U.S. patent application number 11/783255 was filed with the patent office on 2009-12-03 for laser scanning microscope and its operating method.
Invention is credited to Joerg-Michael Funk, Michael Goelles, Frank Hecht, Ralf Netz.
Application Number | 20090296207 11/783255 |
Document ID | / |
Family ID | 38859367 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090296207 |
Kind Code |
A1 |
Goelles; Michael ; et
al. |
December 3, 2009 |
Laser scanning microscope and its operating method
Abstract
Laser scanning microscope and its operating method in which at
least two first and second light distributions activated
independently of each other and that can move in at least one
direction illuminate a sample with the help of a beam-combining
element, and the light is detected by the sample as it comes in,
characterized by the fact that the scanning fields created by the
light distributions on the sample are made to overlap mutually such
that a reference pattern is created on the sample with one of the
light distributions, which is then captured and used to create the
overlap with the help of the second light distribution (correction
values are determined) and/or a reference pattern arranged in the
sample plane or in an intermediate image plane is captured by both
scanning fields and used to create the overlap (correction values
are determined) and/or structural characteristics of the sample are
captured by the two scanning fields as reference pattern and used
to create the overlap in which correction values are
determined.
Inventors: |
Goelles; Michael; (Jena,
DE) ; Netz; Ralf; (Jena, DE) ; Hecht;
Frank; (Weimar, DE) ; Funk; Joerg-Michael;
(Jena, DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
38859367 |
Appl. No.: |
11/783255 |
Filed: |
April 6, 2007 |
Current U.S.
Class: |
359/385 |
Current CPC
Class: |
G02B 21/0036 20130101;
G02B 21/008 20130101; G01B 11/14 20130101 |
Class at
Publication: |
359/385 |
International
Class: |
G02B 21/06 20060101
G02B021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 2006 |
DE |
10 2006 034 906.7 |
Claims
1. Method of operating a laser scanning microscope, in which at
least two independently controlled first and second light
distributions that can move in at least one direction through a
beam-combining element illuminate a sample and the light is
detected by the sample as it comes in, the method comprising the
steps of: causing the two scanning fields created by the light
distribution on the sample to overlap, such that a reference
pattern is created on the sample with one light distribution, which
is then captured by means of the second light distribution and used
to create the overlap and/or a reference pattern arranged in the
plane of the sample or an intermediate image plane is captured by
both scanning fields and used to create an overlap and/or
structural characteristics of the sample are captured by both
scanning fields as a reference pattern and used to create the
overlap, so that correction values are determined.
2. Method of operating a laser scanning microscope according to
claim 1, in which the first light distribution is moved over the
sample to capture a sample image, and the second light distribution
is used to manipulate the sample.
3. Method of operating a laser scanning microscope according to
claim 1, in which the reference pattern is a point
distribution.
4. Method of operating a laser scanning microscope according to
claim 1, further comprising the steps of creating reference points
by a sample manipulation system and capturing the reference points
with an imaging system.
5. Method of operating a laser scanning microscope according to
claim 1, in which the reference pattern consists of at least three
points.
6. Method of operating a laser scanning microscope according to
claim 1, further comprising the steps of creating a light point on
the sample.
7. Method of operating a laser scanning microscope according to
claim 5, further comprising the steps of employing light reflected
by the sample points.
8. Method of operating a laser scanning microscope according to
claim 1, further comprising the steps of capturing
frequency-converted light, wherein the frequency-converted light is
created through a non-linear or linear interaction of the
illuminating light with the sample.
9. Method of operating a laser scanning microscope according to
claim 8, in which at least one luminescence point is created on the
sample.
10. Method of operating a laser scanning microscope according to
claim 8, with creation through inelastic Light scattering.
11. Method of operating a laser scanning microscope according to
claim 1, further comprising the steps of creating reference
patterns from points with light-inducing sample modification.
12. Method of operating a laser scanning microscope according to
claim 1, in which grids are used as reference patterns.
13. Method of operating a laser scanning microscope according to
claim 1, in which a statistical structure distribution of the
sample itself serves as the reference pattern.
