Method and apparatus for image plane modulation pattern recognition

The detection of optical stripes on an image of a glass slide of material of interest. An automated microscope system with an illuminator having a spatial intensity homogenizer provides spatially uniform illumination of a microscope slide specimen. Periodic optical patterns formed from the illumination system are detected by a computer system. The computer forms a high transmission mask of an image of the biological specimen. Edges in the image are detected and a low transmission area process is performed. Horizontal stripes are enhanced. A final thresholding generates a binary image of the patterns. Stripes in the patterns are detected an a stripe flag and strip area are calculated.

The present invention relates to a method and apparatus for the generation 
and detection of liner modulation patterns in the image plane of 
coverslipped microscope specimens. The detection of these patterns 
indicates the presence of flawed or contaminated coverslips. More 
specifically, the invention relates to the generation of periodically 
structured light in the aperture space of an illumination system for an 
automated cytology instrument. This periodically structured light in 
combination with artifacts on the coverslip of the specimen produces 
modulation patterns in the image plane of the specimen. The method and 
apparatus of the invention detects these modulation patterns related to 
flawed or contaminated coverslips. 
BACKGROUND OF THE INVENTION 
Automated analysis of biological specimens requires a high degree of 
spatial and temporal uniformity for accurate and repeatable evaluation. 
Those evaluations often measure photometric properties such as nuclear and 
cytoplasm density. In order to accurately and repeatably measure these 
properties, the illumination must maintain a high degree of uniformity 
across the field of view and from collected image to collected image. In 
addition, morphological operations are conducted to segment various 
objects in the field of view for further analysis. This analysis 
determines various feature values related to size, shape and frequency 
content etc. Operations used in such analyses tend to exhibit nonlinear 
behavior due to various thresholding schemes that may be employed. 
Therefore, accurate and repeatable behavior of these processing methods 
also necessitates a high degree of uniformity across the field of view and 
from collected image to collected image. Further, these objects tend to be 
three dimensional in nature and therefore can be affected by shadowing, 
dependent on the angular characteristics of the illumination. Still 
further, the images of those objects can be corrupted by dust and 
fingerprints and the like located on the cover slips of those specimens. 
Automated cytology systems, have another problem caused by substrate 
thickness variation. In practice, the substrate, or microscope slide 
thickness' can vary by as much as 0.9 mm. As the microscope slide is 
introduced into the optical path, it becomes part of the optical apparatus 
for illuminating the specimen. A variation in thickness of this element 
may have an effect on the axial position of the optical stops of the 
system, in particular, the field stop or detector plane. When the slide 
thickness varies, the location of the detector, as imaged in the 
illumination train, tends to move along the optic axis. It often happens, 
that this movement of the detector plane falls onto one or more element 
surfaces in the illumination train. This causes dust or defects in those 
surfaces to be imaged onto the detector, thereby, creating artifacts in 
the image plane and degrading the image of the specimen. 
A final problem which has received little or no mention in the prior art is 
the detection of dust, debris, fingerprints and other contaminants of the 
surface of a coverslip. Even with perfect illumination, artifacts, such as 
those on the coverslips of specimens, can compromise the integrity of 
information contained in the image plane of those specimens. This is due 
to an alteration in the optical wave-front generated by the light 
interaction of the illumination system and the object after that 
wave-front leaves the specimen. If the wave-front is obscured, or 
aberrated, in any way, on it's path to the detector in the image plane, 
the integrity of the information contained in the wave-front, regarding 
the characteristics of the object, may be compromised. The illumination 
system can play a role in the manner in which contaminants on the 
coverslip manifest themselves in the image plane. 
Therefore, in consideration of the problems associated with illumination in 
the automated analysis of pap smears, it is a motive of this invention to 
provide illumination in such a way as to allow the real time detection of 
contaminants on the coverslips of pap smears. 
SUMMARY OF THE INVENTION 
The invention provides a method and apparatus to detect dark bands or 
"stripes" that appear on the image because of dust, dirt or other sources 
of occlusion that fall on or are found on the slide being illuminated. An 
automated microscope system with an illuminator having a spatial intensity 
homogenizer provides spatially uniform illumination of a microscope slide 
specimen. Periodic optical patterns formed from the illumination system 
are detected by a computer system. The computer forms a high transmission 
mask of an image of the biological specimen. Edges in the image are 
detected and a low transmission area process is performed. Horizontal 
stripes are enhanced. A final thresholding generates a binary image of the 
patterns. Stripes in the patterns are detected and a stripe flag and strip 
area are calculated. Since these patterns alter the true information 
content of the image, the stripe detects the horizontal modulation 
patterns. If the degree of the modulations is beyond a predetermined 
level, the slide is considered unsuitable for processing. 
