Radiant energy – Photocells; circuits and apparatus – Photocell controls its own optical systems
Reexamination Certificate
1999-11-16
2003-04-15
Pyo, Kevin (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Photocell controls its own optical systems
C250S201400
Reexamination Certificate
active
06548795
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to automated focusing systems, in particular to extremely rapid automatic focusing of optical scanning systems.
2. Description of the Related Art
With the development of combinatorial chemistry and bioassays, automated imaging is becoming extremely important. A large variety of tests can be conducted with such systems, such as are disclosed in WO 99/08233 and WO 98/47006. Some of these tests, particularly ones based on fluorescence and reflected light, use confocal systems such as those disclosed in U.S. Pat. No. 5,900,949. In such a system, light is applied through the optics of the system to excite a sample to fluoresce or phosphoresce, or simply to reflect the light. The resulting emitted, reflected or scattered light then is detected either through a separate optical system to the side of the light source, as shown in U.S. Pat. No. 5,900,949, or through reflection or emission back through the same initial optical system as the light source, by way of a half-silvered mirror or di-chroic beam splitter.
In a typical scanning system (illustrated in
FIG. 1
) a focused beam of light moves across a sample and the resultant reflected or fluorescent light is detected. A fluorescent system typically includes a source of light
10
at the proper wavelength, &lgr;
ex
, to excite the sample or a dye in the sample. This light is focused through source optics
12
and deflected by mirror
14
via scan lens
26
onto sample
16
. Light that fluoresces or is reflected from the sample returns to detection optics
18
via half silvered mirror or di-chroic beam splitter
15
. Alternatively, the emitted or fluoresced light can be detected from the side of the system, as shown in U.S. Pat. No. 5,900,949. Light passing through detection optics
18
then is detected using a CCD or equivalent element
20
, the output from which is provided to computer
22
for analysis. Motor
24
is used to move mirror
14
to scan the excitation beam across the sample
16
. The excitation beam, motor, optics and the rest of the system then are controlled by computer
22
to scan relevant portions of sample
16
.
In a true confocal system, the system will reject light that is not substantially in focus. As illustrated in
FIG. 2
, the light in such a confocal system typically will be deflected by mirror
14
through scan lens
26
. A confocal system typically has a very small depth of field d, as illustrated in FIG.
2
. Sample
16
is in scan field
29
, that is, in the depth of field d, for a scan across sample
16
, traversing the range of scan. The focal length of the system is f, and the relative sizes of the values are f>>d>>&lgr;. The range of scan may vary from tens of micrometers to centimeters, depending on the system.
For a truly flat and level surface in a confocal system, once the collection system and the sample are brought into focus, no more focusing along the +z or −z axis (up or down, as shown in
FIG. 2
) is required. If the light beam is scanned, the assumption is that the design of the system is such that rotation of mirror
14
does not move the light beam out of the nominal plane of focus, i.e., scan field
29
is essentially flat in the area where the sample is located.
As will be apparent, the sample must be kept continuously in focus during a scan. One technique for doing this automatically or manually brings the sample into focus below a stationary focused beam, only once, and then scans the sample by moving it on an x-y translation stage. The distance from the sample to the objective then remains constant since the sample does not move up or down, throughout the scan. This method is used by several imaging manufacturers.
Autofocus systems for optical scanners often use a half-blocked or obscuration technique to bring the sample into focus, such as is shown in
FIGS. 3
a, b, c,
and described in detail in section 31.4 of the Optical Society of America's Handbook of Optics Vol. 1 (on CD-ROM), published by McGraw Hill (1996). In such a system, light
41
reflected or emitted from sample
16
passes through a lens
26
. Most of the light passing through lens
26
then will be directed to the detection optics for analysis, but, as shown in
FIG. 1
, some of the light will be directed by a low reflection beam splitter
40
to the autofocusing system
42
, shown in detail in the upper portions of
FIGS. 3
a, b, c.
(For clarity of illustration, beam splitter
40
is omitted from
FIGS. 3
a, b, c.
)
If sample
16
is in focus, as shown in
FIG. 3
b,
light
41
is collimated, so that light
47
passing through lens
46
is properly focused on focal point
49
. If sample
16
is too far from lens
26
(the −z direction), light
41
tends to converge too much, as shown in
FIG. 3
a.
If sample
16
is too close to lens
26
(the +z direction), light
41
tends to diverge too much, as shown in
FIG. 3
c.
In a typical autofocusing system, half of the beam of light
41
is blocked by knife edge
44
. The remaining portion of light
41
passes through lens
46
to become light
47
and impinges upon photodetector
48
. Photodetector
48
typically has halves A, B centered on the focal point
49
of the photodetector
48
, with each half A, B serving as an independent detection region.
When properly focused, as shown in
FIG. 3
b,
the light
47
impinges upon the center
49
of the photodetector
48
, between halves A, B, or at the very least impinges equally upon halves A, B. In contrast, when sample
16
is too far from lens
26
, as shown in
FIG. 3
a,
more of light
47
impinges on photodetector portion B than on A, and similarly, as shown in
FIG. 3
c,
when sample
16
is too close to lens
26
, more of light
47
impinges on photodetector A than on B. Therefore, the position of sample
16
relative to lens
26
can be determined by analyzing the relative signal strengths being generated by photodetector portions A and B. This can be done through any suitable method, but is conveniently done by subtracting the values of the outputs of the two portions A and B of the photodetector in circuit
51
to generate a Focus Error Signal (FES)
50
.
In theory, the absolute value of FES
50
is indicative of the distance by which sample
16
is out of focus, while the positive or negative value of FES
50
indicates the direction in which the sample
16
is out of focus. When sample
16
is in focus, light
47
either impinges on photodetector center
49
, or at least is equally balanced between portions A, B, with the result that the value of FES
50
is 0 and no z-axis adjustment is needed (it will be understood that the value need not be exactly 0—some range around 0 will normally be considered equivalent to 0). If more of light
47
impinges on half B of the detector (as shown in
FIG. 3
a
), FES
50
is a positive signal, indicating that the z translation stage is off in the −z direction, so the stage should be moved in the +z direction to bring the system into focus. If sample
16
is too close to lens
46
, more light
47
hits A than B, and FES
50
is negative, indicating that the stage is out of focus in the +z direction, and should be moved in the −z direction to bring the system into focus. Z-axis translation stages responsive to such an FES signal in this fashion are commercially available.
SUMMARY OF THE INVENTION
While working with new materials to hold samples, the inventors encountered problems with focusing the prior art systems when the sample surface itself undulates significantly. As illustrated in
FIG. 4
, if the surface of sample
16
is not smooth, portions of the surface may be out of focus, even though sample
16
as a whole stays at the same distance from the lens
26
. This may be true of both scanning beam and scanning stage systems.
Specifically,
FIG. 4
illustrates a set of DNA oligonucleotide probes
30
deposited on the surface of substrate
16
. Such probes often are chemical systems which combine with immobilized clipped DNA fra
Atkinson Matthew R. C.
Knudson Orlin B.
Dahl Philip Y.
Dennis II Charles L.
Pyo Kevin
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