U.S. patent application number 11/783290 was filed with the patent office on 2008-03-20 for method and arrangement for the controlled actuation of a microscope, in particular of a laser scanning microscope.
Invention is credited to Ralf Engelmann, Joerg-Michael Funk, Frank Hecht, Ralf Netz, Bernhard Zimmermann.
Application Number | 20080068709 11/783290 |
Document ID | / |
Family ID | 38859369 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080068709 |
Kind Code |
A1 |
Zimmermann; Bernhard ; et
al. |
March 20, 2008 |
Method and arrangement for the controlled actuation of a
microscope, in particular of a laser scanning microscope
Abstract
Method for actuation control of a microscope, in particular of a
Laser Scanning Microscope, in which, at least one first
illumination light, preferably moving at least in one direction, as
well as at least one second illumination light moving at least in
one direction, illuminate a sample through a beam combination, a
detection of the light coming from the sample takes place, whereby,
at least one part of the illumination light is generated through
the splitting of the light from a common illuminating unit,
characterized in that, by means of a common control unit, a
controlled splitting into the first and the second illumination
light takes place, in which the intensity of the first illuminating
light, specified by the user or specified automatically, is
assigned a higher priority (is prioritized) compared to the
specified value for the second illumination light, and an
adjustment for the second illumination light takes place until a
maximum value is obtained, which is determined by the value
specified for the first illumination light.
Inventors: |
Zimmermann; Bernhard; (Jena,
DE) ; Netz; Ralf; (Jena, DE) ; Hecht;
Frank; (Weimar, DE) ; Funk; Joerg-Michael;
(Jena, DE) ; Engelmann; Ralf; (Jena, DE) |
Correspondence
Address: |
Law Offices;Jacobson Holman
Professional Limited Liability Company
400 Seventh Street, N.W.
Washington
DC
20004-2218
US
|
Family ID: |
38859369 |
Appl. No.: |
11/783290 |
Filed: |
April 6, 2007 |
Current U.S.
Class: |
359/385 |
Current CPC
Class: |
G02B 21/00 20130101;
G02B 21/06 20130101; G02B 27/0972 20130101; G02B 21/0032 20130101;
G02B 21/0076 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 034 914.8 |
Claims
1-34. (canceled)
35. Method for actuation control of a microscope in which, a first
illumination light moves at least in a first direction, and a
second illumination light moves at least in a second direction, the
first and second illumination lights illuminating a sample through
a beam combination, the method comprising the steps of: generating
at least one part of the first and second illuminating lights by
splitting the light from a common illuminating unit, in which the
intensity of the first illuminating light, specified by a user or
specified automatically, is assigned a higher priority compared to
the specified value for the second illumination light, and an
adjustment for the second illumination light takes place until a
maximum value is obtained, which is determined by the value
specified for the first illumination light; and detecting the light
coming from the illuminated sample.
36. The method for actuation control of a microscope according to
claim 35, further comprising the steps of moving the first and
second illumination lights through the sample.
37. The method for actuation control of a microscope according to
claim 35, wherein before the adjustment of the second illumination
light, a change in the distribution ratio of the light from the
illumination unit takes place.
38. The method for the actuation control of a microscope claim 35,
wherein, besides the splitting, intensity modulation takes
place.
39. The method for actuation control of a microscope according to
claim 35, further comprising the steps of providing a first imaging
system and a second manipulating system.
40. The method for actuation control of a microscope according to
claim 39, whereby the imaging system is chosen from the group
consisting of a wide-field microscope, a point scanning, a line
scanning microscope, a microscope scanning with point-distribution;
and a Nipkow microscope.
41. The method for actuation control of a microscope according to
claim 35, wherein the first and second illuminating lights have
same wavelengths and are divided.
42. The method for the actuation control of a microscope according
to claim 35, wherein common illumination from the illumination
lights takes place in the same or different regions of the
sample.
43. A light raster microscope comprising: a beam combiner for
bringing together at least one first illumination light moving in a
first direction as well as at least one second illumination light
moving in a second direction, for the illumination of a sample; at
least one detection unit for the detection of the light coming from
the sample; an illumination unit for the generation of at least one
part of the illumination light by means of splitting the light into
the first and the second illumination lights; an actuation control
unit for controlling the splitting of the illumination light of the
illumination unit by assigning priority to the intensity of the
first illumination light specified by a user or automatically
compared to the value specified for the second illumination light
and making adjustment for the second illumination light until a
maximum value is obtained, the maximum value being determined by
the value specified for the first illumination light.
44. The light raster microscope according to claim 43, further
comprising means for the adjustment of the intensity of at least
the first or the second illumination unit.
45. The light raster microscope according to claim 43, further
comprising an imaging first system and a manipulating second
system.
46. The light raster microscope according to claim 45, wherein the
imaging system is selected from the group consisting essentially of
a wide-field microscope, a point-scanning, a line-scanning
microscope, a microscope scanning with point-distribution, and a
Nipkow microscope.
47. The light raster microscope according to claim 45, whereby the
manipulating system is a point-scanner.
48. The light raster microscope according to claim 43, wherein a
tunable laser is split into at least two channels.
