Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Title
MICROSCOPE WITH ADAPTIVE OPTICS
Field of the Invention
The invention relates to the expansion of current microscopes by
adaptive optics in the observation beam path and/or illumination beam path of
a
microscope. By adaptive optics is meant optically active component assemblies
for
wavefront modulation. The adaptive optics purposely change the phase and/or
the
amplitude of the light in such a way that a displacement and shaping of the
focus in
the object space and a correction of possible aberrations is achieved. The
possible
areas of use include confocal microscopy, laser-assisted microscopy,
conventional
light microscopy, and analytic microscopy.
Prior Art
The present: invention proceeds from the following prior publications:
- U.S. 4,408,874; W. Zinky, L. Rosenberg; 1981/1983: "Mechanically or
pneumatically deformable optical element for astigmatic magnification
adjustment 'for imaging systems in lithography".
- EPO 0098939 B1; ,3. Arnaud; 1983/1987: "Deformable optical element
for astigmatic correction". The thickness of the mirror membrane
varies over i:he surface, so that the membrane adopts a previously
calculated shape when subjected to external bending forces.
- EPO 03073:54 B1; H. Choffat; 1988/1992: "Ring arrangement of
bimorphic piezo layers for axial precision adjustment of components,
e.g., microscope objectives".
- U.S. 5,142,132; B. MacDonald, R. Hunter, A. Smith; 1990/1992:
"Adaptively ~:,ontrolled optical system for wafer fabrication (stepper)".
The adaptive element controls the focus and corrects aberrations. The
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error signal for correction is obtained from the light reflected back from
the wafer through interference with the original light. An exact method
for correction of aberrations is not indicated.
- DP DE 3404063 Cc!; A. Suzuki, M. Kohno; 1984/1993: "Curved
transparent membrane in the beam path of an imaging system for
correction of imaging errors, especially lateral focus offset".
- U.S. 5,504,575; R. Stafford; 1993/1996: "Spectrometer based on
spatial light modulator and dispersive element". Fibers and optical
switches/flexible mirrors are used to switch the light to the detector
after passing through the dispersive element.
- EPO 167877; Bille, Heidelberg Instruments; applied for 1985:
"Ophthalmoscope with adaptive mirror".
The description of the present invention is based on the following
terminological definitions:
- "Wavefront modulator", within the meaning of the invention, is a device
for deliberately influencing the phase and/or the amplitude of a light
wave. Based on a reflecting optical element (deformable mirror,
electrostatic control, or controlled by a piezo array, or as a bimorphic
mirror) or a i:ransmitting optical element (LCD or similar unit). It can be
built in a continuous or segmented manner. In particular, the
segments can be adapted for controlling the respective problem.
- "Aberrations in the microscope": The aberrations in the microscope
objective occurring in defocused operating mode can basically be
categorized as correctable or not correctable. Causally, the
aberrations can be divided into aberrations caused by the objective,
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aberrations caused by the additional imaging optics of the microscope,
and) finally, those caused by the preparation itself.
- "Controlling the wavefront modulator": The controlling of the wavefront
modulator is carried out by a computer with appropriate software. The
required correcting variables are either calculated beforehand (offline)
or are calculated from measured quantities (online, e.g., through a
wavefront sensor or' by measuring the point brightness in the
intermediatE~ image).
Description of the Invention:
In conventional light: microscopy, as well as in laser-assisted
microscopy, the focus of the objective must be displaced with high precision
along
the optical axis as well as laterally. In conventional microscopes, this is
carried out
by mechanical displacement of the object stage or objective. In addition, in
case of
illumination by laser radiation, displacements are also necessary in the
object space.
Consequently, there is a reed for three-dimensional focus control in the
object
space.
It is the object of the invention to achieve the axial displacement of the
focus in the object space without changing the distance between the objective
and
the object.
