OJournal of Microscopy, Vol. 115, Pt 3, April 1979, pp. 257-269. Revised paper accepted 28 November 1978
A scanning microinterferometer with correction of errors caused by overlapping ‘ghost’ images
by J. E. D E J O S S E L I N D E J O N G , J. LOEVE*, H. RICHTER*,H. D E S T E R K E t ~, of Cell Biology and Genetics, and “Central and J. S. P L O E MDepartment Research Workshop, Medical Faculty, Erasmus University, Rotterdam; and ?Department of Histochemistry and Cytochemistry, Medical Faculty, University of Leiden, The Netherlands
SUMMARY
An experimental setup has been built to correct the errors caused by overlapping ‘ghost’ images when scanning and integrating measurements are carried out with the Jamin-Lebedef interference microscope. First a reference field with the object, producing the ‘ghost’ image, is scanned and the values of the optical path difference (OPD) of each point in the reference field are stored in a computer. Subsequently, the OPD of the object to be measured is calculated for each point by adding the measuring result to the average OPD of five points, located around the corresponding point in the reference field. The applicability of the setup has been tested by measurements with Sepharose beads, Hela cells and the Zeiss microinterference refractometer. The difference between measurements on the same object with and without overlapping ‘ghost’ image was about 10%. The reproducibility was tested by repeated measurements on the same cell whereby a standard deviation of 1.1% was found. INTRODUCTION
The interferometric measurement of the optical path difference (OPD) in microscopic objects is a useful parameter in cytochemistry since it is directly related to the dry mass of these objects (Barer, 1952; Davies & Wilkins, 1952). For the measurement of the integrated optical path difference (IOPD) in heterogeneous microscopical structures several scanning microinterferometers have been developed with a double interference microscope using the shearing system (Caspersson et al., 1954; Svensson, 1957; Lomakka, 1963; Carlson, 1970a, b; Carlson et al., 1970; Smith, 1972; Boguth, 1974). However, the small distance between the object and reference beam and the resulting double images limits the scope of these instruments when relatively large objects like tissue sections or closely packed smaller objects such as cultured cells have to be measured. For this purpose a double beam instrument with wide separation of both light beams such as the Leitz interference microscope (Grehn, 1958; Horn, 1958) is more suitable and this instrument has successfully been used in investigations, on the dry mass distributions in tissue sections (Galjaard, 1962; Galjaard & Szirmai, 0022-2720/79/0400-0257 502.00
0 1979 The Royal Microscopical
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J . E. deJosselin deJong et al. 1965). Also a scanning device has been built for this double beam instrument (de Josselin de Jong et al., 1973) which proved to be useful for dry mass determinations of cultured human fibroblasts and amniotic fluid cells (Galjaard et al., 1974a, b; van der Veer et ul., 1978). However, the time required for dry mass measurements of large numbers of cells is quite long and the critical stability sometimes requires time-consuming adjustment procedures. The present communication deals with the construction of an experimental setup for scanning microinterferometry with the Jamin-Lebedef interference microscope which enables relatively rapid measurements on large numbers of objects without difficulties caused by overlapping double images. 1.
GENERAL P R I N C I P L E AND OUTLINE O F THE INSTRUMENT
In the Zeiss Jamin-Lebedef interference microscope (Gahni, 1962; Piller, 1962) equipped with a 40-times objective, the two interfering light beams pass through the microscopical preparation with a mutual distance of f 175 pm. If this instrument is used for measurements in preparations with closely packed cells, there is a good chance that the ‘reference beam’ passes through a neighbouring cell thus causing a ‘ghost’ image which overlaps the image of the cell to be measured (Fig. 1). The principle of the present instrument is based on measurements of both the optical path difference of the cell to be investigated and of the cell causing the overlapping ‘ghost’ image. This is based on the following considerations: If: ‘OPD cell’ = the OPD between a point in the cell the dry mass of which has to be determined and the free background; ‘OPD ref. cell’ = the OPD between a point in the cell causing an overlapping ‘ghost’ image because it is passed by the reference beam and the free background; ‘OPD cell/ref. cell’ = the OPD between
Fig. 1. Images and ‘ghost’ images of cultured Hela cells. The image of cell 1 is partly covered by the ‘ghost’image of cell 2. Also the optical path difference of the cell in the middle cannot be measured because of an overlapping ‘ghost’
image.
258
Microinterferometry, correction ‘ghost’ images
+
points in the two mentioned cells. Then: ‘OPD cell’ = ‘OPD cell/ref. cell’ ‘OPD ref. cell’. Hence it is possible to calculate the ‘OPD cell’ by addition if the ‘OPD ref. cell’ is known. With the setup described a rectangular ‘reference field’ containing the ‘reference cell’ is scanned first. The OPDs of all measured points (that is the OPD between these points and the free background) are stored in a computer memory in a twodimensional array (the ‘reference array’). Then the scanning stage is shifted over a distance similar to the difference between the relative positions of the interfering light beams. Subsequently a rectangle of the same dimensions now containing the cell which is overlapped by the ‘ghost’ image of the previous cell is scanned. The values of all points are also stored in a two-dimensional array. The OPD of a point in the second cell is calculated by addition of the value of this point stored in the second array and the average value of five points located around the corresponding point in the ‘reference array’. The results are stored in a new ‘reference array’. The stage is now shifted again and the described procedure is repeated till the cell to be investigated has been scanned. The optical part of the setup consists of a Zeiss universal microscope with a 0.5 pm scanning stage and the Jamin-Lebedef interference equipment with the Pol.Int. I1 40/0.65 objective. With a C 5 x projective a magnification of 200 x is attained. As a light source an Osram H.B.O. 100 W2 lamp is used. The analyser is a permanently rotating polarizer, according to Lomakka (1963), driven by a selfstarting 50 Hz a.c. motor, which is placed at our disposal by Carl Zeiss. The measuring equipment consists of the Leitz U.V. microscope photometer attachment containing an E.M.I. 9558QA photomultiplier that has adequate sensitivity for green light (546 nm) and which is insensitive to the polarization direction of the light. In this system no mirrors or optical components affect the polarized light. The diameter of the measuring field is 0.3 mm (1.5 pm in the object plane) or 0.6 mm (3 pm in the object). The power supply for the photomultiplier is an Oltronix B 2.2K-20HR. The actual measurements are carried out by a phase angle detector. A push-button keyboard is used for steering the scanning stage, selecting the scanning field and starting the scanning procedure. A DEC PDP 11-45 computer with Laboratory Peripheral System (LPS-11) is used for storage and calculation of the data and for the control of the scanning process. The results are printed on a line printer and displayed on a storage display, which is also used for interactive communication. 2.
PHASE ANGLE DETECTION
The intensity of the light, after it has passed the rotating polarizer, varies sinusoidally as a function of the angle of rotation. The phase angle of this curve is a linear function of the phase difference between the interfering light beams. For the measurement of the phase angle, a disc with a slit rotates together with the analyser between a photoelectric cell and a small red lamp. In this way an electric pulse (the analyser pulse) which correlates with a certain position of the analyser, is generated 50 times per second. A second signal is produced by a phase discriminator, which generates a pulse (the ‘discriminator pulse’) each time the photomultiplier signal reaches a maximum value thus indicating the top of the sinusoid. In Fig. 2 the relation between these pulses is shown in a time diagram. Once the measurement of a certain point has been finished, the next analyser pulse (the ‘starting pulse’) generates a trigger signal for the movement of the stage to the next point. The third analyser pulse opens an electronic gate allowing clock pulses to stream into a couriter until the gate is closed by the ‘discriminator pulse’. The number of counted clock pulses is proportional to the phase angle to be measured. 259
J. E. de Josselin de Jong et al. analyser pulses
starting pulse:stage starts movement t o next point
gate is opened
registrated photomultiplier signal discriminator pulse gate is closed
counted clock pulses
next s t a r t i n g pulse
Fig. 2. Time diagram of the electrical signals generated during the measurements.