49. The light raster microscope according to claim 43, wherein
before the splitting, combining with at least one or more lasers
takes place.
50. The light raster microscope according to claim 43, further
comprising means for adjusting the intensity and/or the wavelength
and/or the polarization of at least one of the first and second
illumination lights.
51. The light raster microscope according to claim 43, further
comprising means for optically coupling one of the split
illumination canals with another light raster microscope and/or an
optical manipulation unit.
52. A light raster microscope system comprising: a first light
raster microscope; and a second light raster microscope and/or an
optical manipulation unit, each of the raster microscopes and/or
the manipulation unit illuminating a sample simultaneously and/or
alternately, wherein the illumination unit from the first and/or
the second light raster microscope and/or the manipulation unit is
optically split and serves in each case the purpose of illuminating
the other light raster microscope and/or the manipulation unit.
53. The light raster microscope system of claim 52 wherein at least
one of the first and second raster microscopes and the manipulation
unit comprises: a beam combiner for bringing together at least one
first illumination light moving in a first direction as well as at
least one second illumination light moving in a second direction,
for the illumination of a sample; at least one detection unit for
the detection of the light coming from the sample; an illumination
unit for the generation of at least one part of the illumination
light by means of splitting the light into the first and the second
illumination lights; and an actuation control unit for controlling
the splitting of the illumination light of the illumination unit by
assigning priority to the intensity of the first illumination light
specified by a user or automatically compared to the value
specified for the second illumination light and making adjustment
for the second illumination light until a maximum value is
obtained, the maximum value being determined by the value specified
for the first illumination light.
54. Light raster microscope according to claim 52, further
comprising optical fibers for optically coupling with the
respective second system.
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
[0003] Not Applicable
BACKGROUND OF THE INVENTION
[0004] (1) Field of the Invention
[0005] The present invention relates to a method and apparatus for
the controlled actuation of a microscope, in general, and to
controlled actuation of a laser scanning microscope having multiple
light sources, in particular.
[0006] (2) Description of Related Art
[0007] Confocal microscopy is, among other things, the tool for
defined controlled actuation of micro-objects. Based on that,
numerous methods for examination and influencing of microscopic
objects were proposed, thus, for instance, by Denk in U.S. Pat. No.
5,034,613, by Liu in U.S. Pat. No. 6,159,749, or by Karl Otto
Greulich in "Micromanipulation by Light in Biology and Medicine" in
1999.
[0008] A combination comprising an image-forming point scanning or
line scanning system and a "manipulator" system is increasingly
finding more and more interest in the professional circles.
[0009] The interest in the observation and analysis of fast
microscopic processes has brought forth new devices and methods
(for example Carl Zeiss Line Scanner LSM 5 LIVE), which, in
combination with the above mentioned methods of manipulation, lead
to new insights. Thereby, the simultaneous microscopic observation
of radiation-induced manipulation of the samples with spatial
resolution by means of a suitable imaging system stands especially
in the foreground (See for example U.S. Pat. No. 6,094,300 and DE
102004034987 A1). Therefore the modern microscopes attempt to offer
as many flexible and optically equivalent decoupling and coupling
ports as far as possible (See: DE 102004016433 A1).
[0010] The availability at the same time of at least two coupling
ports for independent scan systems is thereby of special importance
in order to avoid limitations in the temporal resolution due to the
slowness of mechanical switching processes. Besides the tube
interface, other coupling ports on the sides of the microscope
stand are possible (preferably in the extended infinite space
between the microscope objective and the tube lens; the so-called
"sideports") as well as on the rear side of the stand (typically
optically modified incident light axis or transmitted light axis
with suitable tube lens; the "rearports") as well as on the bottom
side (the "baseport").
[0011] Thereby, arrangements with a common direction of the
incident light (either reflected or transmitted light) or with a
direction opposite to incident light (transmitted light and
reflected light) are possible in principle. Apart from the
viewpoint of the applicability, a common direction of incidence is
frequently preferred from the device-technical viewpoint.
[0012] In that case, use of at least one element is necessary,
which combines the beam paths of both devices in the space between
the scanners of the scan systems that are to be operated
simultaneously and the objective. Thereby, according to the
state-of-the-art, a diverse variety of beam-combining elements are
conceivable, such as, for example, the optomechanical components,
like suitably coated beam combiner flat plates and beam combiner
wedges, beam combiner cubes and polarization splitters. Conceivable
are further beam combining acousto-optical modulators and
deflectors.
[0013] In the following, reference is made in particular to DE
102004034987 A1, which is incorporated by reference herein as if
reproduced in full and which forms a part of the subject matter of
the present publication.
[0014] FIG. 1a shows schematically the design of a device system,
which enables simultaneous operation of a manipulating and an
imaging scan module in a microscope stand. The modules provided
with a common actuation control system (control system, PC) and the
laser or the laser modules are connected optically and controllably
with both the scan modules.
[0015] In FIG. 1b, an embodiment with an inverse stand is shown by
way of example.
