifying a diagnosis or the effectiveness of a particular therapy. In these evaluations, it is considered particularly important to observe heterogeneous portions of the endothelium, such as intracellular and intercellular areas of no reflectance (dark spots), hyper reflective areas (bright spots), empty areas in the cells layer (guttae), bubbles, as well as Descemet&#39;s membrane rupture lines. 
   Such portions of the endothelium can be checked relative to the evolution of the various diseases of the endothelium which are of an inflammatory or dystrophic nature. Quantitative evaluation involves the assignment of a numeric parameter to a selected photographic field, which parameter is used to study variations in the endothelium over time, or for comparison between different patients. 
   The most readily accessible parameter is the average cellular density, obtained for comparison purposes by counting the number of cellular elements. A first evaluation method, in this regard, is accomplished by comparing the cellular dimensions with those of the hexagonal reticules that correspond to determined densities. According to a second method, counting of the number of cellular elements is, instead, performed by using fixed or variable reticules. 
   While beneficial, neither method provides information as to the evolution of the cellular dimensions. Such information can be obtained, however, by identifying, in addition to the dimension of the average cellular area and its variability, the perimeters of the cellus as well. This information is obtained through observation using an endothelium reflection microscope, which was first introduced in opthalmologic practice in 1960 by David Maurice who, by modifying a metallography microscope, obtained photographic images of a rabbit&#39;s corneal endothelium. Using the same principles, a microscope was developed subsequently that was able to photograph the endothelium without contacting the eye. 
   Generally speaking, reflection microscopes of the non-contact type are derived from high magnification microscopes with normal slit lamps. These microscopes are based on the principle of visualization of a selected structure in relation to its ability to reflect an incident ray of light used for illumination. In the most commonly used technique (i.e., triangulation), the observation angle is about 45°, the microscope being oriented such that the bisecting axis of the angle of view is perpendicular to the plane tangential to the corneal surface. 
   Non-contact endothelium microscopy is particularly suitable for applications where contact with the cornea can be dangerous, such as immediately after surgery or in cases where the structure of the cornea is extremely fragile. By integrating the microscope with techniques of image analysis, the apparatus also provides a quantitative description of endothelium tissue, in the form of average cellular density and specific morphometric parameters. 
   In one conventional arrangement, a non-contact endothelium microscope apparatus is provided, which includes an optical unit with an illuminating system, for obliquely illuminating through a slit a surface portion of a patient&#39;s eyeball, and frontal eye observation, optical system, wherein an alignment-use indicator light for positional adjustment of the imaging optical axis is projected toward the patient&#39;s 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 surface portion on the TV camera based on slit illuminating light from which the eyeball surface has been illuminated. 
   In addition, a photo-detector is arranged so as to detect a position at which the enlarged-imaging optical system has been focused on the subject surface portion, via a reflected optical path other than that through which the enlarged image has been formed by the enlarged-imaging optical system. The optical unit is automatically moved, in response to the location of the indicator light displayed on a video monitor, both in a transverse direction and toward the eye, so that the location “chases” a specified position on the screen. In this manner, when the photo-detector detects focusing, the enlarged visual image of the subject portion of the cornea is photographed via the TV camera. 
   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. 
   While this system has been found workable, placement of a focusing detection, photo-detector along a supplementary reflected optical path, renders the apparatus complicated, and thus costly for providing and maintaining reliable results. 
   OBJECTS AND SUMMARY OF THE INVENTION 
   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. 
   Another object of the present invention is to provide an apparatus that achieves a higher quality endothelium image than those of conventional arrangements while reducing or eliminating the need for electronic components, thereby providing greater reliability, completeness and flexibility of use. 
   In accordance with one aspect of the present invention, there is provided a method for operating an endothelium reflection microscope apparatus. The apparatus includes an optical head which comprises: an illuminating system, for obliquely illuminating, along a side projection axis through a slit, an eyeball surface of a patient&#39;s eye; an eye-front observation optical system along a central channel in which alignment-use indicator light for positional adjustment of the imaging optical center is projected toward the eye and the resulting reflected light spot is received and imaged by a camera comprising a digital optical sensor; and an enlarged-imaging optical system arranged along a side reflection axis for enlarged observation or photographing of the subject part by the digital camera based on slit illuminating light with which the eyeball surface has been illuminated. The apparatus further comprises a drive for moving the optical head along three Cartesian directions comprising an advancement direction (Z-) generally parallel to the central channel and transverse alignment directions (X-, Y-), and a CPU controller for automatically controlling the drive, the illuminating system, and the eye- front optical system. 