According to the invention, this displacement is carried out at the
wavefront of the beam path. In this respect, the axial displacement of the
focus in
the object corresponds to a spherical change in the wavefront; the lateral
displacement corresponds to a tilting of the wavefront. According to the
invention,
aberrations in the beam path are also compensated by changing the wavefront.
These manipulations are carried out in a pupil plane of the beam path.
In conventional light microscopy in the observation beam path, in order
to achieve an axial displacement of the focus in the object space without
changing
the distance from the objective to the object, the wavefront in the pupil of
the
objective or in a plane equivalent to the pupil plane must be spherically
deformed.
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Such deformation can be achieved through a wavefront modulator, namely a
wavefront-phase modulator.
Fig. 1 and Fig. 1 a show a schematic imaging beam path of an optical
light microscope with an observed object, an objective, and a tube lens for
generating an intermediaire image which can be viewed by eyepieces) not shown.
A
wavefront modulator, according to the invention, is arranged between the tube
lens
and objective. The wavefront which is curved after the objective is corrected
by the
wavefront modulator by compensating for the aberrations of the objective.
Calculation: have shown that with radii of curvature of the wavefront in
the pupil of between -3.Orn and 1.5m, the focus can be displaced by more than
1.5mm. This depends on the objective that is used; in the present case, the
data
refer to the Epiplan-Neofluar 20x/'0.5. Displacements in the range of several
tens of
micrometers are sufficient in most cases. As mathematical calculations have
further
shown, the interval of a possible focus displacement decreases as the
magnification
of the objective increases. However, since the objective is not calculated or
designed for this spherically deformed wavefront in the entrance pupil,
aberrations
through the objective during defocusing cannot be prevented.
A focus displacement of the kind mentioned above without mechanical
influence of the objective has several advantages. First, any mechanical
influencing
of the object by the microscope objective is eliminated by the fixed working
distance
between the front lens of the objective and the object. Accordingly, it is
possible for
the first time to carry out :;ectionwise image recording with different depth
positions
of the observation plane vvith a static water-immersed object. Previously, a
technique of this kind failed as a result of the mechanical deformation of the
object
and its surrounding medium through mechanical pressure on the preparation.
The fixed working distance at the microscope also offers advantages in
the analytic examination of specimens in the biomedical field. When using
microtiter
plates, a correction of abs~rrations caused by the microtiter plate can be
compensated. The microtiter plate can be included optically in the beam path
and
the microscope objective can be partially (e.g., the front lens) integrated
therein.
Fig. 1 b sho~nis a construction of an optical light microscope with
deformable mirrors which correct the wavefront in the direction of the tube
lens. A
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first modulator arrangemE~nt and a second modulator arrangement are included
in
the imaging via a beam splitter between the objective and tube lens. In
addition,
optics for pupil adaptation are provided in front of each modulator
arrangement. The
above-mentioned arrangE:ments will be described more fully in connection with
Fig.
7.
A correction of aberrations due to the preparation and the medium
surrounding the specimen is also possible by means of a suitable deformation
of the
wavefront through the wavefront modulator. This is shown in Fig. 2. The
wavefront
which is distorted by aberrations its corrected by the wavefront modulator
arranged
between the objective and the tube lens. However, the spherical components in
the
wavefront correction are not sufficient for this purpose; aspherical
components must
be included. Annular actuators are sufficient for rotationally symmetric
aberrations
(all terms of higher-order spherical aberration). For angle-dependent
aberrations,
segmented actuators must be used (Fig. 4). These segmented actuators can
either
be integrated together in lrhe same wavefront modulator or two independent
modulators can be used in different pupil planes. In the first case, the
number of
actuators is quadratically scaled, in the latter case linearly scale, with the
required
resolution, which means a reduction in the complexity of control electronics.
Currently okr~tainable phase modulators are limited with respect to
amplitude and with respect to the maximum phase gradients that can be
generated.
This in turn limits the pos;>ibilities for correction far away from the
working point of
the objective. A conceivable alternative consists in combining adaptive optics
with
conventional glass optics. The latter serve to generate a large phase gradient
or
large wavefront amplitudes, and precision tuning is achieved by means of the
adaptive optics.