[0016] In a preferred embodiment, the electronic actuation of the
microscope stand and the coupled manipulation and the imaging
module are suitably equipped using a real-time electronic control
system with an integrated real-time computer for the processing of
the high data rates. Thereby, such embodiments are conceivable in
which the scan systems of the manipulations and the imaging modules
coupled with the microscope stand can be actuated in synchronous or
asynchronous manner. Thus simultaneous scan modes of both the
modules are possible in which manipulation and imaging in the
different regions of the sample (ROIs; "regions of interest"
DE19829981 C2) with variable scanning rates takes place as in FIG.
1c.
[0017] Both for the manipulating system as well as for the imaging
system, the useful spectral range can be extended, depending on the
respective application, from the ultraviolet to the infrared
spectral range. Manipulation wavelengths typically found in the
applications are, for instance, 351, 355 and 364 nm
(photo-uncaging), 405 nm (photoconversion, Kaede, Dronpa, PA-GFP),
488 and 532 nm (photobleaching, FRET, FRAP, FLIP) as well as
780-900 nm (multiphoton bleaching, for example MPFRAP, 2-photon
uncaging; and direct multiphoton stimulation).
[0018] Since in many applications, both the manipulating as well as
the imaging system employ the same laser wavelengths, it is
reasonable to feed both the scan modules with a common laser
source. In DE 102004034987 A1 different suitable arrangements for
variably adjustable division of the beam between two independent
scan modules are described: [0019] a. Laser-specific, variable beam
splitting with a rotatable .lamda./2-plate and polarization beam
splitters (ref. FIG. 2): [0020] By using a motorized rotatable
.lamda./2-plate before each laser and a polarization beam splitter
cube in the combined beam path of all lasers, a variable, loss-free
beam splitting into two illumination canals takes place. Thereby,
by rotating the .lamda.2-plate by an angle .THETA., the
polarization of the incident polarized laser is rotated by angle
2.THETA.. The horizontally and the vertically polarized components
of the field amplitude are split by the subsequent polarization
beam splitter cube (Glan-Taylor prism). Thereby the horizontally
polarized light is transmitted and the vertically polarized light
is reflected. By rotating the .lamda./2-plate from 0.degree. to
45.degree. the polarization of the incident beam is rotated from
0.degree. to 90.degree. and the beam intensity is thus divided
continuously and variably between the split partial beams. The
intensity of the split laser beams can be modulated in any of the
illumination canals individually with the help of an appropriate
light modulator (for example graduated, acousto-optical modulators
like Pockels cells). When different laser sources are used in which
their beams are combined as in FIG. 2, this method of variable beam
splitting is particularly practicable, if the individual beam
combiners of the laser module are largely independent of the
polarization. [0021] In addition to that, the fact that a finite
switching time is necessary for the rotation of the .lamda./2-plate
must be taken into account. Therefore a limitation from the
viewpoint of the applications arises in the case of this method
precisely then, when the manipulation and the fast imaging take
place sequentially at time intervals of less than this switching
period for the same wavelength and, in addition to that, the sum of
laser power required for both partial processes exceeds the total
available. The described method can be employed with advantage
especially then, when the same laser line can be used
simultaneously in the manipulating as well as in the imaging
system. This is true particularly in photobleaching applications,
such as, for instance, FRET, FRAP and FLIP. [0022] b. This
application-related limitation can however be eliminated, if, in
lieu of the rotatable .lamda./2-plate, fast electrooptic or
magnetooptic polarization rotators (for example Pockels cells,
Faraday rotators or LC retarders) are used, which have switching
periods in the microsecond range or shorter (FIG. 2). [0023] c. A
variable, wavelength-specific beam splitting into two illumination
canals can be done also with two AOTFs (acousto-optical tunable
filter) arranged successively one after the other as in FIG. 3,
whereby, for instance, the 1st order of diffraction of the first
AOTF is used for the coupling in the imaging system, whereas the
0th order of diffraction is coupled in through a second AOTF in the
manipulator module (FIG. 3). [0024] The imaging should thereby not
be impaired by switching over of the bleaching ROI. [0025] This
method has the disadvantage in applications that in case of
simultaneous manipulation and imaging, the second manipulator AOTF
must be adjusted simultaneously through software control with the
switching of the first AOTF (for example switching off of the laser
power of the imaging system at the reversal points of the raster
scan). [0026] d. A variant of c. without functional limitations can
be realized when an AOTF is exclusively used for variable beam
splitting between two illumination canals and the laser power can
be adjusted separately in both canals through two other AOTFs (FIG.
4). [0027] e. A simple economical method for beam splitting can be
realized with the help of a neutral graduating wheel with different
positions or a continuously coated neutral filter wheel or a
neutral slider (graduated filter).
[0028] FIG. 5 shows an embodiment of a microscope system by way of
example, which enables real-time microscopic imaging with a line
scanner (right) that takes place simultaneously with the
manipulation of the sample (point scanner left). In this way, both
of the independent scan systems use the laser sources A-D jointly,
whereby the power is divided in a variably tunable ratio between
these two modules according to method a described above. The
unification of the optical axes of the manipulating and the imaging
system takes place in the region of the finite space between the
microscope objective and the tube lens by means of a beam combiner.
A systematic description of numerous other embodiments can be found
in DE102004034987 A1.