14. Method of operating a laser scanning microscope according to
claim 1, in which a coordinate transformation is used to determine
correction values.
15. Method of operating a laser scanning microscope according to
claim 13, with an affine transformation having at least three
reference points.
16. Method of operating a laser scanning microscope according to
claim 4, in which a point-scanning or line-scanning system, or a
scanning point distribution system or a Nipkow system is used as
the imaging system.
17. Method of operating a laser scanning microscope according to
claim 4, in which the manipulating system is a point scanning
device, and the scanning takes place preferably in two
directions.
18. (canceled)
19. Laser scanning microscope according to claim 27, having an
imaging system and a manipulating system.
20. Laser scanning microscope according to claim 19, in which the
imaging system is a point-scanning system, a line-scanning system,
a scanning point distribution system or a Nipkow system.
21. Laser scanning microscope according to claim 19, in which the
manipulating system is mapped and the scanning takes place
preferably in two directions.
22. Laser scanning microscope according to claim 27, with at least
one laser as the light source.
23. Laser scanning microscope according to claim 27, in which a
movement takes place over the sample in at least one scanning
direction.
24. (canceled)
25. Laser scanning microscope according to claim 27, in which at
least one of the beam deflecting devices is provided with Galvo
scanners.
26. Laser scanning microscope according to claim 23, in which one
coordinate transformation takes place through the modification of
gain and offset values of the associated triggering unit.
27. A laser scanning microscope comprising: first and second light
distributions; a beam-combining element for combining the first and
second light distributions that are controlled independently of
each other and that can move in at least one direction; scanning
means interposed between the beam combining element and the sample
for causing first and second scanning fields to cause the combined
light distributions to illuminate the sample; at least one detector
to detect the light coming from the sample; and overlapping means
for superimposing the first and second scanning fields on the
sample through the use of at least one reference pattern by
overlapping the first and second scanning fields.
28. Laser scanning microscope of claim 27, wherein the overlapping
means comprises: means for creating a first reference pattern on
the sample with one light distribution; means for capturing the
reference pattern with the second light distribution; and means for
determining and adjusting correction values and for creating the
overlap of the first and second light distributions.
29. Laser scanning microscope of claim 27, wherein the overlapping
means comprises: means for capturing a reference pattern arranged
in the sample plane or in an intermediate plane of the two scanning
fields; and means for determining and adjusting correction values
and for creating the overlap of the first and second scanning
fields.
30. Laser scanning microscope of claim 29, wherein the overlapping
means comprises: means for capturing the structural characteristics
of the means for determining and adjusting correction values, and
means for creating the overlap with the first and second scanning
fields.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Not Applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISC
[0003] Not Applicable.
BACKGROUND OF THE INVENTION
[0004] (1) Field of the Invention
[0005] The present invention relates to methods of operating a
microscope, in general, and to a method of operating a laser
scanning microscope having at least two independently controlled
light distributions, in particular.
[0006] (2) Description of Related Art
[0007] Confocal laser microscopy is, among other things, the tool
for the defined control of micro objects. Versatile methods of
examining and influencing microscopic objects were recommended on
this basis--e.g., Denk in U.S. Pat. No. 5,034,613, TPA, Liu in U.S.
Pat. No. 6,159,749, Tweezer or Karl Otto Greulich in
"Micromanipulation by Light in Biology and Medicine" 1999. A
combination of a point-scanning or line-scanning imaging system and
a "manipulator" system has evoked increasing interest in the
specialized world.
[0008] Interest in observing and analyzing fast microscopic
processes has created new devices and processes (e.g., line scanner
LSM 5 LIVE), whose combination with the manipulation methods
mentioned above leads to new insights. In this context, the
simultaneous microscopic observation of a light induced, locally
resolved sample manipulation with the help of a suitable imaging
system occupies the foreground (U.S. Pat. No. 6,094,300 and DE 102
004 03 4987 A1). Modern microscopes therefore try to offer the
maximum possible number of flexible and optically equivalent
coupling and decoupling positions (DE 102 004 01 6433 A1).