3.
CALCULATION OF T H E OPTICAL P A T H DIFFERENCE
During the standard measuring routine the following three phase angles are measured: 010: when both light beams are passing through the free background; a1:when the ‘reference beam’ passes through the free background and the measuring beam passes through the ‘reference cell’; 012: when the ‘reference beam’ passes through the ‘reference cell’ and the ‘measuring beam’ passes through the cell to be measured. In the time diagrams in Fig. 3 the relation between these phase angles and the OPDs is shown under different ‘optical circumstances’. The OPD is expressed in degrees (360” corresponds with 1X). In Fig. 3(a) and Fig. 3(b) the factor (“2-010) is equal to the difference between the ‘OPD cell’ and ‘OPD ref. cell’. In this case: ‘OPD cell’ = ‘QPD ref. cell’ + ‘OPD cell/ref. cell’
- (q-010)+ (012-
+ ff2 -2010. Fig. 3(c) shows the situation when + ‘OPD ref. cell’) is greater than 360”. In a0)= 011
(010
this case a wrong ‘discriminator pulse’ is registered one wavelength too early. This difficulty can be avoided if the sum of a0 and the measured OPD does not exceed 360’. Because this is limiting the maximum measurable OPD, the interference equipment has to be adjusted in such a way that 010 is relatively small. However, when “0 is very small, irregularities in the free background or instrumental deviations sometimes may cause this phase angle to become negative. This would cause the ‘registered’ 010 to vary between either small values or values of about 360”. In the present instrument a0 is always about 36” (O.lh), which means 260
analyser pulse
6
w
a1
I ~
iIIIIIIIIIIIIIIIi I (IIIIIIIIIIIIIIIIIIIIII
OPD r e f . c e l l OPD c e l l l r e f . c e l l
discriminator pulses
'3
f i g . 3 a OPD c e l l > OPD r e f . c e l l OPD c e l l = ( a 1 - a o ) + ( a 2 - a o ) analyser 7
+I ,
illlllllllt
.
t
pk
+
a1-ao
I11111111
1,,,,11,111111111111,1,11,1,1
counted clock p u l s e s
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I
-
fig 3 c
ao+OPD ref c
-,
pulse
OPD r e f . cell
t
discriminator pulses
i
counted clock p u l s e s
~
ilIIIIIIIIIIIIIIi
illllllllllllllllllllllllllllllli
f i g . 3 b O P D c e l l < OPD r e f . c e l l OPD c e l l = ( a l - a o ) + ( a 2 - a 0 ) .
w
2
Fig. 3. Relation between the phase angles and OPD under different circumstances. ao=b through the free background. al=reference beam passes through the free background through the reference cell. a2 =reference beam passes through the reference cell and the the cell to be measured.
J. E. de Josselin de Jong et al. that the maximum measurable OPD is about 0.89, allowing small deviations during the measurements. In Fig. 3(d) a situation is illustrated where ‘OPD cell/ref. cell’ is smaller than - ao. This means that, theoretically, the ‘discriminator pulse’ should be registered before the ‘analyser pulse’. In practice a ‘discriminator pulse’ is registered one period later and the ‘QPD cell’ has to be calculated as follows : ‘OPD ref. cell’
= 011- a0
‘QPD cell/ref. cell‘ = 0 1 ~ 0 1~ A Hence ‘OPD cell’
= “2
+ a1 -
2010 - h
+
Usually this situation can easily be recognized because (01.2 a1 - 2010) will be greater than 1A. However, when ‘OPD cell’ is zero or less ‘OPD cell/ref. cell’ will be equal to or less than - ‘OPD ref. cell’. In that case the wrong discriminator pulse might be recorded while the factor (az “1 - 2010) is less than 1A. Therefore it is verified after each measurement whether (az 011 - 2010) is greater than a limit the value of which itself is smaller than 1h. In this case l h is subtracted. As a limit a value of 0.8% is chosen because this fulfils the demands under the circumstances illustrated in Fig. 3(c). When measurements without ‘reference cells’ are carried out, ‘OPD cell’ =
+
+
a1 - O10.
4.
S T A N D A R D P R O C E D U R E FOR M E A S U R E M E N T
The standard procedure for measurement consists of the following steps : - the optical system is put in the adjustment mode; - the upper limit (U) of the measurable OPD is chosen; - the location and dimensions of the scanning field are fixed; - the operator answers the question ‘reference cells ?’ on the display. When the answer is ‘yes’, the stage runs to the reference field. This can be repeated as often as is necessary and possible with respect to the preparation. - by marking three points an L-shaped figure is chosen in the background; - the optical system is switched to measuring conditions; - the phase angle of a point in the background is verified and if necessary adjusted to & 36” by tilting the condensor; - after a start button has been pressed, the L-shaped figure in the background is scanned and the average phase angle of the measured points on this line is taken as an; - if ‘reference cells’ have to be measured the first ‘reference field’ is scanned (011); - the ‘OPD ref. cell’ is calculated (011- 010); - the next field with the next cell is scanned ( “ 2 ) ; - for every point the following calculations are made: S = ‘OPD ref. cell’ + a2 - a0 If S > U then S’=S-h If S < U then S’=S - the last scanned cell becomes the ‘reference cell’ with ‘OPD ref. cell’ = S’; - the next field with the next cell is scanned and so on until the cell to be measured is scanned. The IOPD of this cell is calculated by addition of the OPDs of the points in the field which has been scanned last.
5.
T E S T I N G OF T H E V A L I D I T Y O F T H E P R O C E D U R E
With the method described the error in the measurements caused by overlapping
262
Microinterferometry, correction ‘ghost’ images ‘ghost’ images, is corrected by adding the OPD of the objects producing these ‘ghost’ images, to the measured value. This approach is only valid if the same but opposite OPD is measured in the image and ‘ghost’image. T o verify if this condition has been fulfilled, point measurements have been carried out with the Zeiss microinterference refractometer. This consists of an object slide with a spherical segment with a diameter of about 140 pm which has been covered with a drop of immersion oil and a coverglass. The OPDs have been determined in the middle of the spherical segment with the phase difference between the light beams in the free background set to different values by tilting the condensor. The results shown in Fig. 4 indicate that the measured OPDs vary in a reproducible way with the phase difference in the background. Further, the curve for the ‘ghost’ image is very similar to that for the normal image, but it is shifted in a horizontal direction over a distance of about 0.4h. To a high degree this contributes to a difference between the absolute values of the OPDs, measured in the image and ‘ghost’ image. To test the complete equipment Sepharose beads of different sizes (40-70 pm diameter) mounted in Depex have been measured both when their image was overlapped by the ‘ghost’ image of another bead and when their image was completely free from ‘ghost’ images. This has been achieved by locating the positions of the beads and by repeating the measurement after the object had been turned through 180”. The measurements have been carried out with a measuring spot of 3 pm (in the object plane) and a distance between the measuring points of six stage steps ( 3 pm). The maximum OPD in the largest bead was about 0.4h. The results of the two series of measurements are shown in Table 1. The measured IOPD has been plotted also against the diameter of the beads. The results are shown in Figs. 5 and 6. For both series of measurements the correlaI
0.41
OPD imaae
-0.41
-0.40
-0.39 -0.38
sum
- 0 01 -1 A
-0 5 A OA 05A background phase difference
1A
Fig. 4. The OPD of the Zeiss microinterference refractometer measured in the image and the ‘ghost’ image and the sum of these OPDs plotted for various phase differences measured when both light beams are passing the free background.