The stripe is best revealed in the image background with high 
light-transmission. This processing step isolates the area of the image 
having high light-transmission, higher than 210 in pixel value. It applies 
a global threshold of 210 to the original image and creates a binary image 
mask of the area having pixel values greater than 210. The high 
transmission image mask is then shrunk by two morphological erosion 
operations. The structuring elements include a square, 5 by 5 pixels in 
size, and a diamond shaped kernel, a center pixel and its four adjacent 
pixels in horizontal and vertical directions. The resulting high 
transmission mask is designated as Ihigh.sub.-- trans. 
An edge detection process is applied to the original image to detect the 
edge gradient of the image. It enhances the high frequency information 
content of the horizontal modulation patterns. First, the invention 
applies a grayscale morphological dilation to the original image. The 
structuring element used is a vertical line that is 19 pixels long. Then, 
the original image is subtracted from the dilated image. The difference 
image is the edge detection result Iedge. 
To focus on only the edge information contained in the high transmission 
area of the image, the edge image, Iedge, is combined with the high 
transmission image mask Ihigh.sub.-- trans using an and operation. The and 
operation preserves the Iedgevalue of the pixels included in the 
Ihigh.sub.-- trans mask and assigns zero to the pixels that are not in the 
Ihigh.sub.-- trans mask. This results in a high transmission edge image, 
Ihigh.sub.-- edge. 
Other objects, features and advantages of the present invention will become 
apparent to those skilled in the art through the description of the 
preferred embodiment, claims and drawings herein wherein like numerals 
refer to like elements.

DETAILED DESCRIPTION OF TEE PREFERRED EMBODIMENT 
In a presently preferred embodiment of the invention, the system disclosed 
herein is used in a system for analyzing cervical pap smears, such as that 
shown and disclosed in U.S. patent application Ser. No. 08/571,686, filed 
Dec. 13, 1995, which is a file wrapper continuation of abandoned U.S. 
patent application Ser. No. 07/838,064, entitled "Method For Identifying 
Normal Biomedical Specimens", by Alan C. Nelson et al., filed Feb. 18, 
1992; U.S. Pat. No. 5,528,703, which is a continuation in part of 
abandoned U.S. patent application Ser. No. 07/838,395, entitled "Method 
For Identifying Objects Using Data Processing Techniques", by S. James 
Lee, filed Feb. 18, 1992; U.S. patent application Ser. No. 07/838,070, now 
U.S. Pat. No. 5,315,700, entitled "Method And Apparatus For Rapidly 
Processing Data Sequences", by Richard S. Johnston et al., filed Feb. 18, 
1992; U.S. patent application Ser. No. 07/838,065, now U.S. Pat. No. 
5,361,140, entitled "Method and Apparatus for Dynamic Correction of 
Microscopic Image Signals" by Jon W. Hayenga et al.; and allowed U.S. 
patent application Ser. No. 08/302,355, filed Sep. 7, 1994 entitled 
"Method and Apparatus for Rapid Capture of Focused Microscopic Images" to 
Hayenga et al., which is a continuation-in-part of application Ser. No. 
07/838,063 filed on Feb. 18, 1992 the disclosures of which are 
incorporated herein, in their entirety, by the foregoing references 
thereto. 
The present invention is also related to biological and cytological systems 
as described in the following patent applications which are assigned to 
the saute assignee as the present invention, filed on Sep. 20, 1994 unless 
otherwise noted, and which are all hereby incorporated by reference 
including pending U.S. patent application Ser. No. 08/309,118, to Kuan et 
al. entitled, "Field Prioritization Apparatus and Method," pending U.S. 
patent application Ser. No. 08/309,061, to Wilhelm et al., entitled 
"Apparatus for Automated Identification of Cell Groupings on a Biological 
Specimen," pending U.S. patent application Ser. No. 08/309,116 Meyer et 
al. entitled "Apparatus for Automated Identification of Thick Cell 
Groupings on a Biological Specimen," U.S. patent application Ser. No. 
08/667,292, filed Jun. 20, 1996, which is a file wrapper continuation of 
abandoned U.S. patent application Ser. No. 08/309,115 entitled "Biological 
Analysis System Self Calibration Apparatus," U.S. patent application Ser. 