BRIEF SUMMARY OF THE INVENTION
[0029] The present invention relates to a method and apparatus for
actuation control of a microscope, in particular of a Laser
Scanning Microscope, in which, at least one first illumination
light, preferably moving at least in one direction, as well as at
least one second illumination light moving at least in one
direction, illuminate a sample through a beam combination. A
detection of the light coming from the sample takes place. At least
one part of the illumination light is generated through the
splitting of the light from a common illuminating unit. A common
control unit accomplishes a controlled splitting of the
illumination light into the first and the second illumination
lights. The intensity of the first illuminating light, as specified
by a user or specified automatically, is assigned a higher priority
(is prioritized) compared to the specified value for the second
illumination light, and an adjustment for the second illumination
light takes place until a maximum value is obtained, which is
determined by the value specified for the first illumination
light.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0030] FIG. 1a is a schematic diagram of system which enables
simultaneous operation of a manipulating and an imaging scan module
in a microscope stand;
[0031] FIG. 1b is a schematic drawing of an inverse microscope
stand;
[0032] FIG. 1c is a schematic diagram illustrating regions of
interest and variable scanning rates;
[0033] FIG. 2 is a schematic diagram of two independent scan
modules with variable beam splitting.
[0034] FIG. 3 is a schematic diagram showing beam splitting using
two AOTFs;
[0035] FIG. 4 is a schematic diagram of variable beam splitting
using multiple AOTFs;
[0036] FIG. 5 is a schematic diagram of a microscope system which
enable real-time microscopic imaging with sample manipulation;
[0037] FIG. 6 graphically shows a selection of spectrally possible
properties of beam combiners;
[0038] FIGS. 7a-c are flow charts illustrating implementation of
actuation control;
[0039] FIGS. 8a-c are schematic diagrams showing the derivation of
a beam combiner design embodying the present invention;
[0040] FIG. 9 graphically illustrates the relationship between the
P.sub.SV, mani, sample and the beam combiner reflectivity RSV;
[0041] FIG. 10 is a screenshot of a user interface for a user for
the bleaching as the manipulation; and
[0042] FIG. 11 is a screenshot for the imaging process with an
imaging scan module.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In describing preferred embodiments of the present invention
illustrated in the drawings, specific terminology is employed for
the sake of clarity. However, the invention is not intended to be
limited to the specific terminology so selected, and it is to be
understood that each specific element includes all technical
equivalents that operate in a similar manner to accomplish a
similar purpose.
[0044] If the manipulating as well as imaging systems compete for
the power of the laser line in such a manner that it is as high as
possible in the simultaneous operation in this type of microscope
system, it is an advantage if the power requirement of the imaging
system has a higher priority compared to the manipulator module. In
commercial laser scanning microscope systems with only one scan
module, typically the laser power for the manipulation process and
the subsequent imaging can in each case be adjusted through the
operating interface of the control software. This takes place, for
example, using the corresponding software slider. In contrast to
that, in the methods for simultaneous, variably tunable division of
a laser line between two independent scan systems, shown in FIG. 2,
3 and 4, besides the specification of the power for the
manipulating and the imaging systems, adjustment of the splitting
ratio between the two split up branches of the beams is also
necessary.
[0045] According to the invention, the beam-splitting ratio as well
as the subsequent intensity modulation are so optimally adjusted
that, on one hand, the laser power requirement of the imaging
system is fulfilled (higher priority) and, on the other hand, the
manipulating system also receives laser power that is as high as
possible at the same time. This makes it necessary to provide a
method for optimal management of the laser power that is as
automatic as possible, in which the user of the device only needs
to define the laser powers necessary for imaging and manipulation
in the customary manner (as in LSM systems with only one scan
module) and, against that, the control software takes care on its
own of the optimal tuning of the components shown in FIGS. 2, 3 and
4.
[0046] Implementation of this principle of the actuation control,
shown in the flow charts in FIGS. 7a-7c for the layouts for the
variable splitting of the beam shown in FIGS. 2, 3 and 4, solves
the problem of the management of the laser power that is as
automated and optimal as possible in the simultaneous operation of
two independent scan modules.
[0047] This principle is explained as follows on the basis of the
variable splitting of the beam by means of a rotatable
.lamda./2-plate and intensity modulation of the two split partial
beams by means of an AOM (acousto-optic modulator).
[0048] The AOMs correspond, for instance, to the attenuators in the
beam paths to the manipulator or the line scanner shown in FIG. 2
and 5, whereby the rotatable .lamda./2-plates are arranged behind
the lasers and exercise influence in both paths.
[0049] The principle of the controlling actuation shown generally
in FIGS. 7a.-c. can thereby be employed in analogous manner, if the
other elements as in FIGS. 2, 3 and 4 and the above described
methods a. to e. are used for beam splitting and intensity
modulation.
[0050] As already explained above, in most of the applications, the
power for the light required by the imaging system has the first
priority. The imaging system (for example the line scanner in FIG.
5) is therefore denoted also as the "Master" system following the
nomenclature selected in FIG. 7.
