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Surface finish interference microscopy of biological objects
262 SURFACE
FINISH
INTERFERENCE
BIOLOGICAL
MICROSCOPY
OF
OBJECTS1
S. IVERSEN Cancer Research Department,
The Royal Beatson Memorial
Hospital,
Glasgow, Scotland
Received June 30, 1958
WITH th e surface finish interference microscope furnished with suitable dielectric objectives, as for example in the Baker instrument, it is possible to measure the height, or depth, of fixed stained or unstained biological objects to within a fraction of the wavelength of the illuminating light. When a plane surface is viewed in this microscope the field will be seen traversed by equally spaced parallel fringes. As any linear separation between two adjacent fringes always indicates a difference in vertical direction of J/2 (for green mercury light = 0.273 p), the width and linear separation of the fringes will depend upon the degree of tilt in relation to the optical axis, and the fringe direction will depend upon the direction of the tilt. If the surface has flaws and defects, a deformation of the fringes will take place. The pattern of this deformation will depend upon the shape of the object and its orientation in relation to the optical axis. The extent of the fringe deformation gives an absolute measurement of the difference in vertical direction between the adjacent surface and the measured point. Thus the tail of the fixed, unstained mouse spermatozoon in Fig. 1, has a height ( = thickness) at the point C of 112 x ACIAB = 0.273 x 0.83 = 0.23 p. If the object is spherical with a radius Rp, the fringe deformation will form a system of concentric circles, the number of which will be R/0.273, and their radius will be l/Re - (0.273 x r~)~, where n is an integer having the values 1, 2, 3, . . . R. Fig. 2 shows the appearance of nuclei from films of suspensions of kidney and liver nuclei from the same mouse, isolated simultaneously using the method given by Frazer and Davidson [ 11. The films of suspensions were dried at room temperature, fixed for two hours in acetic acid-ethanol (1:3), rinsed for 6 hours in running tap water and then dried at room temperature. Fig. 2 A and B show liver nuclei, 2 C and D kidney nuclei, and 2 E a (Feulgen stained) liver nucleus seen under greater tilt. From the interference patterns it can be deduced that nuclei prepared in this way rest flatly on the slide with an upward convexity. The height at any point of the nuclei, viewed at normal incidence, can be determined by counting the number of fringes from the periphery to the point in question. This can be done either densitometrically or visually. In both cases the accuracy will depend upon the accuracy with which fractional fringes can be determined. The distance between a dark and a light fringe is l/2 fringe ( = l/4 12);if therefore the fractional fringe is determined to the nearest dark or light fringe the maximum percentage error will be 25/n, where n is the number of dark and light fringes counted. As the occurrence of the nuclear maximum thickness being an exact multiple of L/2 is a rare event, the occurrence of fractional fringes will be the rule. It is, however, 1 This investigation Experimental
was supported
Cell Research 1.5
by a grant
from the British
Empire
Cancer Campaign.
Surface finish interference microscopy
263
E Fig. 2.
Fig. 1. Fig. l.-Fixed,
unstained
mouse spermatozoon.
Total magnification
1150 x .
Fig. 2.-&B, fixed, unstained mouse liver nuclei. Polar axis parallel to the optical axis. C- D, fixed, unstained mouse kidney nuclei. Polar axis parallel to the optical axis. E, fixed, Feulgen stained mouse liver nucleus. Polar axis not parallel to the optical axis.
possible to place the nucleus in such a way on the stage that, while retaining normal incidence, a light or a dark fringe will fall on the centre of the nucleus. In this case the only occurring fractional fringe will be at the periphery, where the curvature is greatest. Fig. 3 is a scatter diagram of the visually determined maximum thickness (to the nearest l/2 fringe) and diameter (to the nearest l/4 mm) measured on enlargements giving a total magnification of 1150 x of 36 randomly chosen kidney nuclei and 36 liver nuclei from the preparations mentioned above. As it is seen from this diagram the kidney nuclei have a smaller diameter (average 7.32 ,u; st.e. =0.1393) than the liver nuclei (average 12.17 p; st.e. =0.3100). As the degree of variation in diameter amongst the two types is not comparable (F = 4.95), a f-test for the difference between the two means is not applicable. The maximum thickness of the two types seems to be similar as judged by the diagram. The average maximum thickness for the kidney nuclei is 2.875 fringe ( co.785 ,u); st.e. =0.0877; and for the liver Experimental
Cell Research 15
264
S. Iversen
FRINGES 5-
.
.
4-
.
00
:
0; 0
3-
..
ogoog~oo~o
l
00
2-
000
0.
0
00000
.
0.
.
. ...
.
:
.
.
..:
l
. .
.
;
.
.
.
IDIAMETER
Fig. 3.-Scatter diagram kidney nuclei. Dots: Liver (mm).
X f 150.
of maximum nuclear thickness and nuclear diameter. Open circles: nuclei. Ordinate: number of fringes. Abscissa: Nuclear diameter x 1150
nuclei 3.222 fringe ( = 0.88 ,u); st.e. = 0.1152. The degree of variation is comparable (F = 1.73), and a t-test for the difference between the two means shows this to be insignificant. The kidney nuclei have thus a greater curvature than the liver nuclei which, if preparational selection can be excluded, indicates that the two types of nuclei have either different masses or different structure. REFERENCE 1. FRAZER, S. C. and DAVIDSOS, J. N., Ezpfl.
Experimental
Cell Research 15
Cell Research 4, 316 (1953).