   The method includes an alignment procedure in which the optical head is moved along the alignment directions (X-, Y-) so that the reflected light spot and the camera optical sensor are mutually centered, and an endothelium image acquisition procedure in which the optical head is moved along the advancement direction (Z-). The image acquisition procedure comprises the steps of:
         (i) constantly checking the grey level inside a check area of the camera sensor, during its progression along the advancement direction (Z-), the check area being displaced generally toward a border of the sensor corresponding to the entry side of reflection of the slit light, shifting in response to movement of the optical head in the advancement direction (Z-);   (ii) when the grey level reaches a predetermined threshold value, triggering a delay time (Δt); and   (iii) when the delay time (Δt) lapses, enabling acquisition of one or more images of the endothelium by the digital camera.       

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     A specific, illustrative apparatus for morphometric analysis of the corneal endothelium by direct image acquisition, according to the present invention, is described below with reference to the accompanying drawings, in which: 
       FIG. 1  shows schematically an optical pathway according to a first embodiment of the present invention; 
       FIG. 2  shows schematically an optical pathway according to a second embodiment of the present invention; 
       FIG. 3  illustrates schematically a hardware configuration of an apparatus according to one aspect of the present invention; 
       FIG. 4  shows a first image displayed on a monitor screen during image acquisition procedures, according to one aspect of the present invention; 
       FIG. 5  shows a second image displayed on a monitor screen during image acquisition procedures according to  FIG. 4 ; 
       FIG. 6  represents schematically selected reflections obtained using an apparatus according to the present invention; 
       FIG. 7  is a flowchart showing a first procedure for image acquisition using an apparatus, according to one aspect of the present invention; 
       FIG. 8  is a flowchart showing a second procedure for image acquisition using an apparatus according to the present invention; and 
       FIG. 9  is a flowchart showing a third procedure for image acquisition using an apparatus according to the present invention. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
   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. 
   High speed camera  2  is connected directly to a central processing unit (CPU)  3 . The CPU includes a controller  4 , e.g., a 65XX type controller produced by National Instruments Corporation (Austin, Tex., U.S.A.) or equivalent. Controller  4  operates a power driver board  5 , such that the signal coming from the CPU is sufficient to power electric DC motors  6 , as described in more detail below. 
   One purpose of the motors is to position microscope  1  and the associated camera  2 , upon their automatic control by CPU  3 , so that center portion  7  of the eye to be examined may readily be found. This is accomplished by reflecting light from an infrared, light emitting diode (LED)  8  into the corneal surface, the LED being mounted to the mobile head of the apparatus, which comprises optical head or microscope  1  and camera  2 . 
   The aforementioned electronic components are preferably connected one another according to known arrangement. Alternatively, as shown in  FIG. 1 , an optical scheme may be used wherein a second LED  9  with associated optics  10  is arranged in proximity to infrared LED  8  in order to provide a fixation point in association with a semireflecting mirror  11  and a semireflecting mirror  12 , as necessary, to center the patient&#39;s eye relative to the microscope and obtain the triangulation necessary to conduct the test. These components, like those forming the optical scheme, are triangulation elements for the endothelium test, as are known and already in use for such applications. 
   In accordance with one aspect of the present invention, the optical scheme comprises a side projection axis  13 , a side reflection axis  14  and a central channel  15 . In the embodiment of  FIG. 1 , a halogen lamp  16  is arranged, transversely to side projection axis  13 , with a lamp condenser  17  and a slit  18 . Along the side projection axis, a semireflecting mirror  19  is also positioned for receiving the light beam generated by the halogen lamp, a beam that can be generated by a halogen lamp, and the beam of light that can be generated by a photoflash or photoflash lamp  20  located at the beginning of side projection axis  13 . On the same axis, following the photoflash lamp  20  is a photoflash condenser  21 , a slit  22  and, beyond semireflecting mirror  19 , an optical unit  23  that concentrates the beam at center portion  7  of the patient&#39;s eye. In the arrangement illustrated in  FIG. 2 , lamp  16 , condenser  17 , slit  18 , semireflecting mirror  19 , and photoflash lamp  20  are replaced with a stroboscopic lamp  36  having the same function as, and activated analogously to, to the previous optical scheme. 
   A side reflection optical unit  24 , arranged along side reflection axis  14 , concentrates the reflected beam and the endothelium image on a mirror  25 , from which the beam and image signal are reflected to central channel  15  passing through a filter  26  and a magnifying optical unit  27 . The beam, and the endothelium image conveyed thereby, joins the central channel at a point where a dichroic mirror  28  is located. 