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J. E. de Josselin de Jong et al. Table 1. IOPD of Sepharose beads measured with and without overlapping ‘ghost’ images (in arbitrary units). The numbers are also mentioned in Figs. 5 and 6. IOPD with ‘ghost’ image 11973 15016 17804 28394 28760 36227 37100 39736 43680 47095 488 11 56064
Bead no. 1 2 3 4 5 6 7 8 9 10 11 12
IOPD without ‘ghost’ image 10045 11185 16700 24017 30211 37164 38775 38406 49795 52929 45933 58131
tion coefficient was 0.98. The standard error of estimate o f y on x (5’’. x) was 3786 for the measurements without ‘ghost’ image and 3434 for the measurements with ‘ghost’ image. The same measurements have been carried out with Hela cells mounted in 0.97/, NaC1. I n this case the measuring spot had a diameter of 1.5 pm and the distance between the measuring points was also 1.5 pm. A difference between the measurements with and without overlapping ‘ghost’ image of about lo:(, of the latter has been found. The measured Sepharose beads and Hela cells were always situated in the preparations in such a way, that only one ‘reference field‘ needed to be scanned with the measurements with ‘ghost’ image.
p
50
0 L c
0 40 L
0
30 40 I
50
60
I
1
t
10
1
20
70 1
-
1
30
d31104
diameter d ( p m )
Fig. 5. IOPD of Sepharose beads measured without ‘ghost’ image plotted against their diameter.
264
Microinterferometry, correction ‘ghost’ images h
v)
.= 6 0 c
c
3
50
L A
!?
c
0
40
L
0 v
30 0
;2 0 c
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C ._
10 50 I
30 4 0 I
I
10
60 1
70
I
20
I 30
-
I
d3/104
diameter d ( p m )
Fig. 6 . IOPD of Sepharose beads measured with ‘ghost’ image plotted against
their diameter. To test the reproducibility of the scanning procedure itself, the same Hela cell was scanned ten times. A standard deviation of 1.1 was found. The scanning time for a Hela cell is about 100 s and when a reference cell has to be measured this time is doubled. The adjustment procedure can be limited to the resetting of the scanning area, which takes about 45 s. 6.
DISCUSSION
The similarity of the two first curves in Fig. 4 and their shifting with respect to each other possibly can be explained as follows: The curve for the ‘ghost’ image has been shifted with respect to the curve for the image over a distance of approximately 0.4X, which corresponds to the OPD of the measured object. We consider a point on the curve for the image and the corresponding point on the curve for the ‘ghost’ image, indicating the measuring results for measurement (a) in the image and measurement (b) in the ‘ghost’ image. The difference between the background phase differences for measurements (a) and (b) is equal to the OPD of the measured object. The background phase difference is directly related to the background phase angle. Hence, also the difference between the background phase angles for measurements (a) and (b) is equal to the OPD. The measured OPDs are equal to the difference between the background phase angles and the phase angles measured in the image. In the normal image the phase angles are larger than those of the background and a positive OPD is measured. In the ‘ghost’ image it is just the reverse and a negative OPD is measured. Hence the background phase angle for measurement (b) in the ‘ghost’ image is equal to the phase angle measured in the image for measurement (a). Consequently, for the measurements (a) and (b) the measured OPDs are equal to the difference between exactly the same phase angles, which possibly is an explanation for the correspondence of the mentioned points on the
265
J. E. de Josseliiz de Jong et al. two curves in Fig. 4. T o examine whether the deviations illustrated in this figure are caused by the interference system itself or by the rotating analyser, the analyser housing has b-en turned 45” around the optical axes, and the measurements were repeated. Deviations in the analyser would cause a shifting of the second curve with respect to the plotted background phase difference. No shifting would be obtained if the variations in the measured OPD are only due to the interference system. The experimental results showed a completely transformed curve with the second measurement. This means that the variations are caused by the rotating analyser and the interference system together. Goldstein & Hartmann-Goldstein (1974) have discussed the deviations caused by the interference system. They also have plotted the measured OPD against the background phase difference and have found a sinusoidally fluctuating deviation, which could be diminished by reducing the diameter of the aperture diaphragm, but could not be avoided completely. A second cause of the variation probably is introduced by variations in the angular velocity of the analyser. However, these systematic errors will not influence the results very much if comparative measurements are carried out, provided that the interference system is not readjusted during the measurements. Difficulties may be caused by the astigmatism of the ‘ghost’ image. With an opened aperture diaphragm the ’ghost’ image has two pronounced directions corresponding with the diagonals in the field of view. However, if the aperture diaphragm is closed completely almost no asymmetric distribution of light is visible in the ‘ghost’ image. In order to compare the quality of the ‘ghost’ image with that of the normal image, both images of Sepharose beads with different sizes (50-80 pm) have been scanned along the mentioned diagonals. The results showed that the difference between the diameters of the ‘ghost’ image and the normal image is about 4 pm on both sides of the image along one diagonal. No difference has been found along the other diagonal. Another critical point is the size of the measuring diaphragm (Goldstein & Hartmann-Goldstein, 1974). When the distance between parts with a different OPD is smaller than the diameter of the measuring spot, the phase angle of a curve, which is the sum of various sinusoids, has to be determined. If errors are to be avoided, this phase angle ought to be the average of the phase angles belonging to the different OPDs of the parts concerned and the contribution of each part has to be proportional to its area. In the most simple case the measuring spot covers an image divided in two parts with different OPDs. If one part has an area of x units and the other part an area of (1 - x) units the corresponding sinusoids can be expressed as y l = x sin w t and y2=(l -x) sin ( w t p), (Goldstein & Hartmann-Goldstein, 1974). If the first curve has a phase angle 01 and the abovementioned condition ought to be fulfilled, the phase angle of the resulting curve ought to be equal to:
+
+
x .a (1 - X)(“
+p) = + (1- x)p) O1
In reality, however, this phase angle is, according to Goldstein & HartmannGoldstein (1974), equal to: a
(1-x) sin p + arctan x+(1-x)cOsp
In Fig. 7 the differences between the above-mentioned functions is plotted against p for various values of x. Goldstein & Hartmann-Goldstein (1974) have shown that the errors arising when the edge of the object is crossing the diaphragm compensate each other for each scan line provided that the distance between the measuring points is smaller than
266
Microinterferometry, correction 'ghost' images
252' W
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216'
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xzo.5
144'
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108'
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a
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36"
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Fig. 7. Difference between the desired phase angles and the measured phase angles, when two object parts with surfaces x and 1 - x and a mutual OPD are measured together, plotted for various values of x and 9. 360" corresponds with l h .