No. 08/678,124 filed Jul. 11, 1996, which is a file wrapper continuation 
of abandoned U.S. patent application Ser. No. 08/308,992, to Lee et al. 
entitled "Apparatus for Identification and Integration of Multiple Cell 
Patterns," pending U.S. patent application Ser. No. 08/309,063, for which 
the issue fee has been paid, to Lee et al. entitled "Method for 
Cytological System Dynamic Normalization," pending U.S. patent application 
Ser. No. 08/309,248, for which the issue fee has been paid, to Rosenlof et 
al. entitled "Method and Apparatus for Detecting a Microscope Slide 
Coverslip," U.S. patent application Ser. No. 08/309,077 now U.S. Pat. No. 
5,566,249 to Rosenlof et al. entitled "Apparatus for Detecting Bubbles in 
Coverslip Adhesive," pending U.S. patent application Ser. No. 08/309,931, 
to Lee et al. entitled "Cytological Slide Scoring Apparatus," pending U.S. 
patent application Ser. No. 08/309,250 to Lee et al. entitled "Apparatus 
for the Identification of Free-Lying Cells," pending U.S. patent 
application Ser. No. 08/309,209 to Oh et al. entitled "A Method and 
Apparatus for Robust Biological Specimen Classification," pending U.S. 
patent application Ser. No. 08/309,117, to Wilhelm et al. entitled "Method 
and Apparatus for Detection of Unsuitable Conditions for Automated 
Cytology Scoring." 
It is to be understood that the various processes described herein may be 
implemented in software suitable for running on a digital processor. The 
software may be embedded, for example, in the central processor 540. 
Refer now to FIGS. 7A, 7B and 7C which show a schematic diagram of an 
automated cytological analysis system employing the apparatus of the 
invention. The cytological system comprises an imaging system 502, a 
motion control system 504, an image processing system 536, a central 
processing system 540, and a workstation 542. The imaging system 502 is 
comprised of the uniform illuminator 10 of the invention, imaging optics 
510, a CCD camera 512, an illumination sensor 514 and an image capture and 
focus system 515. The image capture and focus system 516 provides video 
timing data to the CCD cameras 512, the CCD cameras 512 provide images 
comprising scan lines to the image capture and focus system 516. An 
illumination sensor intensity is provided to the image capture and focus 
system 516 where an illumination sensor 514 receives the sample of the 
image from the optics 510. In one embodiment of the invention, the optics 
may further comprise an automated microscope. The illuminator 508 provides 
illumination of a slide. The image capture and focus system 516 provides 
data to a VME bus 538. The VME bus distributes the data to an image 
processing system 536. The image processing system 536 is comprised of 
field-of-view processors 568. The images are sent along the image bus 564 
from the image capture and focus system 516. A central processor 540 
controls the operation of the invention through the VME bus 538. In one 
embodiment the central processor 562 comprises a Motorola 68030 CPU. The 
motion controller 504 is comprised of a tray handler 518, a microscope 
stage controller 520, a microscope tray controller 522, and a calibration 
slide 524. The motor drivers 526 position the slide under the optics. A 
bar code reader 528 reads a barcode located on the slide 524. A touch 
sensor 530 determines whether a slide is under the microscope objectives, 
and a door interlock 532 prevents operation in case the doors are open. 
Motion controller 534 controls the motor drivers 526 in response to the 
central processor 540. An Ethernet communication system 560 communicates 
to a workstation 542 to provide control of the system. A hard disk 544 is 
controlled by workstation 550. In one embodiment, workstation 550 may 
comprise a Sun Spark Classic (TM) workstation. A tape drive 546 is 
connected to the workstation 550 as well as a modem 548, a monitor 552, a 
keyboard 554, and a mouse pointing device 556. A printer 558 is connected 
to the ethernet communication system 560. 
During the time when the field of view processors 568 are obtaining image 
data the image capture and focus system 516 is adjusting the pixel values 
by an amount proportionate to the output of the strobe intensity sensor 
91. The invention provides a dynamic adjustment of the output of the CCD 
arrays to compensate for variations in slide illumination. In an alternate 
embodiment of the invention a running average of strobe intensities is 
computed and the average value is used to compensate for variations in 
slide illumination. A running average of 300 samples of the illumination 
intensity may be used, the 300 samples may be stored in the memory of the 
FOV processors. 
The central computer 540, running a real time operating system, controls 
the microscope and the processor to acquire and digitize images from the 
microscope. The flatness of the slide may be checked, for example, by 
contacting the four corners of the slide using a computer controlled touch 
sensor. The computer 540 also controls the microscope stage to position 
the specimen under the microscope objective, and from one to 15 field of 
view (FOV) processors 568 which receive images under control of the 
computer 540. 
