u) TECHNICAL REPORT AD NATICK/TR-91/004 · 9 SPONSORING MONTORING AGFV.CS' NAMEIS) AND ADDRESS(Eý....
Transcript of u) TECHNICAL REPORT AD NATICK/TR-91/004 · 9 SPONSORING MONTORING AGFV.CS' NAMEIS) AND ADDRESS(Eý....
u) TECHNICAL REPORT ADNATICK/TR-91/004
N eA COMPUTER PROGRAM FORN •MEASURING FIBERS WITH THE
ZEISS CSM 9500 SCANNING ELECTRON MICROSCOPE
Deborah Sakelakos
Samuel H. Cohen DTICNovember17 199 t L.L 1- r_-
November 1990 DEClIL1990
Final Report ý0June 1990 to September 1990
Approved for public release, distribution unlimited
US ARMY NATICK RESEARCH, DEVELOPMENT ANDENGINEERING CENTERNATICK, MA 01760-5000
SOLDIER SCIENCE DIRECTORATE
ILU
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form AoprovedREPORT DOCUMENTATION PAGE oMB No 0704-0188
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1. AGENCY USE ONO~ ted.i Diant; 2 REPORT DATE 3 REPORT TYPE AND DATES COVERED
1 5 P nal- .Timi 1QQ() +-n~ qinn QQ4 TITLE AND SUBTITLE 5 FUNDING NUMBERS
A Comrputer Progjran for Measuring~ Fibers with the FE: 612786Zeiss CSM 950 Scanning Electron Microscope
6 0,UTihOF, -I.R 1L162766AH91BIC0oDeborah Sakelakos, and Samiuel H. Cohen9BCO
S7PERF~ORMING ORGANIZATION NAME(S. AND ADDRESS(ES; 8 P[RFORMiNG ORGANIZATION
IU.S. Anrmy Nati ck Retah _rn Ct REPORT NUMBER
Kansas StrieetATI!4: SI'RNC-YSENTC/R9/0Natick, Massachusetts 01760-5020 AJc/T-104
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AGENCY REPORT NUMBER
1l1..SU.FPpEK'iN1f~l . r N.L CI
Deborah Sake-Lakos was a pdcir-QipdiL iii ti-ie FI'90 UJ.S. Azii-ity Hg CO1ShMathematics Faculty Program ftuicded by the Army Researchl office.
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13 ABSTRACT (tA',3
17A c=upulter program. designed for the stereological measurement of fibers, waswritten for use with a ZEISS CSM 950 Scanning Electron Microscope (SE2M). T1hisprogjram utilized the ZEISS' build-uin Kontron Image Analyzer to access Surenentsof a variety of paramteters. for fiber analysis of iimges frcin a video cam~era, lightmicroscope or SE2A.
14~ SUJBj!(T TERM. N NM31
, 0 P PACI'
Ccmrputer program, ZEISS L-.12 950, Kontron 1mage Analyzer 26Scanniing Electron Microscope, Fibexs, Images, Parameters 16 FF, ci CO D.
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S-3aca'a co~rn 298 BzcK ':eV '-89)
TABLE OF COOrrMS
LIST OF FIGURES iv
LIST OF TABLES v
PREFACE vii
IN•ODEUCrION 1
ND1•OES 1
RES= 12
CONCLUSIONS 12
REFERENCE 20
-Accesson For
Tr i tributioi__
Availobility Codes
i iivail nd/or.Wt Special
LUST OF FIGURES
1. Drawing of 1i Fibers to be Measured 2
2. Histogram of Area of 11 Fibers 15
3. Histogram of Perimeter of 11 Fibers 16
4. Histogram of Feret X of 11 Fibers 17
5. Histogram of DCircle of 11 Fibers 18
iv
iv
LIST OF TABLES
~rag
1. Image Analysis Progrwn- for Measuring Fibers 13
2. Statistical Data (in Pix ,is) for 11 Fibers Measured 19
V
PREFACE
In this report we describe the individual cmjponents ard seuenRc of
instructions of a cutputer program designed for the sterelogical
measurerent of fibers. Utilizing on-screen menu onnanrds, we were able to
design and execute a step-by-step analysis of a fiber measurement
program. There was same difficulty in writing the program due to a lack
of clear instructions in the operator's manual; however, conversations
with company representatives helped us overcae these problems.
Vii1
C
A CaX fl FpRGRAM FOR • MEAURI FIBER
WrIn THE ZE=SS CSM 950 SCANING ELBCqWN MICROSCOPE
The objective of this report is to describe a computer pzomrn
designed for measuring stereological features of fibers by means of a
Kontron image analysis system, which is built into a Zeiss CSM 950
Scanning Electron Microscope (SEM).