the diameter of the measuring diaphragm and the OPD is smaller than 0% It will be seen that the errors also compensate each other when the border between two areas with a mutual OPD less than 0.5X is passing the diaphragm. However, with the relatively slow scanning instrument described here the distance between the measuring points will mostly be equal to the diameter of the measuring spot. Hence, the errors mentioned above only compensate each other if there are enough scan lines to cancel them. out statistically. Also it has to be taken into account that images and 'ghost' images are measured together. Therefore relatively large phase angle differences may appear within a short distance. Ideally the diameter of the measuring spot should be equal to the resolution of the microscope. However, in interference microscopy the aperture diaphragm has to be small as well (Davies & Deeley, 1956; Hale, 1958; ROSS,1967; Goldstein & Hartmann-Goldstein, 1974) and the same is true to limit the astigmatism of the 'ghost' image. A small aperture diaphragm results in low light intensities and hence the minimum size of the measuring spot will partly b t determined by the minimum permissible signal to noise ratio. Because of the properties of the unsharp and slightly astigmatic 'ghost' image it is impossible to carry out measurements in this image with a high resolution. Therefore the measuring result of every point in the cell to be measured is corrected with the average value of five points in the 'reference cell'. Consequently the instrument can only be used for integrating measurements.
267
J. E. deJosselin deJong et al. Based on the standard deviation found with repeated measurements on a Hela cell a lower Sy.x might have been expected for the measurements without ‘ghost’ image on the Sepharose beads. Probably the diameter of the beads is not the ideal indication for their content. The differences between the measurements with and without ‘ghost’ image may partly be caused be adjustment procedures after turning the preparation through 180”. Because of a gradient in the thickness of the mounting medium (‘wedge effect’) the interference system had to be readjusted each time the preparation was turned in order to obtain the same ‘background phase difference’. During the standard procedure of measurement the ‘wedge effect’ will only influence the measurements if the wedge has a lenticular component (Ross, 1967). With respect to the measurements with ‘ghost’ images the setup can only be used for integrated measurements of objects of the order of 15 pm or larger. It then offers the possibility of dry mass determinations in closely packed microscopic objects without time-consuming adjustment procedures. The instrumental setup may, for instance, contribute to cytochemical studies on individual cultured cells, where the activity of several lysosomal enzymes varies with the dry mass of the cell (van der Veer et al., 1978). Also in the interpretation of enzyme activities after somatic cell hybridization (Galjaard et al., 1974b) dry mass determinations of cultured cells may be important. If there is an object-free strip on two sides of the microscopical preparation the cells just beside these strips are not overlapped by ‘ghost’ images. The OPD of these cells can easily be measured and can be used to correct possible errors caused by their ‘ghost’ images when other cells are measured. If the ‘wedge effect’ can be avoided in this way the whole preparation can be scanned without interruptions. Together with automatic image analysing systems this introduces the possibility of automatic classification by dry mass measurements on large numbers of cells. ACKNOWLEDGMENTS
The authors wish to thank Professor Galjaard for critically reading the manuscript and Ir. F. A. van Hall, head of the department for small computers and data communication of the C.R.I. in Leiden, for giving us the opportunity to use the computer system of the Institute. We wish to thank Professor P. van Duijn for critical and stimulating discussion. The support of Ing. R. van der Baan, Drs B. Hesper and H. Hofstra (C.R.I.) and J. Bonnet (Histochemistry, Leiden) is gratefully acknowledged. We wish to thank Dr J. Gham and Dr W. H. G. Cebulla (Carl Zeiss, Oberkochen, Germany) for their advice and critical comment. Illustrations have been made by J. Fengler, T. van 0 s and W. J. Visser. References Barer, R. (1952) Interference microscopy and mass determination. Nature, 169, 366. Boguth, W. (1974) Scanning-Interferenzmikroskopie mit dem Mikroskop-Photometer. Microsc. Acta, 76, 28. Carlson, L. (1970a) A scanning, recording and integrating microinterferometer. Histochemie, 21, 289. Carlson, L. (1970b) Cytochemical determination of dry muss. Doctorate Thesis, Institute of Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institute, Stockholm. Carlson, L., Caspersson, T., Lomakka, G. & Silverbige, S. (1970) A rapid scanning and integrating microinterferometer for large-scale population work. In : Introduction to Quantitatiae Cytochemistry (Ed. by G. L. Wied and G. F. Bahr), Vol. 2, p. 117. Academic Press, New York. Caspersson, T., Carlson, L. & Svensson, G. (1954) A scanning interference microscope arrangement. Exp. Cell Res. 7, 601.
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Microinterferometry, correction ‘ghost’ images Davies, H.G. & Deeley, E.M. (1956) An integrator for measuring the dry mass of cells and isolated components. Exp. Cell Res. 11, 169. Davies, H.G. & Wilkins, M.H.F. (1952) Interference microscopy and mass determination. Nature, 169, 541. Gahm, J. (1962) Die Untersuchung anisotroper Praparate mit der Interferentzanordnung nach Jamin-Lebedeff. Zeiss-Mitt. 2, 389. Galjaard, H. (1962) Histochemisch en interferometrisch onderzoek van hyalien kraakbeen. Doctoral Thesis, University of Leiden. Galjaard, H. & Szirmai, J.A. (1965) Determination of the dry mass of tissue sections by interference microscopy. J. roy. microsc. SOC.84, 27. Galjaard, H., van Hoogstraten, J.J., de Josselin de Jong, J.E. & Mulder, H.P. (1974a) Methodology of quantitative cytochemical analysis of single or small numbers of cultured cells. Histochem. J. 6, 409. Galjaard, H., Hoogeveen, A., Keyzer, W., de Wit-Verbeek, E. & Vlek-Noot, C . (1974b) The use of quantitative cytochemical analysis in rapid prenatal detection and somatic cell genetic studies of metabolic diseases. Histochem. 3. 6, 491. Goldstein, D. J. & Hartmann-Goldstein, I.J. (1974) Accuracy and precision of a scanning and integrating microinterferometer. 3, Microsc. 102, 143. Grehn, J. (1958) Das Durchlicht-Interferenz-Mikroskop- ein Instrument des Biologen. Umschau Wiss. Techn. 19, 602. Hale, A. J. (1958) The Interference Microscope in Biological Research. Livingstone, Edinburgh. Horn, W. (1958) Micro-Interferentz 11. In:Jahrbuch fur Optik und Feinmechanik. Pegasus Verlag, Wetzlar. de Josselin de Jong, J.E., Boender, W., Carlson, L. & Galjaard, H. (1973) A scanning device for the double beam Leitz interference microscope. Histochemie, 35, 127. Lomakka, G. (1963) A rapid scanning and integrating microinterferometer. Acta Histochem. Jena, Suppl. 6, 393. Piller, H. (1962) Durchlicht- Interferentzmikroskopie nach dem Jamin-Lededeff-Prinzip. Zeiss-Mitt. 2, 309. Ross, K.F.H. (1967) Phase Contrast and Interference Microscopy f o r Cell Biologists. Edward Arnold, London. Smith, F.H. (1972) A laser-illuminated scanning microinterferometer for determining thc dry mass of living cells. Microscope, 20, 153. Svensson, G. (1957) A scanning interference microphotometer. Exp. Cell Res. 12, 406. van der Veer, E., Kleijer, W.J., de Josselin de Jong, J.E. & Galjaard, H. (1978) Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods, combined with interference microscopy. Hum. Genet. 40, 285.