During slide processing, the computer 540 detects horizontal modulation 
patterns exhibited in 20.times. FOVS. The modulation patterns result from 
dirty or scratched coverslips and appear most often on plastic coverslips. 
Oily fingerprints on the surface of a coverslip can also generate the 
patterns. The patterns have appeared predominantly in the horizontal axis 
correlating with the direction of the scratches observed on the coverslip. 
If a slide with horizontal scratches is rotated 90 degrees in the object 
plane, the patterns move with the rotation and appear in the vertical 
direction. 
Refer now to FIG. 8 which shows one embodiment of the invention to process 
stripes. The spatial frequency of the patterns in most cases is higher 
than the spatial frequency of the scratches appearing on the coverslip. 
This also generates the light and dark bands of periodicity in the 
aperture space of the condenser. The most dramatic patterns studied to 
date exhibit a modulation of .+-.5 counts on a 200 count average 
background. Most patterns observed are closer to a .+-.2.5 count 
modulation. 
Since these patterns alter the true information content of the image, it is 
likely that the performance of the a slide classification system would be 
affected. The method and apparatus of the invention is designed to detect 
the horizontal modulation patterns. If the degree of the modulations is 
beyond a predetermined level, the slide should be considered unsuitable 
for processing. 
The computer system 540 obtains a 20.times. image before trying to 
recognize the existence of stripes. The invention generates a stripe flag 
706 and a stripe area count 708. The stripe flag 706 is "true" when a 
significant amount of horizontal stripe, greater than or equal to 1,000 
pixels, are detected in the FOV; otherwise, the stripe flag is set to 
"false." 
The stripe area 708 specifies the area of the detected stripe in the FOV. 
When the stripe flag 706 is "true," the stripe area is the number of 
detected stripe pixels; when the stripe flag 706 is "false," stripe area 
is set to zero. The stripe flag 706 and the stripe area available to 
determine the next level of processing for data from the 20.times. FOV. 
The invention compensates for "stripes" that appear in the image if dust or 
dirt fall on the slide. A stripe pattern will appear on the 20.times. 
image 702 and will result in a modulation of the image in a stripe 
pattern. A stripe flag 706 and a stripe area count 708 are shown in FIG. 
8. The stripe flag 706 is "true" when a significant amount of horizontal 
strip, in one embodiment pixels greater than or equal to 1,000, is 
detected in the FOV 702, otherwise, the stripe flag 706 is set to "false". 
The stripe area 708 specifies the area of the detected strip in the FOV 
702. When the stripe flag 706 is "true", the stripe area 708 is the number 
of detected stripe pixels; when the stripe flag 706 is "false", the stripe 
area 708 is set to zero. The stripe flag 706 and the stripe area 768 are 
used during FOV integration 702. 
Refer now to FIG. 9 which shows the stripe detection method of the 
invention comprising six processing steps. The stripe is best revealed in 
the image 712 background with high light-transmission. This processing 
step 714 isolates the area of the image 712 having high 
light-transmission, in one embodiment higher than 210 in pixel value. It 
applies a global threshold of 210 to the original image and creates a 
binary image mask of the area having pixel values greater than 210. The 
high transmission image mask is then shrunk by two morphological erosion 
operations. The structuring elements include a square, 5 by 5 pixels in 
size, and a diamond shaped kernel, a center pixel and its four adjacent 
pixels in horizontal and vertical directions. The resulting high 
transmission mask is designated as I.sub.high.sbsb.--.sub.trans 717. 
Edge detection 716 is applied to the original image 712 to detect the edge 
gradient of the image. It enhances the high frequency information content 
of the horizontal modulation patterns. First, a grayscale morphological 
dilation is performed on the original image; the structuring element used 
is a vertical line that is 19 pixels long. Then, the original image 712 is 
subtracted from the dilated image. The difference image is the edge 
detection result I.sub.edge 719. 
The low transmission area rejection step 718 focuses on only the edge 
information contained in the high transmission area of the image 712, the 
edge image, I.sub.edge 719, is combined with the high transmission image 
mask I.sub.high-trans 717 using an AND operation. The AND operation 
preserves the I.sub.edge value of the pixels included in the 
I.sub.high.sbsb.--.sub.trans mask and assigns zero to the pixels that are 
not in the I.sub.high.sbsb.--.sub.trans mask. This results in a high 
transmission edge image, I.sub.high.sbsb.--.sub.edge 721. In the 
horizontal stripe enhancement step 720, the high transmission edge image, 
I.sub.high.sbsb.--.sub.edge 721, comprises both horizontal and vertical 
edges. To remove the vertical edges and maintain only the long horizontal 
edges, a grayscale morphological opening operation is applied to remove 
the vertical and short horizontal edges. The structuring element used is a 
horizontal line of 41 pixels and the horizontal stripe enhanced image is 
called I.sub.hse 725. 