7The prngram was written to penrit interaction between the menu program
displayed on the TV monitor ani the keybo-ard or rouse. This increased
capability penzits the operator to change parameters within the
measureme-nt pnxociwct deperdaing on the type of fi>ar sample.
since research presently being conducted in these laboratories on
ballistic fibers, including biotiechnologically'derived fibers, requires
information about fiber size, shape, etc., developing a menu- Iled
conput•.r program facilitates that purpose. . ,
MEIHODS
The sazple model use, was a drawing of 11 fibers (Fig. 1) which were
visualized with a Dage MfI Model CD 725 video camera with a Fuji C6 X
17.5 TV zoom lens. ahe images fror the IV camera were transferred to a
Zeiss CSM 950 SEM with a built-in Kontron image analysis system. The CSM
950 utilizes a CP/M operating system having a Z80 processor with a
FIGURE 1. Drawing of 11 Fibers to be Measured.The par-ameters measured (in pixels)were Area, Perimeter, Feret X(maximum projected length in thehorizontal direction), and DCircle(area of fibers within a circlemeasuring 100 pixels in diametex).
2
20 ?.yte hard disk, 64 10byte ranzgon aooass x0ry (RAM) and a 16 KQ6yte
video RAM.
The image processing system performs evaluations in a series of
steps. First, the image fram a TV camera, SEM, light iricroscpe or any
other internal or ex>ternal source is digitized and stored. Next, the grey
image is processed for improved contrast and signal/noise ratio. Then,
certain features are extracted, i.e., unwanted or overlapping features are
eliminated, and features such as size, shape, etc., are analyzed.
Finally, t-he derived data are processod statistically and a printout or
photograph is obtained.
There are several major functions, which am .- ouped dependiiug upon
the specific sequence of a stardard measuring pneh.-.. The functions
cu t2 -
1. Input - defines the type of iiipt device and controls image
digitization and storage.
2. Calibrate - controls the scaling factors, i.e., inches, microns,
etc.
3. Enhance - rescales the grey levels, thereby enhancing image
quality. Image noise reduction fi-ters are used.
4. Image Edit - contains functions for the editing of images.
5. Segment - extracts the ilmoge backgrrain- (distinguishes between
objects and background).
6. Multiphase - enables further processing of binary (two grey
levels) and multiphase (more than two grey levels) images.
3
7. Parameters - permits the selection of object-spacific (single
objects in a field of view), or field-specific (area of a visual field
minus the area occupied by objects).
8. Evaluate - evaluates objects and outputs the results.
9. Advanced - perfonrs geometric and arithmetic transformation of
images, i.e., improves the quality of an image using Fast Fourier
Transform methodss.
10. Utilities - has general auxiliary functions.
11. Peripheral - permits control of peripheral devies, remote
control, etc.
All the above functions have subfunctions that are displayed on the
video monitor in the form of a menu. Dialogue with the program, allowing
the operator to assign or delete nunmerical values as ell as to control
all of the system functions, is accomplished by utilizing the keyboard or
a miouse.
The following is the selection sequence for the operator in order to
obtain the desired program.
A. Calibrate
1- .Scle - Xs al I reAsurement data are indicated in the selected unit of
calibration, the most inportant prerequisite for every geometric
reasurement is calibrating the measuring system. The calibration
procedure is carried out according to the instructions in the operator's
manual (Anonymous, 1986).
a. SCRO, the number of the scale factor to be activated, is set at I.
4
B Ealuate
1. Reset - Before starting a new measuring sequence, e.g., after loading
a new program, scue cc~rditions such as data bufffering, identification
rrber and scanning stage can be reset. The system then has the same
status as after initialization.
a. Single (on) - Clears the buffer into which all the data fromn
several fields are accumulated.
b. Class (on) - Clears the data sumned up over several evaluated
fields to provide average values (e.g., nean area percentage).
C. Select Paratcrter
1. Area - Object specific area.
a. NCIS (20) - Number of classes when classification is selected.
ClassificzItion refc-r to a r--oe f .•_asu-emenent parameter values. i.e.
area, perimeter, etc. into which classes (groups) of fibers would fit.
b. MO3DX (1-3) - Presentation and scaling xmode of the X-axis in the
histogram of the results.
c. MODY (1-10) - Presentation and scaling mode of the Y-axis in the
histogram of the results.
d. Single (on) - selection of a single list.
e. Class (on) - Selection of a classification.
f. Lcw 0.000 - IU&o-r bound of classification.
g. High 5000 - Upper bound of classification.