[0051] The software slider in the operating software represents
(analogous to the software interface of "stand alone" LSM systems)
the total power for the light demanded by the respective scan
module (image forming as well as manipulating systems). Screenshots
of a user interface for the user are shown in FIG. 10 for the
bleaching (as the manipulation) and in FIG. 11 for the imaging
process (with the imaging scan module as described above). Thereby,
the power for the individual wavelengths, expressed as percent
units, is given in each case by the user in the lower part
(excitation). It comprises, as in the generalized FIG. 7a, the
quantity of light made available by the variable beam splitting
(see box) (.lamda./2-plate & polarization beam splitter cube)
and the AOM (beam modulation box): P .times. ideal , Master = R
.times. .lamda. / 2 T .times. AOM , Master .times. P actual , Slave
= T .lamda. / 2 T AOM , Slave .times. 1 .gtoreq. P actual , Master
+ P actual , Slave .times. 1 .apprxeq. R .lamda. / 2 + T .lamda. /
2 ##EQU1## whereby R.sub..lamda./2 and T.sub.AOM represent the part
of the light reflected by the polarization beam splitter cube and
the part of the light transmitted by the AOM. Thereby the
designations "Master" and "Slave" stand for the "imaging" or the
"manipulating" scan system. The "Master" part of the imaging system
after the polarization beam splitter (R.sub..lamda./2) is obtained
here from the angular position .THETA. of the .lamda./2-plate
R.sub..lamda./2=cos.sup.2(2.theta.)
[0052] In the present invention, the strategy for the control is so
arranged as in FIG. 7b that the .lamda./2-plate, as the beam
splitting element, moves as little as possible: TABLE-US-00001 IF
P.sub.ideal, Master > P.sub.actual, Master then IF R.sub.N2 >
P.sub.ideal, Master then USE T.sub.AOM, Master ELSEIF USE R.sub.N2
ENDIF ELSEIF (P.sub.ideal, Master < P.sub.actual, Master USE
T.sub.AOM, Master ENDIF
[0053] Pideal is the value specified by the user, on response yes
to the comparison in the first box, it goes to the next query, on
response no, the attenuator (AOM) of the master part must be
adjusted.
[0054] In the next comparison, on no, the lambda half plate of the
master system is adjusted, on yes, the attenuator (AOM) of the
master system.
[0055] However, in the control, the power demanded by the
manipulating system ("Slave") comes to an expression as in FIG. 7c.
That means that in principle the unused part of the remaining power
(1-P.sub.ideal, Master) is available to the "slave" system for the
manipulation of the sample. TABLE-US-00002 IF P.sub.ideal, Slave,
> P.sub.actual Slave then IF T.sub.N2 > P.sub.ideal, Slave
then USE T.sub.AOM, Slave ELSEIF USE T.sub.N2, BUT T.sub.N2, MAX
.ltoreq. (1 - P.sub.ideal, Master) USE T.sub.AOM, Master ENDIF
ELSEIF (P.sub.ideal, Slave < P.sub.actual, Slave USE T.sub.AOM,
Slave ENDIF
EXAMPLES
[0056] To illustrate the actuation control processes shown in FIGS.
7a-c with reference to five different user settings, shown in
succession, as they may be found in the applications of the systems
shown in FIGS. 1 and 5.
[0057] The examples 1)-5) follow successively one after the other,
whereby the reaction without the manipulating system is described
first (ref. FIG. 7b). After that, the final result taking into
account the additional power requirement of the manipulator system
as in FIG. 7c is explained.
[0058] 1) Imaging 100%, manipulation 0%, .fwdarw.R.sub..lamda./2=1,
T.sub.AOM,Master=1, T.sub..lamda./2=0 [0059] the .lamda./2-plate is
set to R.sub..lamda./2=1, that is, the master (imaging) receives
the entire laser energy when the transmission of the corresponding
attenuator is maximum (T.sub.AOM,Master=1), the attenuator is
arranged in sequence after the .lamda./2-plate;
[0060] 2) Imaging 50%, manipulation 40% [0061] The imaging demands
50% of the available energy, thus a maximum of 50% remains for the
manipulation [0062] However the manipulation asks for only 40%, so
that the manipulation can also actually receive its 40% [0063] For
that the .lamda./2-plate must be regulated, because at that moment
all the energy flows in the direction of the imaging system
R.sub..quadrature./2=1, the .lamda./2-plate is thereby regulated as
little as possible and hence moves according to
T.sub..quadrature./2=0.4.fwdarw.R.sub..lamda./2=0.6 (the total is
1). [0064] But now the imaging system receives too much energy (60%
because R.sub..quadrature./2/2=0.6 and T.sub.AOM,Master=1), that
is, it must now be slightly attenuated: T.sub.AOM,Master=0. 83
[0065] Final result:
T.sub..lamda./2/2=0.4.fwdarw.R.sub..lamda./2=0.6.fwdarw.T.sub.AOM,Master=-
0. 83, T.sub.AOM,Slave=1.0
[0066] 3) Imaging 50%, manipulation 70% [0067] The manipulation
demands 70%, but can have only 50%, because the power requirement
of 50% for the imaging system has a higher priority, that is,
increase by 10% from 40% to 50% is possible, for that the
.lamda./2-plate must be moved slightly, from R.lamda./2=0.6 to
R.sub..lamda./2=0.5; after that the attenuators of both systems are
each adjusted to give 100% transmission.
