Optics: measuring and testing – Shape or surface configuration – Triangulation
Reexamination Certificate
2002-08-13
2004-05-04
Pham, Hoa Q. (Department: 2877)
Optics: measuring and testing
Shape or surface configuration
Triangulation
Reexamination Certificate
active
06731390
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
The invention relates to a process and device for depth selection in microscope images.
b) Description of the Related Art
It is known to carry out depth measurements in microscope images as well as to suppress image portions located outside of the focal plane by means of structured illumination (AXIOMAP by ZEISS, WO 97/06509, WO 98/45745).
In U.S. Pat. No. 5,493,400 (DE 9308486U), an inclined grating projection is generated by means of wedge-shaped glass bodies with changeable orientation.
These methods are based on methods derived from interference microscopy in which periodic structures projected in the object plane are converted to interference patterns and evaluations which are made possible in this way are carried out.
For this purpose, gratings are imaged in the object planes and displaced by integral fractions of the grating constant either continuously over a certain period of time (phase shift method) or stepwise (phase step method). An image is acquired in every time interval or of every position of the grating by means of a pixel-synchronous CCD camera and the images are calculated together.
The conditions are assessed most simply when a grating with a cos
2
-shaped or sin
2
-shaped intensity variation is shifted three times by ¼ grating constant in the direction of the grating periodicity, e.g., in the x-direction. (The method functions in principle with shifts by each integral fraction <½ of the grating constant).
The image of the initial position is interpreted as an image with sinusoidal intensity modulation vertical to the direction of the grating bar or line (sin x), the next as cos x, and the following as −sin x and the last as −cos x.
Therefore, information is provided for every image point representing the brightfield intensity multiplied by the respective grating phase. Further relevant information can be derived from this output data.
The four images can be described as follows:
1. I
xy1
=0.51
obxy
*(1+m*sin x)
2. I
xy2
=0.51
obxy
*(1+m*cos x)
3. I
xy3
=0.51
obxy
*(1−m*sin x)
4. I
xy4
=0.51
obxy
*(1−m*cos x),
where I
obxy
is the intensity (reflectivity, transmission, fluorescence) contributed by the object location x, y to the imaging, and m is the modulation factor of the grating imaging. Subtracting 3 from 1 and 4 from 2 gives:
I. I
xy
=I
obxy
*m*sin x
II. I
xy
=I
obxy
*m*cos x.
Like components vanish with this procedure.
Conversely, by adding 3 to 1 and 4 to 2, the modulation of the image content with the angle functions vanishes in both cases. Brightfield images A
1
and A
2
are obtained:
A
1
: I
xy
=I
yx3
+I
xy1
=I
obxy
,
A
2
: I
xy
=I
xy4
+I
xy2
=I
obxy
.
i.e., two complete brightfield images are contained in these four partial images.
The modulation m with the angle functions has impressed information on these images about the distance from the exact focal plane. The modulation factor m of the grating imaging changes depending on the utilized grating constant, objective aperture, wavelength and the distance from the focal plane.
When using an objective, a light source and a grating, the first influencing factors remain constant, i.e., the modulation factor in a given arrangement is a function of focusing.
This modulation factor multiplied by the brightfield information can be calculated from I. and II. by means of Pythagorean trigonometry by individual squaring followed by addition:
m
=
I
obxy
*
sin
2
x
+
cos
2
x
I
obxy
When m is taken solely as a function of location, a “distance image” of the image points from the optimum focal plane is obtained. Omitting division by the brightfield image, information is obtained which is virtually identical to that which can be obtained in confocal microscopes. The object characteristics are presented with decreasing intensity as the distance from the object plane increases, i.e., a determined layer of the object is shown.
Since the modulation factor m≦1, the depth selectivity can be increased by raising to a higher power.
By means of selecting limits, setting threshold values for display, it is possible to represent a layer of varying thickness by itself in a synthetic image in that everything located at a distance from the focus is excluded from the display by the threshold values.
This possibility is limited by the signal
oise ratio, the step errors in the grating displacement, the nonlinearities in detection, and the number of nodes by which the modulated grating is represented by the CCD camera generally used.
In principle, the following information can be obtained from the object from the partial images described above:
1. Complete brightfield images without modulated grating structure (A
1
, A
2
);
2. Distance information of object areas from the exact focus (without direction) (m);
3. Brightfield information of object becoming darker with increasing distance from the focus (confocal images) (A*m);
4. Images in which only a thin layer around the focus is shown as a complete brightfield image (A where m>limit value);
5. By raising m to a higher power, a selectable smaller area around the focus.
The apparatus described in U.S. Pat. No. 4,884,881 is an arrangement belonging to the large group of spinning disk arrangements, as they are called. These arrangements require an intermediate imaging (Relaigh) system. This means that the microscope must be expanded by additional optics and accordingly rendered more expensive.
In order to achieve usable confocal effects in arrangements with a spinning disk, structures must be projected into the object plane in such a way that they have a size in that location which is comparable to the resolution limit of the arrangement. Further, the coverage of a structured mask should be no larger than 5% in order to suppress the “breaking through” of information located outside of the focal plane. This precaution leads to a very low light yield or light efficiency (≦5%). In the this patent, wedge-shaped slits are imaged in the object plane. As a result of this construction, the confocal effect varies in radial direction (with respect to the image of the disk). In the present invention, because of the cos
2
-(sin
2
)-shaped intensity distribution of the pattern, the light efficiency is almost 50% and the achievable confocal effect is not spatially dependent in the displayed field. The device can be added to any reflected-light microscope or fluorescence microscope with an accessible field diaphragm plane in the illumination beam path without requiring additional optics.
U.S. Pat. No. 5,831,736 describes a confocal laser scanning microscope in which not all of the radiation returning from the object is detected, but only an angular area limited to the pupil plane. This area is cut out by a sector diaphragm whose direction can be changed in a stepwise manner. Based on the waveform of the detected signal depending on the orientation of the sector diaphragm, spatially dependent information about the inclination of the object structures can be obtained in addition to the depth information that can be obtained in microscopes of this type; however, because of the reduced aperture on the detection side, this information is obtained with decreased lateral and depth resolution.
In U.S. Pat. No. 5,471,308 plane plates with different inclinations are slid or rotated into the beam path of the projector successively in front of a grating pattern so that a different position of the projected pattern occurs with every one of the inclined plane plates (described in optics texts as plane plate micrometer). The apparatus described in the patent has two disadvantages:
1. Because of the large paths to be traveled and the large masses to be moved, the changing of the plates is carried out too slowly for modern applications.
2. Different optical parts are used which are subject to tolerances. In critical applications, this leads to errors which cannot be compensated in practice.
The aim of the appa
Carl Zeiss Jena GmbH
Pham Hoa Q.
Reed Smith LLP
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