D. Select Para-meter
1. Fetvt - Feret diameters (maxinum projected lengths) in X and Y
direction.
a. NCLS (20) - Number of classes when classification is selected.
5
b. MODX (1-3) - Pre-sentation and scaling mode of the X-axis in the
histogram of the results.
c. MODY (1-10) - Presentation and scaling mode of the Y-axis in the
histogram of the results.
d. Single (on) - Selection of a single list.
e. Class (on) - Selection of a classification.
f. Low (0.000) - Lower bound of classification.
g. High (200.00) - Higher bound of classification.
h. 14CLY (20) - Number of classes when classification is selected.
i. MDYX (1-3) - Pres.,entation and scaling mode of the X-axis in the
histogram of the results.
j. MDYY (1-i0) - Presentation and scaling mode of the Y-axis in the
histogram of the results.
k. LO.Y (0.000) - Lower bound of the classification.
1. HI.Y (1000.0) - Upper bound of the classification.
E. Select Parameter
1. Perim - Object specific perimeter and length.
a. NCLS (20) - Number of classes when classification is selected,
b. DI2DX (1-1) - _resentation aryi scaling rmode of the X-axis in the
histogram of the results.
c:. MODY (1-10) - Presentation and scaling mode of the Y-axis in the
histogram of the results.
d. Single (on) - Selection of a single list.
e. Class (on) - Selection of a classification.
6
f. Low (0.000) - Lower bound of classification.
g. High (600.0) - Upper bound of classification.
h. NCLT (10) - Number of classes when classification is selected.
i. MDLX (1-3) - Presentation and scaling mode of the X-axis in the
histogram of the results.
j. YDLY (1-10) - Presentation and scaling mode of the Y-axis in the
histogram of the results.
k. IOLT (600.0) - Lower bound of classification.
1. HILT (0.000) - Upper bound of classification.
F. Select Parameter
1. DCIRCL - Diameter of area-equivalent circle (area of fibers within a
circle which measures 100 pixels in diameter).
a. NCLS (20) - Number of classes when classification is selected.
b. MODX (1-3) - Presentation and scaling mode of the X-axis in the
histogram of the results.
c. MWDY (1-10) - Presentation and scaling mode of the Y-axis in the
histogramn of the results.
d. Single (on) - Selection of a single list.
e. Class (on) - Selection of a classification.
f. lcw (0.000) - Lower bound of classification.
g. High (100.00) - Upper bound of classification.
G. Select Utilities
1. LAB: label definition - labels are used as markers in the sequence of
a measuring program to which a jump (analogous to a GOl1 function) can be
executed.
a. I!fL#(l) - Identifier of the label, which serves as designation for
a jump.
7
1. TVa4 - Switches the color monitor directly to a TV signal.
a. Online. (on) - Switches the cnline status on or off.
&. Select Utilijty
1. SYNC - The Image Processing Syste can be synchronized externally
(e.g., by using a TV camera) or intexnally. In the internal mode, no TV
input is possible.
a. Internal (off), Internal (on), or External (off).
J. Select Utility
1. Pause - Interrupts run of program.
K. Irnutm
1. TVINP - This function stores:
a. 'IV iput - A TV image iii the i,,izop ir-icaty b- i
b. INP (1) - Image memory into which the inage is to be stored.
L. Choose Seqment
1. DISC2L - This function separates objects from the background, by
setting two thresholds. Either the grey levels inside or those outside
the entered limits are set to grey value 0 (black) and constitute the
background. Deperding on the variable BIUARY, the remaining object points
either keep their original grey levels or are set to white (255).
a. INP (1) - Image meory oontaiining the image to be prooessed.
b. OUT (2) - Image mmiiory into which the discriminated image is
stored.
c. IEVI (0) - Lower discrimination limit.
d. IMV2 (118)- Upper discrimination limit.
8
e. BINARY (on) - The object pixels become 255 (white), i.e. a binary
image is prodaced.