   Starting from the eye to be examined, channel  15  accommodates, in addition, semireflecting mirror  12  and a central optical unit  29  that concentrates the image of the eye and of LED  8  on high speed camera  2 , passing through dichroic mirror  28 . 
   The system is preferably controlled by pulses  30 ,  31  from controller  4 . First pulse  30  transmits an on/off signal to LEDs  8  and  9 , to the photoflash lamp, and to the halogen lamp, whereas second pulse  31  transmits a signal for operating motors  6 . 
   The optical head or microscope is driven by the motors along three Cartesian directions where a low-high direction corresponds with a Y-axis direction, motion in a direction horizontally approaching and moving away from the patient&#39;s eye corresponds to a Z-axis direction, and movement in a transverse sideways direction corresponds to an X-axis direction. 
   Turning now to  FIGS. 4-9 , the microscope, according to another aspect of the present invention, operates as follows. Initially, after arranging the optical head at a desired position, the test commences with turning on LED  9 , the LED establishing a fixation point for the patient&#39;s eye. At the same time, infrared LED  8  is switched on, thereby projecting a spot of light onto the corneal surface via reflecting mirror  12 . This spot is detected by camera  2  along central channel  15 . Camera  2  then begins to acquire images, with a resolution of at least around 656× around 400 pixels, taken continuously at a frequency of about 100 Hz. 
   Desirably, data acquisition procedures are carried out with each acquired frame to identify points (pixels) where the grey level is inside a selected predetermined range, so as to eliminate the darker and clearer points of the predetermined range, to identify all the points that belong to the light spot reflected by the cornea, and thus to precisely outline the same spot. 
   Of all the pixels that form the image of the reflected spot, the X and Y coordinates are calculated, with reference to an upper left angle of the image that coincides with the same position on the camera sensor (See point ø in  FIG. 4 ). 
   Subsequently, average, variance and standard deviation of the X, Y coordinates are computed so as to define the center of the reflected spot, and to identify the interference of possible remote luminous signals that could be associated mistakenly with the spot. 
   Driver board  5  is operated continuously so that, through action of electronic motors  6 , the luminous spot created by LED  8  follows and coincides with the center of the camera sensor. In practice, the apparatus, according to the present invention, causes the center position  7  of the eye to coincide with the center of the CCD camera sensor and of the video signal processed by the FireWire IEEE 1394 port and the controller, with a feedback control loop for automatic operation of the electric motors. 
   More specifically, as illustrated generally in  FIGS. 4 and 5 , CPU  3  defines two concentric areas, namely, a bigger area  32  and a smaller area  33 . The bigger area, simply stated, is the area of the image that is deemed useful for testing purposes, the borders of the image being discarded because they are often affected by undesirable external reflections. When the center of the light spot is outside bigger area  32 , further testing is not permitted. Area  32  can be circular in shape, as in the example disclosed, or have a different shape (i.e., be oval, square, etc.) 
   The radius of area  32  may either be defined by the person operating the apparatus, or established as a design parameter, the center of the area coinciding with the center of the CCD camera sensor. Smaller area  33 , on the other hand, is the optimal area for centering, i.e., the target area to be reached by the center of the light spot such that the eye and the camera sensor are centered relative to one another. 
   In this manner, the center of the reflected spot is calculated, namely, the distance between the spot and the center of small area  33  (which can even be as small as a single pixel). The motors are then operated continuously to drive optical head or microscope  1  along the X and Y directions until such distance is minimized, i.e., until the center of the reflected spot is brought (and kept) within area  33 . In practice, the system automatically calculates the center location of the reflected spot relative to the center of area  33  so as to command the motors, accordingly. Through suitable arrangement of driver board  5  and motors  6  in two X-Y directions, movement of the optical head occurs at a frequency equal generally to that with which the frames are taken, i.e., approximately every ten milliseconds. 
   When the reflected spot (image) is deemed centered at the sensor (See step A in  FIGS. 7 and 8 ), lamp  16  is switched on through a suitable TTL signal that activates the driver board. The lamp illuminates slit  18  through lamp condenser  17 , the resulting slit of light projecting on the eye along axis  13  through mirror  19  and lens  23 . The optical head is then moved along the Z-axis direction, until triangulation takes place, i.e., until the slit of light, through the geometric conditions that regulate the optical reflection, can be reflected by the corneal surface via reflection axis  14 . When reflection occurs, the image projected by the slit is superimposed on the image acquired by camera  2  coming from central channel  15 . The aforementioned geometric conditions are such that advancement of the optical head in the Z-axis direction corresponds to a shifting, from left to right (See camera sensor in  FIGS. 4 and 5 ) of the image of the slit reflected by the corneal surface. 