[0009] The simultaneous availability of at least two coupling
positions for independent scanning systems is very important in
this context for avoiding limitations in time resolution due to
slow mechanical control processes. In addition to tubular
interface, there are other coupling positions on the sides of the
microscope stands (preferably in an extended infinite space between
the microscope objective and tube lens; "side ports") as well as on
the rear side of the stand (typically optically modified reflected
or transmitted light axes with suitable tube lens; "rear ports") as
well as the bottom side ("base port"). In principle, arrangements
with a common beaming direction (either reflected light or
transmitted light) or the opposite beaming direction (reflected
light and transmitted light) are conceivable. Apart from the
applicative background, the technical instrument-based view of the
common beaming direction is often preferred.
[0010] At least one element must be used in this case that combines
the beam paths of the two instruments in the space between the
scanners of the simultaneously operated scanning systems and the
objective. According to the prior art, one can think of the most
varied of beam-combining elements such as for instance,
optical-mechanical components like suitably coated beam-combining,
flat plates and beam-combining wedges, beam-combining cubes and a
polarization splitter. Further, beam-combining acoustic-optical
modulators and deflectors are also conceivable.
[0011] The mechanical requirements related to the precision of
location and angle of this beam-combining element are very high. A
faulty installation angle .alpha. causes a beam inclination in the
reflection of 2.alpha. For example, if the beam-combining element
is in the infinite space between a tube lens of focal length
f.sub.TL=164 mm and an objective of the nominal foreground
M=f.sub.TL/f.sub.Obj=40.times. then this leads to an angular
deviation of 2.alpha.=1'(position deviation of the beam-combining
element 0.5') to a deviation .DELTA.=(f.sub.TL/M)*tan 2.alpha.=1.2
.mu.m of both scanning fields in the object plane. In a field of
view 18 (image diagonals) this already corresponds to a deviation
of approximately 0.4% of the lateral length of the scanning field.
In the usual image formats of 512.times.512 or 1024.times.1024,
this corresponds to a deviation of 2-4 image pixels. In addition to
the demanding mechanical requirements related to the mechanical
positioning of the beam-combining element, there are similarly
demanding tolerance specifications related to the mechanical
interfaces of the imaging or manipulation scanning module
(inclination errors and lateral shifting of interface, intermediate
image position in axial direction, and rotation). Further, thermal
influences (heating of the microscope system, and fluctuations in
the environmental temperature) as well as undefined statistical
effects, impose a condition that occurs especially in case of
extremely precise measurements, the cover of the scanning fields in
the manipulating and imaging systems must be adjusted
repeatedly.
BRIEF SUMMARY OF THE INVENTION
[0012] To compensate for the pixel displacement (x, y) between the
manipulating and imaging scanning modules that cannot be controlled
fully through the mechanical tolerance chain, this patent suggests
calibration in such a way that, through various methods, the
position deviations of the scanning fields of the two systems are
determined and the coordinate transformations resulting there from
(scaling, rotation, shift) are computed and considered in the
control of at least one of the scanning systems.
[0013] In this context, it must be considered that the resulting
image cover parameters are influenced by numerous device settings.
An example of this would be the different main beam splitters of a
confocal laser scanning microscope, which in several commercial
devices is arranged on a motorized main beam splitter wheel. If the
excitation beams are reflected on the main beam splitter at less
than 90.degree., minor angular errors are already observable in the
scanning field cover. Examples of other adjustable device
parameters that can influence the scanning field cover crucially
are movable optics (e.g. viewing field or pupil zoom) as well as
non-linear factors and dynamic deviations of the beam deflecting
devices used in the concerned scanning systems (e.g. selected
scanning speed and scanning zoom in devices on the basis of galvo
scanners). Add to this the fact that the wavelength dependency of
the z-deposit is to be calibrated as a function of the excitation
and manipulation wavelengths used in different applications as well
as of the concerned used objective. The z-plane comparison can be
conducted elegantly through moveable collimator optics of the
imaging and/or manipulating system under scrutiny of the color
length fault of the concerned used objective.
[0014] Depending on the concerned application, the spectral use
area can stretch basically from the ultraviolet to the infrared
range for the imaging system as well as the manipulating system.