M. Calibrate
1. MFRAME - This function is used to create a rectangular or circular
measuring frame, which is scsmetimeas necessary to correct errors (eliminate
overlapping images) caused by the finite size of the field of view during
the measuremetnt process.
a. INP (2) - Memory containing the image to be processed.
b. WDSX (508) - X-side of the rectangular frame.
c. WDSY (467) Y-side of the rectangular frame.
d. XO (4) - X position of the (rectangular or circular) frame.
e. YO (6) -- Y position of the (rectangular or circular) frame.
f. RAD (100) - Radius of the circular frame. •
N. Evaluate
1. IDE24T - This function serves two purposes: Identification of
discriminated images and elimination of objects following the conditions
set by the measuring frame (function MFRAME, group Calibrate).
a. INP (2) - Diage memory containing the image to be identified.
b. OUT (3) - Memory containingl the ident if ied and frtme-corrected
imaqe.
c. NRG (1) - Controls the meaning of the measuring frame defined by
MFRAME (function group Calibrate). I1 jis determines whether or not the
fiber touching the rectangular or circular frame on the video monitor
is measured. For example, one might not want to measure a fiber
extending beyond the frame's margin.
9
d. 8-OON (on) - Method of iderti fication, on = 8 CONN.
1. MEASURE - This function ey-xeite- the measurement of selected
parameters such as area, perimeter, etc,.
a. OBJ - Must be switched on if cbject-specific parameters are among
the previously selected parameters (AFEA-TflE).
b. INP (3) - lrage memoxry oontaining the discriminated OBJ image.
c. GRWM (1) - Image memiry containing the original grey image.
d. AIJl (9) - Auxiliary image memry.
e. AUX2 (9) - Auxiliary image irrory.
f. CHAN (1) - Channel selection.
g. SPEC (1) - Distance in pixels between consectiv'e lines of the
"li~n grid". The lines are sinply lines of the image memrory, or pixel
addressez with varying X, but constant V coordinates.
h. FIL# (1) - logical flag ind-icating a buffer overflow.
i. REJECT (on) - Interactive rejection of undesired objects,
preceding an automatic measurement.
P. Evaluate
1. CeTCL -4-4-pu o Clazzi Fj ation T hisz tfunction dsplays thle
results of the classification in the form of a list and/or as a histogram.
a. HIS=I (on) - Provides measured data in histogranm forn.
b. HALT (on) - Presents data on the monitor; the snLm histogram can be
vanipulated.
10
c. NFAC (0.000) - Defines the area (in user units) into which the
absolute counts should be normalized. A value of 0.0 gives normalized
absolate frequencies.
0 . Evaluate
1. CUT'SGL - (Outpuit of single list) This function displays the
individual lists of all neasured data.
a. HALT (on) - Displays lists on the data moriitor after each
nnasureIment.
R. Select Utility
1. Pause - This function interrupts the runnirig of a measuring progran.
1i
RESULTS
The cauiter program designed aid written for the stereological analysis
of several fibers (Table 1) was utilized to measure their Area, Perihieter,
Feret X and D Circle. The irputs to the parameters of this program wre as
follows:
Area (high upper bound of classification) - 5000
Feret X (higher bound of classification) - 200
Feret X (upper bound of classification) - 1000
Perimeter (high upper bound of classification) - 600
DCircle (high upper bound of classification) - 100
The printout of the ccrputer program can be seen in Table 1, the plots for
each parameter can be seen in Figures 2 to 5 and the printout of the
statistical data can be seen in Table 2.
QONCLUSIONS
A carputer program was designed and written to measure certain
stereological parameters of fibers and to generate subsequeit statistical
analysis of the results. Me program was develor-ed to be driven by a series
of menu ccmuw-4s, which allowed for direct interaction beteen the image
appearing on the video monitor and the keyboard.
12
TABLE 1. Image Analysis Program for Measuring Fibers Using the Zeiss CST950 SE24 with a Built-in Kontron Image Analyzer.