When displacing to a greater focus distance, the required convex
wavefront of the pupil results in a vignetting which leads to lower light
efficiency and
a reduction in usable aperture. This limitation is design-related and, in
principle, can
be taken into account in the future in the optical design of an objective.
Further, aberrations occurring in the beam path when the focus is
displaced can result in distortions of the image. In order to correct these
aberrations, non-spherical components can be superposed on the wavefront as
was
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indicated above. According to mathematical calculations, a considerable
improvement can be achieved in the image (Strehl ratio greater than 98%) even
with
small rotationally symmetric components of orders r4 and rs (spherical
aberration of
higher order) at the wavefront.
A further advantage of the process consists in the achromatic behavior
of a reflection-based wavefront modulator. With a suitable coating of the
membrane
mirror, the entire spectral range from low UV to far IR can be phase-
modulated.
Chromatic aberrations (with the exception of absorption effects) are ruled
out. This
results in new methods for chromatic correction in image generation. For this
purpose, the illumination is adjusted sequentially to different wavelengths,
wherein
the wavefront modulator lis adjusted to the suitable optical correction for
each of the
individual wavelengths. In this way, a set of images with optimum chromatic
correction is obtained which, when superposed, give a white-light recording of
high
chromatic correction which cannot be achieved in the same way through the use
of
conventional glass optics. Accordingly, in principle, an objective with a
wavefront
modulator can be corrected in an optimum manner on any number of wavelengths
in
the optical spectrum.
The required wavefronts initially have only a rotationally symmetric
character for displacement of the focus and for correction of spherical
aberrations.
In order to generate such wavefronts in the pupil of the microscope objective,
the
adaptive optics must have a distribution of actuators with spatial frequency
increasing toward the edge (Fig. ~4) because the largest gradient in the
wavefront
occurs at the edge.
Fig. 4 show, various actuator structures with increasing spatial
frequency in Fig. 4a to Fil~. 4c and with segments in Fig. 4d, e.g.) for
correcting
astigmatism and coma.
In camera-assisted image generation, the effect of pixel mismatching
occurs especially with high spatial resolution. In this case, the microscope
image is
displaced toward the camera so that the individual images of the video signal
are
spatially displaced. This problem can be eliminated by a variable tilt
component in
the wavefront of the imaging signal. By means of suitable regulation, the
unsteady
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movements of the image signal can be eliminated and a static image can
accordingly be generated.
Another problem in camera-assisted image recording is field curvature.
The field curvature can be improved during operation) at the expense of other
parameters such as chromatic correction, through the use of a wavefront
modulator
in the imaging beam path.
In conventional light microscopy, a flexible configuration of optics,
improved optical charactE~ristics of the microscope, and new illumination
techniques
can be realized in the illumination beam path by introducing adaptive optics.
In a
similar way to the observation beam path, a wavefront phase modulator can
optimize the imaging of the illumination burner (or of the laser, as the case
may be)
in the object plane. Likev~ise, in the case of critical illumination, an even
illumination
of the object space can be adjusted. Fig. 3 shows a wavefront modulator
between
the collector and conden:~er which are arranged following an illumination
burner.
The illumin~~tion intensity in the object plane can be optimized spatially
with respect to intensity and homogeneity by a wavefront amplitude modulator.
In
principle, a manipulation of the pupil is possible in this way. An oblique
illumination
of the object space can b~e achieved by purposely changing the tilt proportion
of the
wavefront.
In confocal ,microscopy and with the laser scanning microscope, the
applications can be realized even more readily than in conventional light
microscopy
by using laser light for illumination.
When using a laser for illumination, the use of a wavefront modulator is
advantageous already when coupling into the illumination fiber. In this
respect, it is
possible to realize variable adaptation optics whose focal lengths and imaging
scale
ratio are adjustable in dependence on the beam characteristics of the lasers)
and
the utilized fibers) in ordE~r to achieve an optimum in-coupling into the
fiber.