[0068] Final result:
T.sub..lamda./2=0.5.fwdarw.R.sub..lamda./2=0.5.fwdarw.T.sub.AOM,Master=1.-
0, T.sub.AOM,Slave=1.0, P.sub.Slave=0.5 (insted of 0.7)
[0069] 4) Imaging 10%, manipulation 40% [0070] The .lamda./2-plate
can remain as it is, only the attenuators must be readjusted, this
is done fast: T.sub.AOM,Master=0.2, T.sub.AOM,Slave=0.8
[0071] Final result:
T.sub..lamda./2=0.5.fwdarw.R.sub..lamda./2=0.5.fwdarw.T.sub.AOM,Master=0.-
2, T.sub.AOM,Slave=0.8, P.sub.Slave=0.4
[0072] 5) Imaging 10%, manipulation 70% [0073] The imaging (master)
demands 10% of the laser power, that is, the manipulation can
receive 70%; for that the .lamda./2-plate must be moved:
T.sub..lamda./2=0.7.fwdarw.R.sub..quadrature./2=0.3 [0074] After
that the attenuators are adjusted so as to yield the total values
of 10% and 70% respectively
[0075] Final result:
T.sub..lamda./2=0.7.fwdarw.R.sub..lamda./2=0.3.fwdarw.T.sub.AOM,Master=0.
33, T.sub.AOM,Slave=1.0
[0076] The generalized principle of the control shown in FIG. 7a-c
describes a method for optimal management of light power with
simultaneous operation of two independent scanning systems, whereby
[0077] at least one source of light can be divided with a variably
adjustable ratio of R.sub.ST/T.sub.ST between two scanning systems
by means of a beam splitting element ST; [0078] the power
requirement of one scanning system ("Master") is assigned higher
priority than that of the other scanning system ("Slave"); [0079]
suitable intensity modulators are provided for, if necessary,
reducing the intensity of the transmitted light distributed between
the two partial branches T.sub.Master and T.sub.Slave; [0080] the
user of the devices defines only the power required by the two
scanning systems through the interface of the operating SW, and the
control SW determines on its own the optimal settings for the
variable beam splitting and for the intensity modulators of the
master and the slave scan modules.
[0081] FIG. 6 shows a selection of the spectrally possible
properties of beam combiner types relevant from the viewpoint of
applications, whereby the manipulation wavelengths 355 nm, 405 nm,
488 nm and 532 nm can be used both in the direction of transmission
as well as of reflection. Typically, different types of beam
combiners are provided with motorized loading devices for
exchanging, such as, for example, a motorized reflector revolver,
or a reflector slider, in the region of the infinite space between
the objective and tube lens.
[0082] Neutral combiners (for example T20/R80) can be employed
universally as beam combiners for most diverse varieties of
applications and, in addition to that, enable applications in a
simple manner, in which the same laser wavelengths can be used in
simultaneous operation, both of the imaging system as well as of
the manipulation system (in particular photobleaching, FRET, FRAP,
FLIP). On the other hand, neutral combiners often represent a
compromise, especially when the same laser line is used
simultaneously for the manipulation as well as for the imaging,
between the branching ratio for the respective laser wavelength, on
one hand, and maximizing the signal efficiency in the range of the
detection wavelength, on the other hand. Therefore, this demands an
optimal design for the beam combiner, which is explicitly optimized
for simultaneous operation of a manipulating and an imaging system
for the same laser wavelength.
[0083] It is evident from FIG. 6 that simultaneous manipulation of
the sample and imaging can be realized without problems with the
help of a suitable dichroic beam combiner, if both scanning systems
use different laser wavelengths. Thus, for example, the beam
combiner denoted by "T405" has transmission T>0.9 only within a
narrow bandpass range of, for instance, 405 nm.+-.5 nm, whereas
ideally it has mirroring effect with R .apprxeq.1 in all the other
spectral ranges. This beam combiner is thus exclusively suitable
for the manipulation of the sample with 405 nm (for example in
photoconversion of Dronpa, Kaede, PA-GFP), whereby the manipulating
system is arranged in the direction of transmission. Against that,
the imaging system is arranged in the reflection direction, and
allows, in the case of this special beam combiner type,
fluorescence excitation and detection for any wavelength outside
the bandpass range of 405 nm.+-.5 nm. In the present invention,
there is the requirement of bringing together a laser source that
is split between a manipulating system and an imaging system to a
beam combiner, whereby the beam combiner design optimally supports
the management of the laser power implied in FIGS. 7a-c. Since both
scanning systems thereby simultaneously fall back on the same
source of laser wavelength, a dichroic beam-combiner is not
suitable for such an application.
[0084] FIGS. 8a-c elucidate the derivation of a beam combiner
design, which is designed especially for simultaneous operation of
a manipulating system and an imaging system with the same laser
wavelength distributed with a variable ratio. In this way, a
comparison is done with the ideal mirror (FIG. 8a.), on one hand,
and with a neutral combiner (FIG. 8b.), on the other hand. FIG. 8a
shows a microscope system, which is equipped only with an imaging
system, which is arranged in the reflection direction (90.degree.
arrangement) with respect to the optical axis of the objective. The
beams with the fluorescence excitation light of wavelength .lamda.
and the Stokes-shifted fluorescence light of wavelength
.lamda..sub.FL generated in the sample are incident through an
idealized mirror, with the reflectivity being R=1 in the entire
spectral range under consideration. In order to generate a suitable
fluorescence signal in this imaging system, the normalized relative
laser power must be P.sub.0,imag<1. The total available power of
the source of light is 1. In the following considerations, the
power P.sub.0,imag is taken as the reference value in each
case.