Typical manipulation wavelengths used in applications are, e.g.,
351, 355 and 364 nm (photo-uncaging), 405 nm (photo conversion,
Kaede, Dronpa, PA-GFP), 488 and 532 nm (photo bleaching, FRET,
FRAP, FLIP) as well as 780-900 nm (multi-photon bleach, e.g.,
MPFRAP, 2-photon uncaging; direct multi-photon stimulation).
Depending on the combined wavelength as well as the coupling
positions of the imaging system and the manipulating system, there
are numerous types of dichroitic beam-combining elements that are
meaningful from the application point of view. FIG. 1 shows a
selection of spectrally possible properties of beam-combining
element types that are relevant to applications in which the
manipulation wavelengths of 355 nm, 405 nm, 488 and 532 nm can be
used in the transmission as well as reflection direction. Neutral
combining elements (e.g., T20/R80) can be used universally for
different applications and they also enable simple applications in
which the same laser wavelengths are used for the imaging system as
well as the manipulating system (particularly FRAP).
[0015] Depending on the application under consideration, there is a
typical requirement of using different beam-combining element types
in a microscope system. A motorized replacement device is used for
this purpose. It can be, e.g., a motorized reflector revolver in
the area of the infinite space between the objective and tube lens,
as illustrated in FIG. 2. An alternative to the displayed reflector
revolver is, e.g., an appropriate reflector disk. The replacement
device for the different beam-combining element's conditions
further influence factors that affect the coverage of the scanning
fields of the imaging system and manipulation system. Thus, already
negligible mutual deviations of the beam-combining element
alignment lead particularly in the reflection direction to a
measurable scanning field shift.
[0016] Another problem is the ability to mechanically reproduce
(beam-combining element location and beam-combining element
alignment) the scanning position of the replacement device. Thus,
on the one hand, the precision and reproduction capacity
requirements of the replacement device increase as compared to
traditional light-microscopic systems, and, on the other hand,
claims of the practical management of the calibration method
mentioned above. Even the complete replacement of the revolver
device displayed in FIG. 2 can lead to a deviation of the scanning
field cover, requiring a fresh calibration due to residual errors
of the mechanical record.
[0017] In short, there is a need for the very general requirement
of the simplest possible calibration method that allows the
correction of the scanning field cover of the imaging system and
the manipulating system as a function of varying device settings.
This calibration method should particularly be used by the device
user and if possible, it should be possible to execute it
automatically.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0018] FIG. 1 shows a selection of spectrally possible properties
of beam-combining element types;
[0019] FIG. 2 a motorized reflector revolver in the area of the
infinite space between the objective and tube lens;
[0020] FIG. 3a illustrates the non-coinciding scanning fields of a
mapped scanning system (imaging) and a manipulation system
(manipulating) with orientations;
[0021] FIG. 3b illustrates an affine transformation with reference
to the orientation points P1-P3 and their position;
[0022] FIG. 4 provides a schematic overview of the different
calibration methods for the determination of the scanning field
cover;
[0023] FIG. 5a, the imaging system in transmission and the
manipulating system in reflection are coupled or decoupled. In FIG.
5b it is just the opposite.
[0024] FIG. 5c displays a stationary focus of the manipulation
system where at least three such focuses are captured directly in
the direction of reflection in the imaging system.
[0025] FIG. 9 illustrates an exemplary embodiment of a suitably
structured calibration sample.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The invention is described in greater detail in the
following pages with the help of the following schematic
diagrams:
[0027] FIG. 3a illustrates the non-coinciding scanning fields of a
mapped scanning system (imaging) and a manipulation system
(manipulating) with orientations, deviating from each other, of the
schematically represented X/Y orientation as well as orientation
points P1-P3 whose position on both systems are used for
overlapping.
[0028] FIG. 3b illustrates an affine transformation with reference
to the orientation points P1-P3 and their position.
[0029] FIG. 4 displays the different methods that are described
subsequently schematically; at the left the creation of static
reference points by one of the scanning systems, preferably of the
manipulation system (in reflection, fluorescence or two photon
conversion, in general, each sample modification through
illumination (also, e.g., ablation)), at the right the
cross-correlation with the help of image characteristics of the two
scanners.