In the final threshold step 722, the horizontal stripe enhanced image 725 
is thresholded by a value of 2. The pixels having the horizontal stripe 
enhanced image value greater than 2 are assigned a value "255" and the 
remaining pixels are assigned "0". The resulting binary image, 
I.sub.binary 727 corresponds to the detected stripe areas. 
In the stripe detection and measurement step 724, the number of the pixels 
included in the detected stripe image mask is determined by a histogram 
operation. If the total number of the stripe pixels is greater than or 
equal to 1,000, the stripe flag 706 is set to "true." Otherwise, the 
stripe flag 706 is set to "false." The number of stripe pixels measured by 
the histogram operation is stored in the output variable, stripe area 708, 
when the stripe flag 706 is "true." It is set to zero when the stripe flag 
706 is "false". 
Those skilled in the art will recognize that other period optical 
disturbances may be detected by the method of the invention, such as dirt 
and dust on other optical elements in the optical path. In general the 
invention is able to detect the existence of any similar periodic pattern 
what ever its original cause. 
Now refer to FIG. 1 which shows a schematic representation of the one 
embodiment of the invention's illumination. The illumination device of the 
invention comprises a light 10, an optical conditioning system 12, a light 
pipe 40, a mechanical slide with elements 54 and 56 positioned in place to 
intercept the light leaving the light pipe 40, and condenser optics 18. 
This configuration of the device provides for illumination suitable for 
4.times. magnification of biological specimens on a microscope slide 20. 
The optical conditioning system 12 includes, a collimator lens 24, an 
aperture stop 26, a bandpass filter 28, a condenser lens 30, a turning 
mirror 32 and a neutral density filter 34. The optical elements of the 
optical conditioning system 12, except for the neutral density wedge 34 
are positioned along optical axis 36. 
Light 10 comprises a light source 22 and flash power unit 17. The light 
source 22 is positioned to provide illumination to the collimator lens 24. 
In one preferred embodiment, high intensity arc lamp 23 serves as the 
light source 22. Typically the arc in an arc lamp 23 is not stable and 
tends to move from flash to flash causing illumination variation. 
The optical conditioning system 12 includes a collimator lens 24 of focal 
length 29.5 mm, an aperture stop 26, a bandpass filter 28, another lens 30 
with focal length of 100 mm, a turning mirror 32 and a neutral density 
filter 34. The arc lamp 23 is positioned to provide illumination to the 
collimator lens 24. The collimator serves to gather light from the lamp 23 
and concentrate it into beam 25. This beam is directed towards and 
overfills an aperture stop 26. The collimator lens 24 is positioned along 
axis 36 between the light source 22 and the aperture stop 26. The 
collimator lens 24 directs the light beam 25 through the aperture stop 26. 
The aperture stop 26 is chosen so that the light beam 25 will overfill the 
aperture stop 26. 
The beam then passes through a spectral bandpass filter 28 with a passband 
of 10 nm centered on 570 nm. These filter characteristics are chosen to 
provide the maximum contrast between the nucleus and cytoplasm for 
specimens stained with the Papanicolaou stain. Other filters may be used 
depending on specimen characteristics. In one embodiment of the invention 
the spectral bandpass filter comprises a neutral density filter (NDF). 
Since the transmission varies across the NDF 34, placing the NDF 34 after 
the homogenizer 40 would provide either arclets 21 across the aperture 
that vary in intensity or an illumination field that varies in intensity 
at the slide 20. Likewise, placing the NDF 34 before the input aperture 
will result in an illumination whose intensity is dependent on angle. The 
homogenizer conserves the angular distribution of the light so it will not 
have a corrective effect on the angular distribution of the light. Placing 
the NDF 34 after the homogenizer may either create spatial or angular 
intensity variations or combinations of both. 
The neutral density filter 34 may be positioned orthogonal to the light 
beam 25. The neutral density filter 34 has a control input and regulates 
the transmission of light beam 25 passed through the neutral density 
filter 34 according to a control signal 43, providing an attenuated light 
beam 35. The control signal 43 may be provided during calibration of the 
light 10. The neutral density filter 34 in one embodiment is comprised of 
a disk having a clear area in a pie shaped section. The remainder of the 
disk varies linearly in density in a radial direction from 0.0 optical 
density to 3.0 optical density. Optical density is related to transmission 
in the following way where T is transmission and OD is optical density. 