[0085] FIG. 8b shows a microscope system, which enables
simultaneous use of a manipulator arranged in the direction of
transmission and an imaging system arranged in the direction of
reflection. In use, the laser wavelength .lamda., split variably
between the two scanning systems, is used both for the manipulation
of the sample as well as for the fluorescence excitation, whereby
the total laser power of the common source of light is again 1. In
use, the superposition, accurate to the pixel, of the optical axes
of the two scanning systems take place by means of a neutral beam
splitter, which exhibits a constant reflectivity R.sub.NV<1 in
the spectral range of interest. Thus, in the imaging, both the
excitation light of wavelength .lamda. as well as Stokes-shifted
fluorescence signal of wavelength .lamda..sub.FL is reduced in each
case by factor R.sub.NV. The power requirement of the imaging
"Master" system (See FIG. 7) follows from the requirement that the
same fluorescence signal intensity is detected after the neutral
beam combiner as the combiner is arranged in the measurement setup
shown in FIG. 8a. The reduction in the intensity on the excitation
and the emission side taking place in the neutral beam combiner can
thereby each be compensated by a factor R.sub.NV, whereby, compared
to the system in FIG. 8a, laser power that is greater by a factor
1/(R.sub.NV).sup.2 is incident on the neutral combiner. In order to
detect the same fluorescence signal intensity as in the arrangement
in FIG. 8a, the power requirement of the imaging "Master" module is
P.sub.NV,imag=P.sub.0,imag/(R.sub.NV).sup.2
[0086] The remaining laser power (1-P.sub.NV,imag) of the common
source of light of wavelength .lamda. is thus available to the
manipulating "Slave" system according to the actuation control
schema in FIG. 7, whereby, of this remaining manipulation laser
power, again only the part (1-R.sub.NV) is transmitted in the
neutral combiner. The resulting laser power for the manipulation,
which can be maximally available in the object plane, thus amounts
to P.sub.NV, mani, sample=(1-P.sub.NV,imag)*(1-R.sub.NV)
[0087] The optimal reflectivity R.sub.NV of the neutral beam
combiner is obtained by maximizing the resulting manipulating laser
power in the object plane P.sub.NV, mani, sample for the same
fluorescence signal intensity as in the layout in FIG. 8a. Thus one
obtains the following analytical expression for the optimal
reflectivity: R NV = { P 0 , imag + P 0 , imag 2 + ( P 0 , imag 3 )
3 3 + P 0 , imag - P 0 , imag 2 + ( P 0 , imag 3 ) 3 3 } ##EQU2##
[0088] Example: P.sub.0,imag=0.08 (8% excitation power for the
embodiment 8a.) R.sub.NV=0.4939 P.sub.NV, mani, sample=0.3401
[0089] FIG. 8c now shows a beam combiner design optimized compared
to such a neutral combiner. Let this beam combiner have
reflectivity R.sub.SV<1 for the manipulation and fluorescence
excitation wavelength .lamda., whereas let the reflectivity be RFL
in the fluorescence wavelength range .lamda..sub.FL, which is as
nearly equal to 1 as possible. In the calculation of the power
requirement of the imaging "Master" system, again let the losses
appearing on the excitation and the emission side be taken into
account, which are compensated by the correspondingly increased
laser power P.sub.SV,imag of the imaging module. Thereby the laser
power incident on the beam combiner is reduced by factor R.sub.SV,
whereas the reverse fluorescence signal is reduced by factor
R.sub.FL. Therefore, in order to detect the same fluorescence
signal intensity as in FIG. 8a, the imaging system in FIG. 8c
requires the laser power:
P.sub.SV,imag=P.sub.0,imag/(R.sub.SV*R.sub.FL)
[0090] The remaining power (1-P.sub.SV,imag) of the common light
source of wavelength .lamda. is thus available to the "slave"
manipulation system according to the actuation control principle
shown in FIG. 7, whereby, of that, only the part (1-R.sub.SV)
crosses the beam combiner. The resulting laser power for the
manipulation, which can be maximally available in the object plane,
is thus expressed by: P.sub.SV, mani,
sample=(1-P.sub.SV,imag)*(1-R.sub.SV)
[0091] The reflectivity R.sub.SV of the beam combiner for the
excitation and manipulation wavelength .lamda. is now to be so
optimized that for a given fluorescence reflectivity R.sub.FL (in
the ideal case as nearly equal to 1 as possible) and the same
fluorescence signal intensity as in the embodiment 8a, a highest
possible manipulation laser power P.sub.SV, mani, sample in the
object plane is obtained. Analytically one obtains the optimum for:
[R.sub.SV].sup.opt=(P.sub.0,imag/R.sub.FL).sup.1/2
[0092] In FIG. 9, the relationship between the P.sub.SV, mani,
sample and the beam combiner reflectivity RSV is shown. [0093]
Example: P.sub.0,imag=0.08 (8% excitation power for the embodiment
8a.), R.sub.FL=0.85 R.sub.SV=0.3068 and P.sub.SV, mani,
sample=0.4805
[0094] For the same fluorescence signal intensity in the imaging
system, one thus obtains, using this beam combiner, about 30%
higher manipulation laser power in the sample--compared to the
optimized neutral combiner of the embodiment 8b.