[0030] In FIG. 5a, the imaging system in transmission and the
manipulating system in reflection are coupled or decoupled. In FIG.
5b it is just the opposite. FIG. 5c displays a stationary focus of
the manipulation system where at least three such focuses are
captured directly in the direction of reflection in the imaging
system.
[0031] A luminescent light point or a light point created elsewhere
through frequency conversion is captured and used, instead of the
focus, in FIG. 6 a-c.
[0032] The light-inducing sample modifications created by the
manipulation system are captured by the imaging system and used as
points in FIG. 7. This can happen statically one after another or
even during the scanning movement of the two systems (through
activation and deactivation of the manipulator light at different
places).
[0033] FIG. 8 displays a structured calibration substrate detected
by both systems and the position of the lines used for calibration,
either through cross-correlation or interactively by the user
(mutual displacement in the display).
[0034] FIG. 10 shows a separately arranged detector in both systems
(quadrant diode or CCD receiver) directly in the beam-combining
element that captures and evaluates the transmitted or reflected
residual beam for calibration. If a programmable, automatically
triggering beam deflection device is used for the imaging and/or
manipulating system, then the described problem of pixel-precise
scanning field coverage can be solved elegantly with the help of a
suitable coordinate transformation. Thus for instance, in the
absence of an angular distortion corresponding to FIG. 3a, the
coordinate systems of the two scanning systems can be exposed to
the following shifts even in the two-dimensional case: [0035]
Parallel displacement along the translation vector (m.sub.0,
n.sub.0) [0036] Rotated around the angle .PSI. [0037] Narrowed or
stretched along the x- or y-scaling factors.
[0038] In this case, a transformation of the k and j coordinates of
the manipulating system in the concerned m and n coordinates of the
imaging system is possible with the help of an affine mapping
(compare FIG. 3b):
m=m.sub.0+a11k+j (1a)
n=n.sub.0+a.sub.21k+j (1b)
[0039] Thus, if the coordinates of at least three points are
defined in the two independent scanning coordinate systems within
the framework of a suitable calibration, Equations (1a) and (1b)
can be used to convert the coordinates of the two scanning systems
into each other for random scanning field points. A total of six
image cover parameters are to be determined in this calibration
process: Offset (zero position), angle (mutual rotation) and three
stretching parameters. This therefore enables control of the beam
deflecting device of the manipulating system in such a way that a
pixel-precise cover with the object field of the imaging system is
possible (or vice versa).
[0040] This method of implementing a pixel-precise scanning field
cover of the two independent scanning systems presupposes that at
least one system has a programmable, automatically triggering beam
deflecting device.
[0041] This can be based on one of the following scanning
principles: [0042] Galvo mirror or [0043] Deflectable, especially
rotary or tilting mirror, e.g., step motor controlled deflecting
mirror [0044] Polygon mirror [0045] Acoustic-optical deflection
devices, especially acoustic-optical deflectors (AODs) [0046] Moved
perforated mask, especially in the form of a Nipkow disk [0047]
Moved (mono-mode) fibers [0048] Moveable objective or objective
parts [0049] Mechanical x- and y-displacement of a suitable part or
of the whole scanning system, e.g., with the help of
acoustic-optical modulators
[0050] (As the two scanning systems must be independent of each
other in the sense of the invention, a mechanical x- and
y-displacement of the sample is not permissible.)
[0051] In the case of the Galvo mirror that is used frequently in
commercial systems, a transformation, for example, corresponding to
Equation (1a, 1b) is possible through suitable adjustment of the
gain and offset values of the associated triggering
electronics.
[0052] In confocal systems, coverage of the scanning coordinates of
the imaging and manipulating system in three-dimensional space is
possible. As in the plane, a transformation of the two scanning
coordinate systems in space can be undertaken:
x=.phi..sub.1(u,v,w) (2a)
y=.phi..sub.2(u,v,w) (2b)
z=.phi..sub.3(u,v,w) (2c)
[0053] Three-dimensional sample objects are captured in confocal
imaging systems in which z microscopic images of the section planes
x, y are recorded for each different sample depth.