Arrangements based on the same principle can also be used in coupling of
illumination fibers to the microscope optics. Because of the rapidity of the
modulators, time-resolved measurements and multiplexing procedures can also be
realized in order to switch between one or more lasers and different fibers.
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In confocal imaging, the transmission can be adapted dynamically
through the defining pinhole. Both the position and diameter of the focus are
variable within wide limits. The illumination laser, or lasers, can thus be
adjusted in
an optimum manner basE:d on requirements. Further, the contour of the light
distribution of the focus can also be adapted to the pinhole. Not only
rotationally
symmetric apertures but also those having other kinds of outlines or,profiles
such as
lozenge-shaped or rectangular apertures of the type always occurring in
pinholes
realized in practice can accordingly be adapted and optimized to maximum
transmission or minimum diffraction losses. An optimization of this kind can
be
initiated statically by parameters that are calculated beforehand on the one
hand or
can be regulated during operation to determined optimizing parameters.
As in conventional light microscopy, the chromatic correction can also
be adjusted in dependen~:,e on the utilized illumination laser. Sequential
images can
be recorded at different wavelengths) with optimum chromatic correction in
each
instance, through the uses of fast, synchronously controlled wavefront
modulators in
the laser input coupling and in the illumination optics and recording optics.
Brief Description of the Drawings
The invention will be explained more fully with reference to
embodiment examples. Shown in the accompanying drawings are
Fig. 1 and Fig. 1 a the schematic imaging beam path of a light microscope;
Fig. 2 the correction of a wavefront distorted as a result of
aberrations with a wavefront modulator arranged
between the objective and the tube lens;
Fig. 3 a wavefront modulator arranged between the collector
and the condenser for adjusting an even illumination of
the object space;
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Fig. 4 various actuator structures, with increasing spatial
frequency in Fig. 4a to Fig. 4c and with segments in Fig.
4d;
Fig. 5 various constructions of wavefront modulators, including
those with electrostatic (Fig. 5a), piezo-controlled (Fig.
5b) and bimorphic membranes (Fig. 5c) as actuating
elements;
Fig. 6 wavefront modulator with electrostatic membrane mirror;
Fig. 7 the principle of a laser scanning microscope with a short-
pulse laser;
Fig. 8 the basic construction of a pre-chirping unit.
Detailed Description of the Drawings
Fig. 5 shows various constructions of wavefront modulators as are
currently obtainable. For example, transmitting modulators based on LCD, as
shown in Fig. 5d) or reflecting modulators with movable membranes are
available.
These may be distinguished, in turn, according to their type of actuating
elements:
electrostatic (Fig. 5a), pie~zo-controlled (Fig. 5b), or bimorphic membranes
(Fig. 5c)
as actuating elements. A,Ithough the invention is directed generally to
wavefront
modulators, the electrostatic mernbrane mirror is especially emphasized in
this
respect in view of its nurrnerous advantages.
A micro-fabricated monolithic membrane mirror of the type mentioned
above, which is shown in more detail in Fig. 6a and Fig. 6b with membrane M
and
driving electrodes E, is distinguished by excellent flatness and good optical
quality of
the reflecting surface (less than J20), small physical size (2mm to 20mm),
hysteresis-free control with low voltages (less than 100V), high mechanical
cutoff
frequency of the membrane (several MHz), large travel or lift ( ~ 1 OONm), and
therefore small radius of curvature (down to 1 m), and an actuator structure
that is
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variable within wide limits and has a high spatial density. The minimum
actuator
size is ultimately only limited by the condition that it must be greater than
the
distance between the electrode and the membrane.
The great advantage of the electrostatic membrane mirror consists in
the fact that only a constant potential need be applied to the actuator
electrodes for
adjusting a parabolic shape. The parabolic shape of the mirror is given at
constant
driving of the electrodes by the physical behavior of the membrane (constant
surface
force). Accordingly, high dynamics can be achieved in the correcting variable
(mirror
travel) with low dynamics in the control variable, that is, the applied
voltage.