WMJOR SUB INTDGER VARIABLES SCALEFUNCTIONS FUNCMIONS VALUES VALUES
A. CALIJATE SCALESCNO I
B. EVLIT RESET --
PAIRAM
C. PARAMEiLRS APEANCLS 20 SINGLE LOW 0.000MODX 1 CLASS HIGH 5000.0MODY 1
D. PARAMETERS 7ERETNCrl5 20 SINGLE LOW 0.000MDDX 1 CLASS HIGH 200.0MDDY 1 LO.Y 0.000NCLY 20 1IU.Y 1000.0MDYX 1MDYY 1
E. PAPAMEV:6 PEPIMNC=S 20 SINGLE LOW 0.000M)DX 1 CLASS HIGH 600.0MODY 2 LOLT 600.0NCLT 10 LILT 0.000
MDLY 1
F. PARAMETERS DCIRCLNCLS 20 SINGLE L) 0. 000M)DX 1 CLASS HICG 100.0MODY 1
G. U17ILITIES 1ABILB• 1
H. INPUr WVON
1. UrILITIE SYNC(Continued)
13
TABLE 1. Image Analysis Program for Measuring Fibers Using the Zeiss CSM950 SE4 with a Built-in Kontron Image Analyzer. (Continued)
MAJOR SUB INfEGER VARIABLES SCALEFUNCTIONS FUNCTIONS VALUES VALUES
J. UITILITITS PAUSE
K. INPJT TVINPINP 1
L. SEGMENT DISC2L BINARYLEVi 0
LEVl 01EV2 118
M. CALIBRATE NFRAMEfINP 2WDSX 508
WEMSY 467XO 4YO 6RAD 100
N. EVALUATE IDENTINP 2 8-ONN-our 3MAUG 1
0. EVTdLATE MEASURINP 3GRIN 1AUXI 9 OB"AUX2 9
SPAC 1FLG " REJECT
P. EVAIUATE OUTcLS__T NFAC 0.000
Q. EVALUATE WTSGL
HALT
R. UrIITIES PAUSE
TOTAL PiF M LEGfli 340 BYTES
14
CAMi'r 11ABS. FREWNCY MD. FLOW 0
Ok7PTLOW 0
10.0 ClA&SL 20%-IL CLASSE
25% 7MEDIAN 9
8.00 75% 1195% 18OM3JL250.0
6.00 L. BOUND0.000
U. BOUND5000.
4.00 oTNTf YM
1142.MAX1MJM
4254.
2.00 - EAI 2177.
0.00
ARE 901.3
0.00 1000 2000 3000 4000 5000
jIGURE 2. Ristogran of Area Measurenlents fron Fiqy1re 1.The X axis values are in pixels.The Y axIs represents the frequency disti ibution.
15
COLUM 11AB. FPXDU4CY M. Flow 0
0
10.0 CLASSE 20I %-IL C~hSSES
8.00 75% 1195% 19
HOWL30.00
6.00 L. BOUND/ 0.000
U. BOUND600.0
4.00 - MINT_165 .7
,4Afl2MJM
:)47.32.00 -KA
289.4SD
108.20.00 PE...-IOM0
0.00 200.0 400.0 600.00
FIGURE 3. Histogram of Perimeter Measure•ents fram Figure 1.The X axis values are in pixels.•The Y axis represents the frequency distribution.
16
aoums 11ABS. F CY UND. FLOW 0
OVERFLOW 010.0 - CLASSES 20
%-IL CLASSE25% 5
MDIAN 68.00 75% 11
95% 20MDDL
20.0006.00 - L. BOUND
0.000U. BOUND
200.04.00 M l
MAXIMIJM
191.02.00 ?TAN
77.91
____ ____ ____ ____ ____ ___48.38
0.00 •= . . . . . . . --. . . . .. FERET X
0.000 100.0 200.0
FIGURE 4. Histogranr of Feret X Meamurerents frnm Figure-. IThe X axis values are in pixels.The Y axis represents the frequency distribution.
17
0OUNTS 11AES. M2LENCY UND. FLOW 0(•RIUMID 010.0 o-ASSE 20
%-IL CLASSES25% 9
MEDIAN 118.00 75% 12
95% 15MDMX1L
5.0006.00 L. BOUND
0.000U. BOUND
100.00
4.00 MI030 .3
73.60
0.00ME
0.000 20.00 40.00 60.00 80.00 100.0
FIGURE 5. Histogram of an Area Equivalent to a Circle(called a DCircle) within Figure 1.
The X axis values are in pixels.The Y axis represents the frequency distribution.
18
TABLE 2. Statistical Data (in Pixels) for 11 Fibers Measured
.cuAREA PERIM .u- R
1 311i.0 413.4 137.0 62.942 2134.0 270.0 53.0 52.133 2512.0 318.4 58.0 56.554 1731.0 250.8 103.0 46.955 2378.0 302.6 52.0 55.036 1691.0 226.0 41.0 46.407 2196.0 269.5 44.0 52.888 1142.0 169.7 68.0 38.139 12).7.0 196.7 79.0 39.36
10 1582.0 219.2 31.0 44.88Ii 4254.0 547.3 191.0 73.60
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REFERENCE
Anoynois, 1986. "Y•ntron SEi-IPS OperatOr's manual," Volnme II, Release
4.4, 410 pages.
This document reports research undertaken at the
US Army Natick Research, Development and En~in~ejipgCenter and has been assigned No. NATICK/TR-/K I/[gI/in the series of reports approved for publication.
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