[0054] Between recordings of the individual confocal split images,
the sample depth z is varied in each case through a mechanical
displacement of the sample, the objective or the entire microscope
unit. In addition to the customary (micro) mechanical drive
systems, one can also use acousto-optical modulators, especially in
quick imaging systems for z-adjustment.
[0055] A preferred embodiment therefore uses two scanning systems
that are independent in the x- and y-directions as the imaging
system and manipulating system respectively, where at least one
system has a programmable, automatically triggering beam deflection
device so that a pixel-precise scanning cover is possible with the
help of the affine mapping Equations (1a, 1b). In this preferred
embodiment, the scanning process in the z-direction affects both
systems identically; e.g., the sample or the common objective is
displaced in the z-direction. In this case, it must be guaranteed
that the scanning planes of the two independent modules overlap
fully. A mutual adjustment ensures that scanning planes are not
misaligned relative to each other. The comparison of the parallel
scanning planes in the z-direction takes place preferably with the
help of suitable motorized adjustable optics. The collimators
described in DE 19702753 A1 are preferably used. The use of
motorized optics for z-comparison of the two scanning planes enable
the automated correction of chromatic longitudinal errors of the
different objectives used, at the different excitation and
manipulation wavelengths.
[0056] If, however, the two independent scanning modules do not
have any common beaming direction on the sample, an independent
scanning device is required for both systems in general in the
z-direction. To implement a pixel-precise cover of the x, y, and
z-scanning devices in a three-dimensional space in this case, one
must use the generalized Equations (2a-c).
[0057] The determination of the concerned transformation equation
with the help of which the two independent scanning systems can be
superimposed with pixel precision requires a suitable calibration
method. Hence, it has already been mentioned that the affine
mapping Equations (1a, b) can be determined uniquely if the
coordinates of at least three scanning field points are known in
both the scanning coordinate systems.
[0058] FIG. 4 provides a schematic overview of the different
calibration methods for the determination of the scanning field
cover. It has already been explained at the beginning that the
cover of the scanning fields of the two independent scanning
systems depends on different adjustment dimensions. Thus for
example, fine angle deviations between the different main and
auxiliary beam splitters of the imaging system or between the
different beam-combining elements used (compare FIGS. 1 and 2)
result in measurable differences in the cover of the two scanning
systems. Especially in case of frequently used commercial scanning
systems with Galvo mirrors as beam deflection device, the cover of
the two scanning fields also depends on the scanning speed set in
the two systems and the concerned selected scan zoom factor. In a
design model of the invention, the calibration methods illustrated
in FIG. 4 are determined for different setting combinations of the
adjustable sizes of the system that influence the scanning field
cover (e.g., determination of mapping equation (1a, b) for the
different main and auxiliary beam splitters of the system and the
different beam-combining elements of the replacement device
displayed in FIG. 2). This can be undertaken individually for the
concerned adjustable combination by the device user, where suitable
operating software is available. Another preferred design model of
the invention enables the automatic determination of individual
calibration records for all adjustable combinations of all relevant
adjustable sizes, where the control software falls back on the
concerned relevant calibration record as a function of the set
device configuration.
[0059] In a calibration method according to the invention, the
position of the stationary focus of the manipulating scanning
system is determined with the help of the scanning imaging system.
If this procedure is followed for a minimum of three focus
positions of the manipulating system, it is possible to obtain a
clear determination of the transformation equation (1a, b).
Different practical embodiments of this calibration method are
conceivable:
[0060] 1. In the simplest case, the stationary laser focus of the
manipulating system is observed directly with the help of the
confocal imaging system according to FIGS. 5a and b. In this
calibration measurement, the imaging scanning module "scans" the
object plane without beaming an exciting light. The manipulation
focus appears in a dark image background (FIG. 5c) exactly when the
stationary locus of the manipulating system is located within the
detection volume of the imaging system. As the manipulating and
imaging systems typically have the same beaming direction on the
sample, a surface reflex of the manipulator focus is observed in a
mirror located in the object plane, so that at least a small
portion of this reflex (dotted line in FIGS. 5a and 5b) must pass
the beam-combining element in the direction of the imaging system.