Fig. 7 shows a laser scanning microscope with a short-pulse laser,
especially for multiphoton excitation. This will be explained more fully
hereinafter.
In nonlinear processes, the detected signal depends on the nth power
of the excitation intensity, High intensities are necessary for excitation.
These high
intensities are achieved through the use of short-pulse lasers and the
subsequent
diffraction-limited focusing with microscope objectives. Therefore, it is the
aim of the
arrangement to realize the smallest possible (i.e., ideal) focus and the
shortest
possible pulse length in the specimen. In this way, high intensities can be
achieved
in the specimen. Nonlinear processes are, for example, multiphoton absorption,
surface second harmonic. generation (SSHG), and second harmonic generation
(SHG), time-resolved microscopy, OBIC, LIVA, etc.
The invention will be explained more fully in the following with
reference to two-photon microscopy. In this connection, the following prior
art
serves as a point of departure:
WO 91/07651 discloses a two-photon laser scanning microscope with
excitation through laser pulses in the subpicosecond range at excitation
wavelengths
in the red or infrared region. The publications EP 666473A1, WO 95/30166, DE
4414940 A1 describe excitations in or above the picosecond range with pulsed
or
continuous radiation. A process for optical excitation of a specimen by means
of
two-photon excitation is described in DE C2 4331570.
Utility Model DE 29609850 describes coupling of the radiation of short-
pulse lasers into a micro:~cope beam path via light-conducting fibers. In this
case,
an optical arrangement for wavelength-dependent temporal change of laser
pulses
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is provided between the I<~ser and light-conducting fiber and comprises at
least two
optical elements, for exannple, prisms or mirrors. This optical arrangement
can be
used to adjust the time divfference of different wavelengths of the laser
pulses by
changing the distance beirween tree optical elements.
As is well known, two-photon fluorescence microscopy basically opens
up the following possibilities in contrast to conventional single-photon
fluorescence
microscopy:
- Realization of a nonlinear excitation probability I2,,~ = A ~ I~~ with the
following
advantages: three-dimensional discrimination) i.e., depth discrimination,
without the use of ;~ confoc;al diaphragm; bleaching out and destruction of
cells takes place - if at all - only in the focus; improved signal-to-noise
ratio;
use of new detection methods such as non-descanned detection;
- NIR excitation with femtosecond lasers has the following advantages for the
examination of biological preparations: Working in the region of the optical
window for biological preparations (700-1400 nm) due to low absorption; this
method is therefor~s also suitable for the examination of living preparations;
low loading of cell:c due to low mean excitation output; large penetration
depths due to low acatter;
- The excitation of so-called UV dyes without the use of UV light means that
no
UV optics are necE;ssary;
- In two-photon excitation, there are broad-band excitation spectra of the
dyes
and it is therefore possible to excite a wide range of different dyes with
only
one excitation wavelength.
When ultra~~hort pulses pass through a dispersive medium, e.g., glass
or a preparation, the following effects take place in particular:
Group Velocity Dispersion (GVD): Femtosecond laser pulses have a spectral
width of several nanometers. The red-shifted wavelength components propagate
more swiftly through a positively dispersive medium (e.g., glass) that the
blue-shifted
wavelength components. There is accordingly a temporal widening of the pulses
and thus a reduction in pE:ak output or in the fluorescence signal.
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A pre-chirping unit (pair of prisms, pair of gratings or a combination of
the two) is a negatively dispersive medium, that is, blue-shifted wavelength
components propagate faster than red-shifted wavelength components. The group
velocity dispersion can accordingly be compensated by means of a pre-chirping
unit.
Propagation Time C)ifference (PTD): The glass paths differ over the
beam cross section (see Fig. 4). This causes a spatial magnification of the
focus so
that there is a reduction in the resolving capability and peak output or
fluorescence
signal.