269
A scanning microinterferometer with correction of errors caused by overlapping ‘ghost’ images
by J. E. D E J O S S E L I N D E J O N G , J. LOEVE*, H. RICHTER*,H. D E S T E R K E t ~, of Cell Biology and Genetics, and “Central and J. S. P L O E MDepartment Research Workshop, Medical Faculty, Erasmus University, Rotterdam; and ?Department of Histochemistry and Cytochemistry, Medical Faculty, University of Leiden, The Netherlands
SUMMARY
An experimental setup has been built to correct the errors caused by overlapping ‘ghost’ images when scanning and integrating measurements are carried out with the Jamin-Lebedef interference microscope. First a reference field with the object, producing the ‘ghost’ image, is scanned and the values of the optical path difference (OPD) of each point in the reference field are stored in a computer. Subsequently, the OPD of the object to be measured is calculated for each point by adding the measuring result to the average OPD of five points, located around the corresponding point in the reference field. The applicability of the setup has been tested by measurements with Sepharose beads, Hela cells and the Zeiss microinterference refractometer. The difference between measurements on the same object with and without overlapping ‘ghost’ image was about 10%. The reproducibility was tested by repeated measurements on the same cell whereby a standard deviation of 1.1% was found. INTRODUCTION
The interferometric measurement of the optical path difference (OPD) in microscopic objects is a useful parameter in cytochemistry since it is directly related to the dry mass of these objects (Barer, 1952; Davies & Wilkins, 1952). For the measurement of the integrated optical path difference (IOPD) in heterogeneous microscopical structures several scanning microinterferometers have been developed with a double interference microscope using the shearing system (Caspersson et al., 1954; Svensson, 1957; Lomakka, 1963; Carlson, 1970a, b; Carlson et al., 1970; Smith, 1972; Boguth, 1974). However, the small distance between the object and reference beam and the resulting double images limits the scope of these instruments when relatively large objects like tissue sections or closely packed smaller objects such as cultured cells have to be measured. For this purpose a double beam instrument with wide separation of both light beams such as the Leitz interference microscope (Grehn, 1958; Horn, 1958) is more suitable and this instrument has successfully been used in investigations, on the dry mass distributions in tissue sections (Galjaard, 1962; Galjaard & Szirmai, 0022-2720/79/0400-0257 502.00
0 1979 The Royal Microscopical
Society
257
J . E. deJosselin deJong et al. 1965). Also a scanning device has been built for this double beam instrument (de Josselin de Jong et al., 1973) which proved to be useful for dry mass determinations of cultured human fibroblasts and amniotic fluid cells (Galjaard et al., 1974a, b; van der Veer et ul., 1978). However, the time required for dry mass measurements of large numbers of cells is quite long and the critical stability sometimes requires time-consuming adjustment procedures. The present communication deals with the construction of an experimental setup for scanning microinterferometry with the Jamin-Lebedef interference microscope which enables relatively rapid measurements on large numbers of objects without difficulties caused by overlapping double images. 1.
GENERAL P R I N C I P L E AND OUTLINE O F THE INSTRUMENT
In the Zeiss Jamin-Lebedef interference microscope (Gahni, 1962; Piller, 1962) equipped with a 40-times objective, the two interfering light beams pass through the microscopical preparation with a mutual distance of f 175 pm. If this instrument is used for measurements in preparations with closely packed cells, there is a good chance that the ‘reference beam’ passes through a neighbouring cell thus causing a ‘ghost’ image which overlaps the image of the cell to be measured (Fig. 1). The principle of the present instrument is based on measurements of both the optical path difference of the cell to be investigated and of the cell causing the overlapping ‘ghost’ image. This is based on the following considerations: If: ‘OPD cell’ = the OPD between a point in the cell the dry mass of which has to be determined and the free background; ‘OPD ref. cell’ = the OPD between a point in the cell causing an overlapping ‘ghost’ image because it is passed by the reference beam and the free background; ‘OPD cell/ref. cell’ = the OPD between
Fig. 1. Images and ‘ghost’ images of cultured Hela cells. The image of cell 1 is partly covered by the ‘ghost’image of cell 2. Also the optical path difference of the cell in the middle cannot be measured because of an overlapping ‘ghost’
image.
258
Microinterferometry, correction ‘ghost’ images
+
points in the two mentioned cells. Then: ‘OPD cell’ = ‘OPD cell/ref. cell’ ‘OPD ref. cell’. Hence it is possible to calculate the ‘OPD cell’ by addition if the ‘OPD ref. cell’ is known. With the setup described a rectangular ‘reference field’ containing the ‘reference cell’ is scanned first. The OPDs of all measured points (that is the OPD between these points and the free background) are stored in a computer memory in a twodimensional array (the ‘reference array’). Then the scanning stage is shifted over a distance similar to the difference between the relative positions of the interfering light beams. Subsequently a rectangle of the same dimensions now containing the cell which is overlapped by the ‘ghost’ image of the previous cell is scanned. The values of all points are also stored in a two-dimensional array. The OPD of a point in the second cell is calculated by addition of the value of this point stored in the second array and the average value of five points located around the corresponding point in the ‘reference array’. The results are stored in a new ‘reference array’. The stage is now shifted again and the described procedure is repeated till the cell to be investigated has been scanned. The optical part of the setup consists of a Zeiss universal microscope with a 0.5 pm scanning stage and the Jamin-Lebedef interference equipment with the Pol.Int. I1 40/0.65 objective. With a C 5 x projective a magnification of 200 x is attained. As a light source an Osram H.B.O. 100 W2 lamp is used. The analyser is a permanently rotating polarizer, according to Lomakka (1963), driven by a selfstarting 50 Hz a.c. motor, which is placed at our disposal by Carl Zeiss. The measuring equipment consists of the Leitz U.V. microscope photometer attachment containing an E.M.I. 9558QA photomultiplier that has adequate sensitivity for green light (546 nm) and which is insensitive to the polarization direction of the light. In this system no mirrors or optical components affect the polarized light. The diameter of the measuring field is 0.3 mm (1.5 pm in the object plane) or 0.6 mm (3 pm in the object). The power supply for the photomultiplier is an Oltronix B 2.2K-20HR. The actual measurements are carried out by a phase angle detector. A push-button keyboard is used for steering the scanning stage, selecting the scanning field and starting the scanning procedure. A DEC PDP 11-45 computer with Laboratory Peripheral System (LPS-11) is used for storage and calculation of the data and for the control of the scanning process. The results are printed on a line printer and displayed on a storage display, which is also used for interactive communication. 2.
PHASE ANGLE DETECTION
The intensity of the light, after it has passed the rotating polarizer, varies sinusoidally as a function of the angle of rotation. The phase angle of this curve is a linear function of the phase difference between the interfering light beams. For the measurement of the phase angle, a disc with a slit rotates together with the analyser between a photoelectric cell and a small red lamp. In this way an electric pulse (the analyser pulse) which correlates with a certain position of the analyser, is generated 50 times per second. A second signal is produced by a phase discriminator, which generates a pulse (the ‘discriminator pulse’) each time the photomultiplier signal reaches a maximum value thus indicating the top of the sinusoid. In Fig. 2 the relation between these pulses is shown in a time diagram. Once the measurement of a certain point has been finished, the next analyser pulse (the ‘starting pulse’) generates a trigger signal for the movement of the stage to the next point. The third analyser pulse opens an electronic gate allowing clock pulses to stream into a couriter until the gate is closed by the ‘discriminator pulse’. The number of counted clock pulses is proportional to the phase angle to be measured. 259
J. E. de Josselin de Jong et al. analyser pulses
starting pulse:stage starts movement t o next point
gate is opened
registrated photomultiplier signal discriminator pulse gate is closed
counted clock pulses
next s t a r t i n g pulse
Fig. 2. Time diagram of the electrical signals generated during the measurements.