This method is therefore ideally suited if a neutral splitter is
used as a beam-combining element. Due to the typically very high
sensitivity of imaging confocal systems, this calibration method is
ideal in practice, but is also similarly suitable for any
convenient dichroitic beam-combining elements in which even in the
ideal case less than 1% of the reflected (drawn as a dotted line)
manipulation light passes the beam-combining element in the
direction of the imaging system. Further, this method requires an
emission filter attachment in the imaging system which enables a
direct observation of the manipulation wavelength. This is often
not guaranteed in commercial systems, especially in the infrared
and ultraviolet range.
[0061] 2. In a transformation of the calibration method 1
corresponding to FIG. 6 a-c, the stationary focus of the
manipulating system is observed indirectly through the imaging
system. In this context, the imaging system detects a frequency
conversion such as luminescence, non-linear processes, or inelastic
scattering such as Raman, which the stationary focus of the
manipulating system creates in a suitable substrate located in the
object plane or an intermediate image plane. Here too, the imaging
system scans the object plane without beaming exciting light. As
the wavelength of the manipulating system was not observed
directly. Instead of the light produced by it in the range of the
visible spectrum, this additional calibration method is often
better adapted to the spectral properties of the beam-combining
elements and the emission filter in the system than calibration
method 1. Accordingly, calibration method 2 also allows an
adjustment of the scanning field cover in the z-direction--even
while using manipulation light in the ultraviolet or infrared
range--i.e., a spectral range in which the detection optics
(pinhole optics) of commercial imaging systems is typically not
corrected. Ideally, the layer thickness of the calibration
substrate in which the manipulating system creates the luminescent
beam should be as small as possible because otherwise the spot
observed in the imaging system becomes too large due to the absence
of location discrimination with scattered light.
[0062] 3. In another transformation of calibration methods 1 and 2,
a suitable unstructured sample substrate is modified through
illumination with the stationary focus of the manipulating system,
according to FIG. 7. This light-induced sample modification can be,
e.g., bleaching, photo activation or photo conversion of a
fluorescent coloring substance, or even a thermally or mechanically
induced sample change (e.g., laser ablation). Decisive for the
calibration process is that this light-inducing modification is
limited exclusively to the area of the stationary focus of the
manipulating system, and it is stable at least sporadically. After
this laser-induced sample modification is made at a minimum of
three different scanning field positions, the thus structured
sample substrate is measured with the help of the imaging system.
The difference from the calibration methods 1 and 2 is that the
calibration takes place in a two-phase process in which the image
capturing after the sample structuring takes place with the
excitation light of the imaging system, if necessary, also with the
help of samples in which a modification, e.g., through optical
switches can be reversed.
[0063] Decisive for the function of the three described calibration
methods is a correct adjustment of the confocal opening of the
concerned imaging system (e.g., pinhole for point scanners and slot
opening for line scanners). In calibration methods 2 and 3, the
signal light lies typically in the range of the visible spectrum
(i.e., in the detection area typical in most applications). Hence,
these calibration methods have the same requirements with regard to
correct adjustment of the confocal opening as in the commercial
confocal microscopes. In calibration method 1 however, the confocal
opening is to be adjusted in such a way that a direct detection of
laser light can take place in which the spectral range can lie, if
necessary, even in the ultraviolet or infrared range.
[0064] Wavelength dependencies of the detection channel of the
imaging system thus play the most crucial role in calibration
method 1. In a design model of the invention, the three calibration
methods 1-3 were combined with the automatic position optimization
of the confocal opening. This automated adjustment of the confocal
opening can be performed interactively by the device user--a
suitable software interface is available for this--or it can also
be undertaken fully automatically by microscope systems within the
framework of the actual calibration method. The optimum adjusting
positions for the concerned device settings can be stored in the
corresponding calibration records.
[0065] In general, calibration methods 1 to 3 combine a dynamic
scanning process of a module with a static focus positioning of the
concerned other scanning module. In contrast, no spot bleaching is
carried out in most of the applications described at the start.