This effect c;an be compensated by means of a wavefront modulator,
for example, an adaptive mirror. With a modulator of this kind, the phase and
amplitude of the light wave in the excitation beam path can be specifically
influenced. A reflecting optical element (e.g., deformable mirror) or a
transmitting
optical element (e.g., LCD) are possible modulators.
Wavefront distortion through scattering and diffractionlrefraction: This
distortion can be caused by the utilized optics themselves or by the
preparation. As
in the second effect, the wavefront distortion likewise results in deviations
from the
ideal focus. This effect can also be compensated by a wavefront modulator (as
was
already shown).
The effects mentioned above are generally dependent on the depth of
penetration into the preparation. In this regard, the arrangement according to
the
invention compensates for the GVD, PTD and wavefront distortion effects
synchronously as a functiion of the depth of penetration into the preparation
in order
to achieve short pulse lengths and the most ideal small focus in the focus of
the
preparation even with larl~e penetration depths.
A possible construction of the arrangement is shown in Fig. 7 by way of
example. The radiation of a short-pulse laser KPL passes into a pre-chirping
unit
PCU and then travels, via beam splitter ST1 and beam splitters ST2, ST3, to
two
adaptive optical element: AD1, AD2. The first element AD1 (coarse) is used for
coarse adjustment of the wavefront. It is accordingly possible to shift the
focus in
the z-direction. The wavc:front distortions and the PTD effects are
compensated by
the second element AD2 (fine). 'The laser light reaches the object via beam
splitter
DBS, x/y-scanning unit, optics Sl_) TL, and mirror SP and objective OL. The
light
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coming from the object tr;~vels back via beam splitter DBS, lens L, pinhole PH
and
filter EF to a detector, in this case a photomultiplier PMT, for example,
which is
connected in turn with a control unit as are the PCU, AD1 and AD2.
The adjustment of the adaptive elements AD1, AD2 and the pre-
chirping unit) for example, can be effected in this way until a maximum signal
is
present at the PMT. The beam path shown in the drawing is particularly
advantageous for an inverse microscope in which observation takes place "from
below", wherein the advantage consists in that the specimen remains fully
accessible for possible manipulation.
Fig. 6 already showed the basic construction of an adaptive mirror. It
comprises a highly reflecting mernbrane (e.g., silicon nitrate) and a
structure with
electrodes. By specifically contralling the individual electrodes, the
membrane
situated above the latter c:an be deformed and the phase front of the laser
beam can
accordingly be influenceal. The deformations of the phase front which occur
when
the pulses pass through the system and through the specimen can accordingly be
compensated.
The pre-chirping unit can comprise one or more prisms or gratings or a
combination thereof. Fig.. 8 shows possible arrangements with four prisms in
Fig.
8a, with four gratings in Fig. 8b, and with prisms and gratings in Fig. 8c.
Its manner
of operation is explained more fully in Fig 8a with reference to a prism
compressor.
The spectral width of a femtosecond laser pulse is several nanometers. When
the
laser beam passes through the first prism, the beam is broken up into its
spectral
components. Subsequently, the spectral components travel different glass paths
in
the second prism. Red-shifted wavelength components are accordingly retarded
in
relation to the blue-shifted components. The pre-chirping unit accordingly
acts as a
negatively dispersive medium and a compensation of GVD is also possible.
For the first time, through the use of the arrangement described above,
the advantages of the excitation of nonlinear processes can be utilized to
their full
extent and the use of low-power femtosecond lasers is made possible even at
greater depths of penetration in the specimen. High peak outputs can
accordingly
be realized with the use of low mean excitation outputs so that loading of the
biological preparations or specimens can be kept low and a high signal-to-
noise ratio
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and high resolution can be achieved in the axial and lateral directions.
In all of the arrangements described above, the wavefront adaptation
can be advantageously detected and monitored and adjusted in a defined manner
by means of a wavefront sensor which communicates with the microscope beam
path via a beam splitter (not shown).