3.
CALCULATION OF T H E OPTICAL P A T H DIFFERENCE
During the standard measuring routine the following three phase angles are measured: 010: when both light beams are passing through the free background; a1:when the ‘reference beam’ passes through the free background and the measuring beam passes through the ‘reference cell’; 012: when the ‘reference beam’ passes through the ‘reference cell’ and the ‘measuring beam’ passes through the cell to be measured. In the time diagrams in Fig. 3 the relation between these phase angles and the OPDs is shown under different ‘optical circumstances’. The OPD is expressed in degrees (360” corresponds with 1X). In Fig. 3(a) and Fig. 3(b) the factor (“2-010) is equal to the difference between the ‘OPD cell’ and ‘OPD ref. cell’. In this case: ‘OPD cell’ = ‘QPD ref. cell’ + ‘OPD cell/ref. cell’
- (q-010)+ (012-
+ ff2 -2010. Fig. 3(c) shows the situation when + ‘OPD ref. cell’) is greater than 360”. In a0)= 011
(010
this case a wrong ‘discriminator pulse’ is registered one wavelength too early. This difficulty can be avoided if the sum of a0 and the measured OPD does not exceed 360’. Because this is limiting the maximum measurable OPD, the interference equipment has to be adjusted in such a way that 010 is relatively small. However, when “0 is very small, irregularities in the free background or instrumental deviations sometimes may cause this phase angle to become negative. This would cause the ‘registered’ 010 to vary between either small values or values of about 360”. In the present instrument a0 is always about 36” (O.lh), which means 260
analyser pulse
6
w
a1
I ~
iIIIIIIIIIIIIIIIi I (IIIIIIIIIIIIIIIIIIIIII
OPD r e f . c e l l OPD c e l l l r e f . c e l l
discriminator pulses
'3
f i g . 3 a OPD c e l l > OPD r e f . c e l l OPD c e l l = ( a 1 - a o ) + ( a 2 - a o ) analyser 7
+I ,
illlllllllt
.
t
pk
+
a1-ao
I11111111
1,,,,11,111111111111,1,11,1,1
counted clock p u l s e s
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I
-
fig 3 c
ao+OPD ref c
-,
pulse
OPD r e f . cell
t
discriminator pulses
i
counted clock p u l s e s
~
ilIIIIIIIIIIIIIIi
illllllllllllllllllllllllllllllli
f i g . 3 b O P D c e l l < OPD r e f . c e l l OPD c e l l = ( a l - a o ) + ( a 2 - a 0 ) .
w
2
Fig. 3. Relation between the phase angles and OPD under different circumstances. ao=b through the free background. al=reference beam passes through the free background through the reference cell. a2 =reference beam passes through the reference cell and the the cell to be measured.
J. E. de Josselin de Jong et al. that the maximum measurable OPD is about 0.89, allowing small deviations during the measurements. In Fig. 3(d) a situation is illustrated where ‘OPD cell/ref. cell’ is smaller than - ao. This means that, theoretically, the ‘discriminator pulse’ should be registered before the ‘analyser pulse’. In practice a ‘discriminator pulse’ is registered one period later and the ‘QPD cell’ has to be calculated as follows : ‘OPD ref. cell’
= 011- a0
‘QPD cell/ref. cell‘ = 0 1 ~ 0 1~ A Hence ‘OPD cell’
= “2
+ a1 -
2010 - h
+
Usually this situation can easily be recognized because (01.2 a1 - 2010) will be greater than 1A. However, when ‘OPD cell’ is zero or less ‘OPD cell/ref. cell’ will be equal to or less than - ‘OPD ref. cell’. In that case the wrong discriminator pulse might be recorded while the factor (az “1 - 2010) is less than 1A. Therefore it is verified after each measurement whether (az 011 - 2010) is greater than a limit the value of which itself is smaller than 1h. In this case l h is subtracted. As a limit a value of 0.8% is chosen because this fulfils the demands under the circumstances illustrated in Fig. 3(c). When measurements without ‘reference cells’ are carried out, ‘OPD cell’ =
+
+
a1 - O10.
4.
S T A N D A R D P R O C E D U R E FOR M E A S U R E M E N T
The standard procedure for measurement consists of the following steps : - the optical system is put in the adjustment mode; - the upper limit (U) of the measurable OPD is chosen; - the location and dimensions of the scanning field are fixed; - the operator answers the question ‘reference cells ?’ on the display. When the answer is ‘yes’, the stage runs to the reference field. This can be repeated as often as is necessary and possible with respect to the preparation. - by marking three points an L-shaped figure is chosen in the background; - the optical system is switched to measuring conditions; - the phase angle of a point in the background is verified and if necessary adjusted to & 36” by tilting the condensor; - after a start button has been pressed, the L-shaped figure in the background is scanned and the average phase angle of the measured points on this line is taken as an; - if ‘reference cells’ have to be measured the first ‘reference field’ is scanned (011); - the ‘OPD ref. cell’ is calculated (011- 010); - the next field with the next cell is scanned ( “ 2 ) ; - for every point the following calculations are made: S = ‘OPD ref. cell’ + a2 - a0 If S > U then S’=S-h If S < U then S’=S - the last scanned cell becomes the ‘reference cell’ with ‘OPD ref. cell’ = S’; - the next field with the next cell is scanned and so on until the cell to be measured is scanned. The IOPD of this cell is calculated by addition of the OPDs of the points in the field which has been scanned last.
5.
T E S T I N G OF T H E V A L I D I T Y O F T H E P R O C E D U R E
With the method described the error in the measurements caused by overlapping
262
Microinterferometry, correction ‘ghost’ images ‘ghost’ images, is corrected by adding the OPD of the objects producing these ‘ghost’ images, to the measured value. This approach is only valid if the same but opposite OPD is measured in the image and ‘ghost’image. T o verify if this condition has been fulfilled, point measurements have been carried out with the Zeiss microinterference refractometer. This consists of an object slide with a spherical segment with a diameter of about 140 pm which has been covered with a drop of immersion oil and a coverglass. The OPDs have been determined in the middle of the spherical segment with the phase difference between the light beams in the free background set to different values by tilting the condensor. The results shown in Fig. 4 indicate that the measured OPDs vary in a reproducible way with the phase difference in the background. Further, the curve for the ‘ghost’ image is very similar to that for the normal image, but it is shifted in a horizontal direction over a distance of about 0.4h. To a high degree this contributes to a difference between the absolute values of the OPDs, measured in the image and ‘ghost’ image. To test the complete equipment Sepharose beads of different sizes (40-70 pm diameter) mounted in Depex have been measured both when their image was overlapped by the ‘ghost’ image of another bead and when their image was completely free from ‘ghost’ images. This has been achieved by locating the positions of the beads and by repeating the measurement after the object had been turned through 180”. The measurements have been carried out with a measuring spot of 3 pm (in the object plane) and a distance between the measuring points of six stage steps ( 3 pm). The maximum OPD in the largest bead was about 0.4h. The results of the two series of measurements are shown in Table 1. The measured IOPD has been plotted also against the diameter of the beads. The results are shown in Figs. 5 and 6. For both series of measurements the correlaI
0.41
OPD imaae
-0.41
-0.40
-0.39 -0.38
sum
- 0 01 -1 A
-0 5 A OA 05A background phase difference
1A
Fig. 4. The OPD of the Zeiss microinterference refractometer measured in the image and the ‘ghost’ image and the sum of these OPDs plotted for various phase differences measured when both light beams are passing the free background.