Instead, the bleaching process takes place within an extended
"region of interest." All thus far explained calibration methods
have the disadvantage that dynamic effects of the beam deflection
device of one of the scanning modules cannot be determined during
the calibration of the scanning field cover. As explained earlier,
such dynamic effects are encountered especially in Galvo scanners
in which the scanning field cover can depend, for example, on the
concerned selected scanning speed and the concerned scanning
zoom.
[0066] This disadvantage is rectified with the help of a basically
different calibration method illustrated in FIG. 8. In this dynamic
calibration method ("area-based image matching"), a structured
calibration preparation located in the object or a common
intermediate image plane is measured separately by the two
independent scanning systems, in which the triggering of the beam
deflecting device is adjusted by at least one of the two scanning
systems according to Equations (1a, 1b) in such a way that the
images of the structured calibration sample captured by the two
systems are brought for cover. This calibration method can be
executed interactively by the device user, in which a suitable
software interface is used to superimpose the sample images
captured with the help of the two scanning systems. However, a
fully automatic calibration routine is also conceivable in which
the optimal superimposition of the sample images captured with the
help of the two scanning systems is determined through computation,
e.g., with the help of the cross correlation method. If Galvo
scanners are used as beam deflecting devices, the electronic gain
and offset settings of at least one scanning system are adjusted
during the calibration of the scanning field cover.
[0067] A precondition of this calibration method is that both
scanning systems enable the image capturing of the calibration
sample independently of each other. If no detector suitable for the
image capturing is integrated in the manipulating system (e.g., a
cost-effective diode with simple grab electronics), then an
external detector according to FIG. 8 (preferably in the
transmission beam path) must be used for this purpose.
[0068] FIG. 9 illustrates an exemplary embodiment of a suitably
structured calibration sample. This can be, e.g., a reflecting
structure on a glass substrate or vice versa--a transparent
structure on a reflecting substrate. In the calibration, the laser
light reflected or transmitted (during used of an external
detector) from the concerned scanning system on this calibration
sample is used for image capturing. If a line scanner with a bar
mirror as a space filtering element (DE 10257237A1) is used as an
imaging system, then neither the reflected laser light nor the
transmitted laser light can be detected directly. In this case one
can execute the described calibration method in which the
calibration structure is brought in direct contact with a
homogeneous fluorescence medium, where a dark sample structure is
detected in a bright fluorescent background. Another option is to
illuminate the sample structure with the help of a bulb in the wide
field and to scan it with the help of a confocal scanning
system.
[0069] Due to the parallel data capture, this method is ideal,
particularly if a confocal linear scanner is used as the imaging
system. This calibration method has the advantage of being a
dynamic method, i.e., relative changes between the image field
overlap between the two scanning modules can be determined directly
as a function of the scanning speed and scanning zoom. Thus, the
dynamic effects of the concerned beam deflecting device can be
considered in the appropriate calibration records.
[0070] All the thus far described methods of optimizing the
scanning field overlap can be automated with the help of suitable
software in which a constant interaction of the device user is
required. In contrast, the arrangement displayed in FIG. 10 enables
a fully automatic calibration of the scanning field cover without
involving the user. The second output of the beam-combining element
is used here to determine the mutual scanning field cover of the
two scanning systems. Thus, corresponding to FIG. 10, even in case
of dichroitic beam-combining elements, a small portion of the
injected light is reflected or transmitted in the direction of the
second output. If there is a locally triggered flat image detector
(e.g., a CCD or CMOS camera or a quadrant diode), the relative
position deviations between the two scanning modules can be
determined directly and corrected automatically without requiring
any further intervention of the user (such as e.g., the insertion
of a calibration sample in the object plane). The arrangement
displayed in FIG. 10 therefore is ideal particularly for automated
rule processes that enable subsequent device-internal correction of
the concerned optimum scanning field cover in case of fluctuating
environmental influences (e.g., temperature) and variable device
settings (e.g., beam-combining elements, main beam splitters, zoom
optics, objectives, wavelengths).
* * * * *