EQU OD=log (1/T) 
The filter is attached to a drive motor 33 for the purpose of changing its 
rotation to pass either more or less light depending on the needs of the 
system. The neutral density filter 34, NDF 34, is located in the proximity 
(2 mm) of the input aperture 42 because the transmission across the NDF 34 
varies radially around the NDF 34. The light pipe 40 homogenizes this 
variation. 
The light beam 25 provided by an arc lamp may vary due to aging, variation 
in the amount of energy, and other causes. When light beam 25 provides an 
excess of light intensity, the neutral density filter 34 may be rotated 
such that a less transmissive optical region may be used. When the light 
beam 25 provides a lesser amount of light intensity, a more optically 
transmissive region may be used. In one preferred embodiment of the 
invention, the neutral density filter 34 provides variable transmittance 
that changes at a continuous rate. 
The beam then passes through the lens element 30 which creates an image of 
the arc lamp 35 at a 3.times. magnification at a distance of 100 mm from 
the lens element 30. This image is conjugate with the input aperture 42 of 
the light pipe However, before reaching the light pipe 40, the beam passes 
through a neutral density filter and is deviated 90 degrees by a turning 
mirror 32. Those skilled in the art will appreciate the mirror 32 provides 
for a designed footprint and that other configurations are possible 
without deviating from the scope of the invention. The aperture stop 26 in 
conjunction with the lens elements 24 and 30 limit the input numeric 
aperture into the light pipe 40 to NA 0.13. Lenses 24 and 30 in 
combination provide a 3:1 magnification of the arc 23 at the input of the 
light pipe 40. The magnified image 35 of the arc is roughly 1.8 mm at the 
input aperture to the light pipe 40. The input aperture 42 is square with 
a 2.6 mm side dimension. These dimensions were chosen to underfill the 
input aperture to the light pipe 40 to allow for lateral movement of the 
arc without causing obscuration leading to light loss. Those skilled in 
the art will appreciate other ratios of underfill can be used without 
deviating from the scope of the invention. 
Light pipe 40 has an input aperture 42 and an output aperture 44. Light 
entering the light pipe 40 at the input aperture. 42 travels in a 
direction along the optic axis reflecting off the walls until it exits at 
point 41. A pellicle 16 encapsulates the end of the light pipe 40 in a 
sealed and clean space. This prevents dust from landing on the output 
aperture 41 and being imaged onto the specimen 20. The pellicle 16 is 
positioned far enough beyond the aperture 41 to keep dust that may fall on 
it, out of focus at the specimen 20. The lens 56, with a focal length is 
positioned to collimate the output beam of the light pipe 40 originating 
at the output aperture 41. Additionally, the lens 56 images the input 
aperture 42 of the light pipe 40 at a point roughly 21.6 mm beyond the 
principle plane of the lens 56. This point is roughly 1 mm beyond the 
turning mirror 54. The lens 58 with a focal length of 100 mm accepts the 
collimated light beam from lens 56 and creates an image 252 of the output 
aperture 41. The path is deviated twice by 90 degrees with two turning 
mirrors 54. Lens 58 also serves to collimate the input aperture image 250. 
The beam is again deviated 90 degrees by a turning mirror 54 to be 
accepted by the lens 59. Lens 59 with a focal length of 100 mm is 
positioned along the axis to collimate the image 252 of the output 
aperture 41. At the same time, lens 59 forms an image 253 of the input 
aperture 42 at a point 100 mm beyond the principal plane of the lens 59. 
The image 253 is comprised of a plurality of arclet images formed by the 
interaction of the light pipe 40 and the previously disclosed elements. 
The slide 20 itself changes the location of the field stop image due to its 
optical characteristics. Over sizing the stop 44 provides a full zone of 
illumination. A beam splitter 62 with a 70:30 reflection to transmission 
ratio intercepts the beam allowing 30% of the incident light to pass 
through. Seventy percent of the light is deviated 90 degrees to allow the 
image 253 to be formed in the vicinity of the pupil 73 of the condenser 
lens 70. The condenser lens 70, with a focal length of 20 mm, forms a 
secondary image 256 of the exit aperture 41 conjugate with the specimen 
plane 20. Light forming this image first passes through the microscope 
slide 258. The image 256 is 2.6 mm on a side with a numeric aperture of 
0.13. In the preferred embodiment, the output aperture 41 of the light 
pipe 40 serves as the field stop for the illumination system. This stop is 
oversized by 2 times with respect to the CCD image on the slide. This 
prevents vignetting which causes illumination drop-off at the edges of the 
field due to the absence of a full cone angle of illumination at the edge 
of the output aperture 41. 