FINISH
INTERFERENCE
BIOLOGICAL
MICROSCOPY
OF
OBJECTS1
S. IVERSEN Cancer Research Department,
The Royal Beatson Memorial
Hospital,
Glasgow, Scotland
Received June 30, 1958
WITH th e surface finish interference microscope furnished with suitable dielectric objectives, as for example in the Baker instrument, it is possible to measure the height, or depth, of fixed stained or unstained biological objects to within a fraction of the wavelength of the illuminating light. When a plane surface is viewed in this microscope the field will be seen traversed by equally spaced parallel fringes. As any linear separation between two adjacent fringes always indicates a difference in vertical direction of J/2 (for green mercury light = 0.273 p), the width and linear separation of the fringes will depend upon the degree of tilt in relation to the optical axis, and the fringe direction will depend upon the direction of the tilt. If the surface has flaws and defects, a deformation of the fringes will take place. The pattern of this deformation will depend upon the shape of the object and its orientation in relation to the optical axis. The extent of the fringe deformation gives an absolute measurement of the difference in vertical direction between the adjacent surface and the measured point. Thus the tail of the fixed, unstained mouse spermatozoon in Fig. 1, has a height ( = thickness) at the point C of 112 x ACIAB = 0.273 x 0.83 = 0.23 p. If the object is spherical with a radius Rp, the fringe deformation will form a system of concentric circles, the number of which will be R/0.273, and their radius will be l/Re - (0.273 x r~)~, where n is an integer having the values 1, 2, 3, . . . R. Fig. 2 shows the appearance of nuclei from films of suspensions of kidney and liver nuclei from the same mouse, isolated simultaneously using the method given by Frazer and Davidson [ 11. The films of suspensions were dried at room temperature, fixed for two hours in acetic acid-ethanol (1:3), rinsed for 6 hours in running tap water and then dried at room temperature. Fig. 2 A and B show liver nuclei, 2 C and D kidney nuclei, and 2 E a (Feulgen stained) liver nucleus seen under greater tilt. From the interference patterns it can be deduced that nuclei prepared in this way rest flatly on the slide with an upward convexity. The height at any point of the nuclei, viewed at normal incidence, can be determined by counting the number of fringes from the periphery to the point in question. This can be done either densitometrically or visually. In both cases the accuracy will depend upon the accuracy with which fractional fringes can be determined. The distance between a dark and a light fringe is l/2 fringe ( = l/4 12);if therefore the fractional fringe is determined to the nearest dark or light fringe the maximum percentage error will be 25/n, where n is the number of dark and light fringes counted. As the occurrence of the nuclear maximum thickness being an exact multiple of L/2 is a rare event, the occurrence of fractional fringes will be the rule. It is, however, 1 This investigation Experimental
was supported
Cell Research 1.5
by a grant
from the British
Empire
Cancer Campaign.
Surface finish interference microscopy
263
E Fig. 2.
Fig. 1. Fig. l.-Fixed,
unstained
mouse spermatozoon.
Total magnification
1150 x .
Fig. 2.-&B, fixed, unstained mouse liver nuclei. Polar axis parallel to the optical axis. C- D, fixed, unstained mouse kidney nuclei. Polar axis parallel to the optical axis. E, fixed, Feulgen stained mouse liver nucleus. Polar axis not parallel to the optical axis.
possible to place the nucleus in such a way on the stage that, while retaining normal incidence, a light or a dark fringe will fall on the centre of the nucleus. In this case the only occurring fractional fringe will be at the periphery, where the curvature is greatest. Fig. 3 is a scatter diagram of the visually determined maximum thickness (to the nearest l/2 fringe) and diameter (to the nearest l/4 mm) measured on enlargements giving a total magnification of 1150 x of 36 randomly chosen kidney nuclei and 36 liver nuclei from the preparations mentioned above. As it is seen from this diagram the kidney nuclei have a smaller diameter (average 7.32 ,u; st.e. =0.1393) than the liver nuclei (average 12.17 p; st.e. =0.3100). As the degree of variation in diameter amongst the two types is not comparable (F = 4.95), a f-test for the difference between the two means is not applicable. The maximum thickness of the two types seems to be similar as judged by the diagram. The average maximum thickness for the kidney nuclei is 2.875 fringe ( co.785 ,u); st.e. =0.0877; and for the liver Experimental
Cell Research 15
264
S. Iversen
FRINGES 5-
.
.
4-
.
00
:
0; 0
3-
..
ogoog~oo~o
l
00
2-
000
0.
0
00000
.
0.
.
. ...
.
:
.
.
..:
l
. .
.
;
.
.
.
IDIAMETER
Fig. 3.-Scatter diagram kidney nuclei. Dots: Liver (mm).
X f 150.
of maximum nuclear thickness and nuclear diameter. Open circles: nuclei. Ordinate: number of fringes. Abscissa: Nuclear diameter x 1150
nuclei 3.222 fringe ( = 0.88 ,u); st.e. = 0.1152. The degree of variation is comparable (F = 1.73), and a t-test for the difference between the two means shows this to be insignificant. The kidney nuclei have thus a greater curvature than the liver nuclei which, if preparational selection can be excluded, indicates that the two types of nuclei have either different masses or different structure. REFERENCE 1. FRAZER, S. C. and DAVIDSOS, J. N., Ezpfl.
Experimental
Cell Research 15
Cell Research 4, 316 (1953).