263
J. E. de Josselin de Jong et al. Table 1. IOPD of Sepharose beads measured with and without overlapping ‘ghost’ images (in arbitrary units). The numbers are also mentioned in Figs. 5 and 6. IOPD with ‘ghost’ image 11973 15016 17804 28394 28760 36227 37100 39736 43680 47095 488 11 56064
Bead no. 1 2 3 4 5 6 7 8 9 10 11 12
IOPD without ‘ghost’ image 10045 11185 16700 24017 30211 37164 38775 38406 49795 52929 45933 58131
tion coefficient was 0.98. The standard error of estimate o f y on x (5’’. x) was 3786 for the measurements without ‘ghost’ image and 3434 for the measurements with ‘ghost’ image. The same measurements have been carried out with Hela cells mounted in 0.97/, NaC1. I n this case the measuring spot had a diameter of 1.5 pm and the distance between the measuring points was also 1.5 pm. A difference between the measurements with and without overlapping ‘ghost’ image of about lo:(, of the latter has been found. The measured Sepharose beads and Hela cells were always situated in the preparations in such a way, that only one ‘reference field‘ needed to be scanned with the measurements with ‘ghost’ image.
p
50
0 L c
0 40 L
0
30 40 I
50
60
I
1
t
10
1
20
70 1
-
1
30
d31104
diameter d ( p m )
Fig. 5. IOPD of Sepharose beads measured without ‘ghost’ image plotted against their diameter.
264
Microinterferometry, correction ‘ghost’ images h
v)
.= 6 0 c
c
3
50
L A
!?
c
0
40
L
0 v
30 0
;2 0 c
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10 50 I
30 4 0 I
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70
I
20
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diameter d ( p m )
Fig. 6 . IOPD of Sepharose beads measured with ‘ghost’ image plotted against
their diameter. To test the reproducibility of the scanning procedure itself, the same Hela cell was scanned ten times. A standard deviation of 1.1 was found. The scanning time for a Hela cell is about 100 s and when a reference cell has to be measured this time is doubled. The adjustment procedure can be limited to the resetting of the scanning area, which takes about 45 s. 6.
DISCUSSION
The similarity of the two first curves in Fig. 4 and their shifting with respect to each other possibly can be explained as follows: The curve for the ‘ghost’ image has been shifted with respect to the curve for the image over a distance of approximately 0.4X, which corresponds to the OPD of the measured object. We consider a point on the curve for the image and the corresponding point on the curve for the ‘ghost’ image, indicating the measuring results for measurement (a) in the image and measurement (b) in the ‘ghost’ image. The difference between the background phase differences for measurements (a) and (b) is equal to the OPD of the measured object. The background phase difference is directly related to the background phase angle. Hence, also the difference between the background phase angles for measurements (a) and (b) is equal to the OPD. The measured OPDs are equal to the difference between the background phase angles and the phase angles measured in the image. In the normal image the phase angles are larger than those of the background and a positive OPD is measured. In the ‘ghost’ image it is just the reverse and a negative OPD is measured. Hence the background phase angle for measurement (b) in the ‘ghost’ image is equal to the phase angle measured in the image for measurement (a). Consequently, for the measurements (a) and (b) the measured OPDs are equal to the difference between exactly the same phase angles, which possibly is an explanation for the correspondence of the mentioned points on the
265
J. E. de Josseliiz de Jong et al. two curves in Fig. 4. T o examine whether the deviations illustrated in this figure are caused by the interference system itself or by the rotating analyser, the analyser housing has b-en turned 45” around the optical axes, and the measurements were repeated. Deviations in the analyser would cause a shifting of the second curve with respect to the plotted background phase difference. No shifting would be obtained if the variations in the measured OPD are only due to the interference system. The experimental results showed a completely transformed curve with the second measurement. This means that the variations are caused by the rotating analyser and the interference system together. Goldstein & Hartmann-Goldstein (1974) have discussed the deviations caused by the interference system. They also have plotted the measured OPD against the background phase difference and have found a sinusoidally fluctuating deviation, which could be diminished by reducing the diameter of the aperture diaphragm, but could not be avoided completely. A second cause of the variation probably is introduced by variations in the angular velocity of the analyser. However, these systematic errors will not influence the results very much if comparative measurements are carried out, provided that the interference system is not readjusted during the measurements. Difficulties may be caused by the astigmatism of the ‘ghost’ image. With an opened aperture diaphragm the ’ghost’ image has two pronounced directions corresponding with the diagonals in the field of view. However, if the aperture diaphragm is closed completely almost no asymmetric distribution of light is visible in the ‘ghost’ image. In order to compare the quality of the ‘ghost’ image with that of the normal image, both images of Sepharose beads with different sizes (50-80 pm) have been scanned along the mentioned diagonals. The results showed that the difference between the diameters of the ‘ghost’ image and the normal image is about 4 pm on both sides of the image along one diagonal. No difference has been found along the other diagonal. Another critical point is the size of the measuring diaphragm (Goldstein & Hartmann-Goldstein, 1974). When the distance between parts with a different OPD is smaller than the diameter of the measuring spot, the phase angle of a curve, which is the sum of various sinusoids, has to be determined. If errors are to be avoided, this phase angle ought to be the average of the phase angles belonging to the different OPDs of the parts concerned and the contribution of each part has to be proportional to its area. In the most simple case the measuring spot covers an image divided in two parts with different OPDs. If one part has an area of x units and the other part an area of (1 - x) units the corresponding sinusoids can be expressed as y l = x sin w t and y2=(l -x) sin ( w t p), (Goldstein & Hartmann-Goldstein, 1974). If the first curve has a phase angle 01 and the abovementioned condition ought to be fulfilled, the phase angle of the resulting curve ought to be equal to:
+
+
x .a (1 - X)(“
+p) = + (1- x)p) O1
In reality, however, this phase angle is, according to Goldstein & HartmannGoldstein (1974), equal to: a
(1-x) sin p + arctan x+(1-x)cOsp
In Fig. 7 the differences between the above-mentioned functions is plotted against p for various values of x. Goldstein & Hartmann-Goldstein (1974) have shown that the errors arising when the edge of the object is crossing the diaphragm compensate each other for each scan line provided that the distance between the measuring points is smaller than
266
Microinterferometry, correction 'ghost' images
252' W
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Fig. 7. Difference between the desired phase angles and the measured phase angles, when two object parts with surfaces x and 1 - x and a mutual OPD are measured together, plotted for various values of x and 9. 360" corresponds with l h .