Slide 20 is illuminated by a number of arclets of light 21. Each arclet 21 
is schematically illustrated in more detail in FIGS. 6A and 6B. The 
condenser lens 70 provides uniform illumination of the slide 20 by 
focusing the arclets 21 at infinity or near infinity. The slide is 
illuminated with a field of 0.52 millimeters and at a numeric aperture of 
0.45 for a 20.times. configuration where the slide is imaged at 20.times. 
magnification. A condenser lens 70 of 2 mm focal length is positioned with 
its principal plane 20 mm behind the focal positions. Twenty millimeters 
behind the principal plane 71, aperture stop 73 is positioned to limit the 
angular extent of light to the slide (i.e. numeric aperture). The 70/30 RT 
beam splitter 62 provides strobe sensor 65 with 30% of the arclet image 
through a spherical lens 64. Strobe sensor 65 is conjugate in the same 
place as the specimen field. A spherical lens 64 is sized to receive the 
full lateral and angular extent of the arclet illumination. The beam 
splitter 62 may advantageously be positioned 65 mm behind the aperture 
stop 73. 
Thirty-five mm from beam splitter 62 is the principal plane of lens 59. 
Lens 59 images the arclets 21 at the aperture stop 73 and culminates the 
image from the output aperture 44. The output aperture 44 of light 
homogenizer 40 also acts as a field stop and is encapsulated by pellicle 
47 to prevent dust and debris from being imaged onto the slide 20. Because 
dust and debris is prevented from falling on output aperture 44, lenses 59 
and 70 combine to provide a 5-to-1 magnification of the output aperture 44 
of the light pipe 40. In one preferred embodiment, the field stop may be 
2.6 mm on a slide to match the CCD camera size. The output aperture 44 is 
oversized as compared to the CCD image on the slide. This prevents 
vignetting which causes illumination drop off at the edges due to absence 
of a full cone angle at the edge of the output aperture 44. 
Refer now to FIG. 2 which shows one alternate embodiment of the invention 
suitable for 20.times. microscopic illumination. In this embodiment the 
mechanical slide 52 is positioned to allow light to freely pass to the 
lens element 59. The position of lens element 59 was chosen to collimate 
the output of the light pipe 40 for the light beam emanating from the 
output aperture 41 when the mechanical slide is positioned to remove the 
elements 56 and 54 from the optical path. The lens element 59 also images 
the input aperture 42 of the light pipe 40 in the vicinity of the pupil 73 
of the condenser lens 70. The condenser lens 70 with a focal length of 20 
mm creates an image 256 of the output aperture 41 on the specimen 20. This 
image is provides for a field of illumination that is 0.52 mm on a side 
with an NA 0.60. However, the pupil of the condenser lens in the 20.times. 
case is set to 0.45 NA. This coupled with a Numeric Aperture of the 
imaging objective of 0.75 serves to maximize the contrast of the 
frequencies of interest in the specimen. 
Now refer to FIG. 3 which shows an alternate schematic diagram of one 
embodiment of the apparatus of the invention to provide uniform 
illumination of the specimen 20. A light source 22 illuminates an optical 
conditioning system 12 with light. The optical conditioning system 12 
provides a light bundle of a predetermined numeric aperture, predetermined 
spectral frequency bandpass, and predetermined intensity to the light pipe 
40. The optical conditioning system 12 underfills the input of the light 
pipe 40. The light pipe 40 provides a light bundle of homogenized spatial 
content to a transport optical system 50. The transport optical system 50 
collimates the output aperture 41 of the light pipe 40 while imaging the 
input aperture 42 of the light pipe 40 near the pupil 73 of condenser lens 
system 100. Imaging of the input end provides a plurality of images of the 
input end 42 of the light pipe 40 due to internal reflections in the light 
pipe 40 with a primary image of the input 42 centered on the optical axis. 
The plurality of images fill the input of pupil 73 of the condenser lens 
system 100. The condenser lens system 100 images the collimated light 
corresponding to the output aperture 41 onto the specimen 20. An optical 
sensor 122 receives and image of the specimen 20 that has been uniformly 
illuminated. 
Now refer to FIGS. 4A, 4B, 4C, 4D and 5 which show the operation of the 
light pipe 40 to generate arclets 21 and homogenize an input light 
pattern. The light pipe 40 is a solid glass parallel-piped, preferable 
made of BK7. All six surfaces of the parallel piped are polished to 
optical smoothness. The aspect ratio of the light pipe 40 of the in one 
embodiment is 100:1 with a side dimension of 2.6 mm. Other aspect ratios 
may be chosen without deviating from the scope of the invention. Given an 
extreme ray angle defined by the numeric aperture of 0.13, those skilled 
in the art will appreciate that over eight reflections of the extreme ray 
occur inside the light pipe 40. The reflections follow the laws of total 
internal reflection and therefore are practically lossless. 