   To achieve high quality images of the endothelium, it is considered important that the images be captured, and preferably that the cornea be illuminated by photoflash lamp  20 , for the duration of time that the incident beam coming from side projection axis  13  is in the optimal position to create the necessary reflection on the layer of endothelium cells. Accordingly, the apparatus, according to one aspect of the present invention, operates as follows. 
   First, as set forth in  FIG. 5 , a check area or band  34  is established in a left hand side portion of the image taken by the CCD camera sensor. In the example shown, the check area is a band five pixels wide starting from the left hand border of the sensor, but may be displaced less relative to the center, and be smaller in width and length, depending on the circumstances. Absent triangulation, the image in check area or band  34  is generally comprised of a low intensity, grey background with a low intensity value. 
   Check area  34  is checked constantly, during advancement of the optical head in the direction of the Z-axis, against the maximum frequency permitted to be used with the camera (for instance, around 100 frames per second). As best seen in  FIG. 6 , a beam  14 B is reflected by cornea C and, more particularly, by a surface thereof, i.e., the epithelium Cep. Reflected beam  14 B is captured by the camera as a luminous strip  35  (i.e., the aforementioned image produced upon illumination of the slit) moving from left to right. 
   When luminous strip  35  enters the check area, the grey level intensity detected increases to a value greater than a predetermined threshold value; and the corresponding time t 0  is fixed or set as a temporal reference. The grey level intensity detected in the check area is accomplished by calculating the average intensity over all the pixels forming the area. 
   From threshold or reference time t 0  a suitable delay time Δt is selected to control the acquisition of image data. Indeed, given the velocity of the optical head along the Z-axis and, moreover, the thickness of the cornea, it is only with the selected delay, after image  35  reflected by epithelium Cep has been detected in the check area, that an image reflected by the endothelium arrives at an optimal position for image capture by camera  2 . An arrangement of this general description is also shown in  FIG. 6 , namely, where beam  14 A reflected by endothelium Cend produces a strip image  37  displaced rearwardly relative to image  35 , as reflected by epithelium Cep. 
   Generally, the length of time Δt between reference t 0  and the time when the image of the endothelium is captured is fundamental, and is evaluated based on the advancement speed and the average thickness of the cornea. The delay time Δt can, in any case, be adjusted either manually or automatically. Once Δt has been reached, photoflash lamp  20  is turned on, thereby illuminating the cornea, and the image of the endothelium is captured by camera  2 . A number of different images can be taken, in addition, so that the one of best quality can be chosen. The images are then stored in a database for further processing or treatment. After the data acquisition cycle has ended, the apparatus returns to its starting position and awaits the next test to be performed. 
   Optionally, both the time delay, Δt, and position of check area  34  can be varied so as to give to the medical operator the ability to obtain better images, particularly in the case of corneas with specific morphologies. The photoflash lamp, with its supplementary luminous impulse, allows the user to lower the gain of the camera for less “noise” in the images. The photoflash lamp may be actutated upon a selected advance of the optical head relative to the time lapse of Δt, taking into consideration the lag intrinsic to the device. 
   Overall, the apparatus, according to the present invention, advantageously provides testing of the endothelium without the use of sensors, photosensors or placement of other devices in a reflected optical path. It also achieves an endothelium image of much higher quality than those of conventional arrangements, while reducing or eliminating the need for electronic components, thereby providing greater reliability, completeness and flexibility of use. The absence of a photosensor or linear sensor along an optical reflection path, and the use of an acquisition procedure, controlled and realized through simple software-based instructions given to the apparatus, results in higher reliability, lower costs and greater flexibility. Furthermore, by allowing the user to capture a number of frames, and then choose the one of highest quality, increases the quality of endothelium images even more, as compared to known arrangements and conventional focusing techniques. 
   The patients, test data, and captured images are advantageously stored in a database, permitting the medical operator or user to use, review and/or otherwise work on the data collected, even after testing has concluded. In this manner, useful clinical parameters may be readily relied upon and, subsequently, processed so as to determine the number and density of the cells, their shape, their surface, i.e., minimum, maximum and average surface area, their deviation from standard parameters, a variance coefficient, the ratio of cells of various forms, as well as show graphically their distribution, the dimension of cell areas, and their perimeters&#39; distribution. Moreover, because of the automatic control provided, testing can now be performed with significantly less assistance from the user. 
   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.