[0095] If in contrast to the devices shown in FIGS. 8a-c, the
manipulator is instead arranged in the direction of reflection and
the imaging scan system is arranged in the direction of
transmission, the aforementioned argument follows in analogous
manner, whereby in the above mentioned equations the designations
for the transmission T and the reflection R must then be mutually
exchanged.
[0096] To generalize, an optimized beam combiner design for the
superposition of the optical axes of two independent scanning
systems is required, in which both the modules are operated with at
least one common laser wavelength .lamda.. Thereby, at least one of
the two scanning systems is designed as an imaging system and its
power requirement is assigned higher priority compared to the other
scanning system in such a manner that the detected fluorescence
signal intensity is comparable with the corresponding "stand alone"
system. For the wavelength(s) .lamda. commonly used by both the
systems, the branching ratio of this beam combiner is so selected
that for a given fluorescence signal intensity, which would
correspond to the typical intensity in a "stand alone" scanning
system for free passage of the beam without a beam combiner, laser
power that is as high as possible in the sample plane is obtained
for one scanning system. Outside the common wavelength(s) .lamda.
used by the two scanning systems, the beam combiner is so designed
that it is either only reflecting or transmitting as far as
possible. The optimized spectral design of this beam combiner
corresponds therefore to a "bad" bandpass filter in transmission or
reflection.
[0097] In other words, as the control variables for the method
according to the invention serve the grade of the reflectivity
(Rsv, Rfl) or the transmission of the corresponding beam combiner
for the excitation beam and fluorescence beam in the imaging system
with respect to the proportion of the manipulation system or if
specific power is given, the selection of a suitable beam combiner
is optimized as the control variable.
[0098] In FIG. 6, two examples for such types of beam combiners are
shown schematically. The beam combiner "T488-30%" is thereby so
embodied that the imaging system is arranged in the direction of
reflection and the manipulating system in the direction of
transmission. The wavelength 488 serves thereby both the purpose of
the manipulation of the sample as well as of the excitation of
fluorescence. The beam combiner layout is so designed that the
transmission of 488 nm manipulation light is 70% and the reflection
of 488 nm fluorescence signal light is 30%.
[0099] Outside the bandpass range of 488 nm, the beam combiner is
as reflecting as possible as in FIG. 6, so as to enable efficient
signal detection in the direction of reflection. This beam combiner
layout is therefore designed for such imaging applications, which
require relatively low fluorescence excitation power (P.sub.0,imag
approximately 8%) and, at the same time, the manipulation power is
as high as possible for the wavelength 488 nm. In practice such
requirements are of relevance especially in FRAP applications.
Thus, in a special embodiment, beam combiners optimized especially
for FRAP applications are required. In contrast to that, the beam
combiner type "R488-30%," which is schematically depicted in FIG.
6, is optimized for an arrangement in which the imaging system is
in the transmission direction and the manipulation system in the
reflection direction.
[0100] The described invention relates in a general sense to any
type of imaging and manipulating system. Besides the (confocal and
partially confocal) point and line scanners, it can also be of
relevance in particular in multifocal laser scanning systems (for
example, those based on lens arrays, diode laser arrays, with any
type of beam splitting arrangement) and spinning disk
systems/Nipkow systems. Further, in the present invention, the
sample can be scanned with a scanning method according to current
state-of-the-art. Thereby, one of the following can be the
underlying scanning principle of the device for the deflection of
the beam in the imaging or the manipulating system: [0101] Galvo
mirror or [0102] guidable, in particular rotatable and tiltable
mirrors, for example step motor driven deflecting mirrors [0103]
polygon mirrors [0104] acousto-optical deflecting devices, in
particular acousto-optical deflectors (AODs) [0105] movable
aperture masks, in particular in the form of a Nipkow disk [0106]
movable (monomode) fibers [0107] movable objectives or objective
parts [0108] mechanical x- and y-adjustment of a suitable component
or of the entire scanning system, for example by means of
acousto-optical modulators
[0109] However, since both the scanning systems must be independent
of each other in the sense of this invention, a mechanical x- and
y-adjustment of the sample is not admissible.
[0110] Besides the use of microscope systems with coherent light
sources (lasers) and confocal or partially confocal scan modules,
an advantageous application of the invention in analogous manner is
conceivable also in the simultaneous manipulation of the sample
and/or the imaging with the help of (structured) wide-field
illumination systems with incoherent light sources.
[0111] Modifications and variations of the above-described
embodiments of the present invention are possible, as appreciated
by those skilled in the art in light of the above teachings. It is
therefore to be understood that, within the scope of the appended
claims and their equivalents, the invention may be practiced
otherwise than as specifically described.
* * * * *