the diameter of the measuring diaphragm and the OPD is smaller than 0% It will be seen that the errors also compensate each other when the border between two areas with a mutual OPD less than 0.5X is passing the diaphragm. However, with the relatively slow scanning instrument described here the distance between the measuring points will mostly be equal to the diameter of the measuring spot. Hence, the errors mentioned above only compensate each other if there are enough scan lines to cancel them. out statistically. Also it has to be taken into account that images and 'ghost' images are measured together. Therefore relatively large phase angle differences may appear within a short distance. Ideally the diameter of the measuring spot should be equal to the resolution of the microscope. However, in interference microscopy the aperture diaphragm has to be small as well (Davies & Deeley, 1956; Hale, 1958; ROSS,1967; Goldstein & Hartmann-Goldstein, 1974) and the same is true to limit the astigmatism of the 'ghost' image. A small aperture diaphragm results in low light intensities and hence the minimum size of the measuring spot will partly b t determined by the minimum permissible signal to noise ratio. Because of the properties of the unsharp and slightly astigmatic 'ghost' image it is impossible to carry out measurements in this image with a high resolution. Therefore the measuring result of every point in the cell to be measured is corrected with the average value of five points in the 'reference cell'. Consequently the instrument can only be used for integrating measurements.
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J. E. deJosselin deJong et al. Based on the standard deviation found with repeated measurements on a Hela cell a lower Sy.x might have been expected for the measurements without ‘ghost’ image on the Sepharose beads. Probably the diameter of the beads is not the ideal indication for their content. The differences between the measurements with and without ‘ghost’ image may partly be caused be adjustment procedures after turning the preparation through 180”. Because of a gradient in the thickness of the mounting medium (‘wedge effect’) the interference system had to be readjusted each time the preparation was turned in order to obtain the same ‘background phase difference’. During the standard procedure of measurement the ‘wedge effect’ will only influence the measurements if the wedge has a lenticular component (Ross, 1967). With respect to the measurements with ‘ghost’ images the setup can only be used for integrated measurements of objects of the order of 15 pm or larger. It then offers the possibility of dry mass determinations in closely packed microscopic objects without time-consuming adjustment procedures. The instrumental setup may, for instance, contribute to cytochemical studies on individual cultured cells, where the activity of several lysosomal enzymes varies with the dry mass of the cell (van der Veer et al., 1978). Also in the interpretation of enzyme activities after somatic cell hybridization (Galjaard et al., 1974b) dry mass determinations of cultured cells may be important. If there is an object-free strip on two sides of the microscopical preparation the cells just beside these strips are not overlapped by ‘ghost’ images. The OPD of these cells can easily be measured and can be used to correct possible errors caused by their ‘ghost’ images when other cells are measured. If the ‘wedge effect’ can be avoided in this way the whole preparation can be scanned without interruptions. Together with automatic image analysing systems this introduces the possibility of automatic classification by dry mass measurements on large numbers of cells. ACKNOWLEDGMENTS
The authors wish to thank Professor Galjaard for critically reading the manuscript and Ir. F. A. van Hall, head of the department for small computers and data communication of the C.R.I. in Leiden, for giving us the opportunity to use the computer system of the Institute. We wish to thank Professor P. van Duijn for critical and stimulating discussion. The support of Ing. R. van der Baan, Drs B. Hesper and H. Hofstra (C.R.I.) and J. Bonnet (Histochemistry, Leiden) is gratefully acknowledged. We wish to thank Dr J. Gham and Dr W. H. G. Cebulla (Carl Zeiss, Oberkochen, Germany) for their advice and critical comment. Illustrations have been made by J. Fengler, T. van 0 s and W. J. Visser. References Barer, R. (1952) Interference microscopy and mass determination. Nature, 169, 366. Boguth, W. (1974) Scanning-Interferenzmikroskopie mit dem Mikroskop-Photometer. Microsc. Acta, 76, 28. Carlson, L. (1970a) A scanning, recording and integrating microinterferometer. Histochemie, 21, 289. Carlson, L. (1970b) Cytochemical determination of dry muss. Doctorate Thesis, Institute of Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institute, Stockholm. Carlson, L., Caspersson, T., Lomakka, G. & Silverbige, S. (1970) A rapid scanning and integrating microinterferometer for large-scale population work. In : Introduction to Quantitatiae Cytochemistry (Ed. by G. L. Wied and G. F. Bahr), Vol. 2, p. 117. Academic Press, New York. Caspersson, T., Carlson, L. & Svensson, G. (1954) A scanning interference microscope arrangement. Exp. Cell Res. 7, 601.
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Microinterferometry, correction ‘ghost’ images Davies, H.G. & Deeley, E.M. (1956) An integrator for measuring the dry mass of cells and isolated components. Exp. Cell Res. 11, 169. Davies, H.G. & Wilkins, M.H.F. (1952) Interference microscopy and mass determination. Nature, 169, 541. Gahm, J. (1962) Die Untersuchung anisotroper Praparate mit der Interferentzanordnung nach Jamin-Lebedeff. Zeiss-Mitt. 2, 389. Galjaard, H. (1962) Histochemisch en interferometrisch onderzoek van hyalien kraakbeen. Doctoral Thesis, University of Leiden. Galjaard, H. & Szirmai, J.A. (1965) Determination of the dry mass of tissue sections by interference microscopy. J. roy. microsc. SOC.84, 27. Galjaard, H., van Hoogstraten, J.J., de Josselin de Jong, J.E. & Mulder, H.P. (1974a) Methodology of quantitative cytochemical analysis of single or small numbers of cultured cells. Histochem. J. 6, 409. Galjaard, H., Hoogeveen, A., Keyzer, W., de Wit-Verbeek, E. & Vlek-Noot, C . (1974b) The use of quantitative cytochemical analysis in rapid prenatal detection and somatic cell genetic studies of metabolic diseases. Histochem. 3. 6, 491. Goldstein, D. J. & Hartmann-Goldstein, I.J. (1974) Accuracy and precision of a scanning and integrating microinterferometer. 3, Microsc. 102, 143. Grehn, J. (1958) Das Durchlicht-Interferenz-Mikroskop- ein Instrument des Biologen. Umschau Wiss. Techn. 19, 602. Hale, A. J. (1958) The Interference Microscope in Biological Research. Livingstone, Edinburgh. Horn, W. (1958) Micro-Interferentz 11. In:Jahrbuch fur Optik und Feinmechanik. Pegasus Verlag, Wetzlar. de Josselin de Jong, J.E., Boender, W., Carlson, L. & Galjaard, H. (1973) A scanning device for the double beam Leitz interference microscope. Histochemie, 35, 127. Lomakka, G. (1963) A rapid scanning and integrating microinterferometer. Acta Histochem. Jena, Suppl. 6, 393. Piller, H. (1962) Durchlicht- Interferentzmikroskopie nach dem Jamin-Lededeff-Prinzip. Zeiss-Mitt. 2, 309. Ross, K.F.H. (1967) Phase Contrast and Interference Microscopy f o r Cell Biologists. Edward Arnold, London. Smith, F.H. (1972) A laser-illuminated scanning microinterferometer for determining thc dry mass of living cells. Microscope, 20, 153. Svensson, G. (1957) A scanning interference microphotometer. Exp. Cell Res. 12, 406. van der Veer, E., Kleijer, W.J., de Josselin de Jong, J.E. & Galjaard, H. (1978) Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods, combined with interference microscopy. Hum. Genet. 40, 285.
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