referring now to the drawings and , more particularly , to fig1 - 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 fig1 , 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 fig1 , 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 fig2 , 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 fig4 - 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 fig4 ). 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 fig4 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 fig7 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 fig4 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 fig5 , 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 fig6 , 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 fig6 , 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 .
an apparatus and a method for operating an endothelium reflection microscope . the apparatus includes an optical head , which comprises : an illuminating system , 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 is triggered ; and when the delay time lapses , acquisition by the digital camera of one or more images of the endothelium is enabled .