FIG. 4D demonstrates the homogenization effect for a single point of light 
with only three passes of the extreme ray. FIGS. 4A, 4B and 4C show the 
effect of the light pipe 40 coupled with a lens 260 to generate arclet 
images A', B' and C' from a point A in the primary input aperture 42. 
Note, the ray fan from point A passes through the light pipe 40 without 
reflection and forms in image A' by lens 260. The image B' is formed by 
the ray fan from point A that has a ray fan defined by the following 
extreme rays. One extreme ray of the fan originates at point A and 
reflects off the side of the light pipe 40 just before it leaves the light 
pipe 40 at the edge of the output aperture 41. The other extreme ray 
defining the fan originates at point A and reflects off the light pipe 40 
at a point halfway between the input and output apertures 42 and 41 
respectively. If one were to trace these rays backwards in space, shown as 
dotted lines 300 and 301, they would intersect at a point in space 
originating in the same plane as the input aperture 42. They would also 
intersect at a point exactly one aperture width away from point A. 
Likewise the image C' and its complement virtual object C are formed in 
the same manner. Note that the images A',B' and C' are practically all at 
the same intensity level due to the lossless total internal reflections of 
the light pipe 40. Those skilled in the art will also appreciate that an 
additional set of images A", B" and C" etc. will also form on the opposite 
side of the axis from the primed images due to complementary ray fans on 
the other side of the optic axis originating at point A. 
In the preferred embodiment of this invention those skilled in the art will 
appreciate that given the length of the light pipe 40 in combination with 
the input NA, approximately sixteen images, eight on each side of the 
optical axis, of the input aperture 42 will be formed at the condenser 
pupil 73. Those skilled in the art will also appreciate that the prime 
images A', B' and C' will move in a direction opposite in sign from the 
double prime images A", B" and C" as the primary arc image A moves 
laterally on the input aperture 42. This provides for a mitigation of the 
effects of shading due to arc movement at the input aperture 42. 
FIG. 5 shows a perspective drawing of the plurality of arclet images 
filling the input pupil of the condenser by means of the light pipe 40 and 
the lens system 260. Those skilled in the art will appreciate that other 
combinations of the NA and length can be constructed and will not deviate 
from the scope of the invention. 
FIGS. 6A and 6B show the effect of underfill on light intensity stability. 
FIG. 6A shows the arc centered in the aperture stop 26. In FIG. 6B the arc 
has moved to the edge of the aperture. Because magnification of the arc is 
chosen to underfill the aperture stop 26, this movement of the arc does 
not significantly affect the intensity of the light passed through the 
aperture stop 26. When the arc is magnified to fill the aperture stop 26, 
this movement of the arc would cause over half of the arc to be occluded, 
reducing the intensity of light passed through the aperture stop 26 by a 
like proportion. 
In the place of arclets, a mask may be placed in the aperture space of the 
condenser lens. This mask may contain periodic structures of semi-opaque 
and semi-transparent areas. FIG. 13 shows such a mask. This mask will 
generate angular intensity variation in the cone angle of illumination of 
the specimen. This angular variation causes the flaws and contaminants on 
the coverslip to cast shadows of the predetermined spatial frequencies in 
the image plane of the specimen. Those skilled in the art will appreciate 
that a multitude of masks can be used for this purpose and this will not 
deviate from the scope of the invention. 
Those skilled in the art will appreciate the fact that many other light 
sources may be used such as florescent lamps, LASERS, Laser Diodes, Light 
emitting diodes, and continuous arc lamps and photo-luminescent sources 
without deviating from the scope of the invention. 
Those skilled in the art will also appreciate that other detection 
techniques, such as Fourier decomposition of the image, followed by 
identification and measurement of a spectral peak corresponding to the 
frequency of the modulation patterns in the image plane without deviating 
from the scope of the invention. 
The invention has been described herein in considerable detail in order to 
comply with the Patent Statutes and to provide those skilled in the art 
with the information needed to apply the novel principles and to construct 
and use such specialized components as are required. However, it is to be 
understood that the invention can be carried out by specifically different 
equipment and devices, and that various modifications, both as to the 
equipment details and operating procedures, can be accomplished without 
departing from the scope of the invention itself.