The present invention relates to a compensator system and method adapted to compensate for the angular dispersion of electromagnetic beams deflected by at least a pair of acousto-optic deflectors of an optical system, wherein the angular dispersion of each deflected beam is dependent on the deflection angle obtained by the deflecting acoustic frequencies of the acousto-optic deflectors.
Three-dimensional (3D) random access laser scanning technologies have great importance in performing measurements on biological specimens (including scanning, imaging, detection, excitation, etc.) e.g. imaging biological structures or mapping fluorescent markers of cell surface receptors or performing measurements such as uncaging/photosimulation, FRET (Fluorescence resonance energy transfer), FLIM (Fluorescence lifetime imaging), etc.
Commonly used 3D laser scanning microscopes are either confocal microscopes or multi-photon (two-photon) microscopes. In the confocal microscope technology a pinhole is arranged before the detector to filter out light reflected from any other plane than the focus plane of the microscope objective. Thereby it is possible to image planes lying in different depths within a sample (e.g. a biological specimen).
Two-photon laser scanning microscopes use a laser light of lower energy of which two photons are needed to excite a flourophore in a quantum event, resulting in the emission of a fluorescence photon, which is then detected by a detector. The probability of a near simultaneous absorption of two photons is extremely low requiring a high flux of excitation photons, thus two-photon excitation practically only occurs in the focal spot of the laser beam, where the beam intensity overcomes the two-photon threshold. The photon number is increased also by mode-locking the excitation laser causing photons to arrive in high intensity bounces at the sample. Generally a femtosecond pulsed laser is used to provide the required photon flux for the two-photon excitation, while keeping the average laser beam intensity sufficiently low to avoid thermal sample deterioration.
When applying either of the above-mentioned technologies the 3D scanning is conventionally carried out by moving the sample stage e.g. via stepping motors, however this is complicated to implement when using submerge specimen chambers or when electrical recording is performed on the biological specimen with microelectrodes. The movement of the sample stage is a slow process, allowing either minute order imaging speed or very low (kpixel) number of measurement points thus low resolution. Accordingly, in the case of analysing biological specimens it is often preferred to move the focus spot of the laser beam instead of moving the specimen. This can be achieved by deflecting the laser beam to scan different points in a focal plane (XY plane) and by displacing the objective along its optical axis (Z axis) e.g. via a piezo-positioner to change the depth of the focal plane. XY scanning is conventionally achieved by deflecting the laser beam within a given focal plane (XY plane) via mechano-optical deflecting means such as deflecting mirrors mounted on galvanometric scanners.
The inertia of the mechanical scanning components (i.e. the scanning mirrors and the microscope objective) presents certain limitations with regard to the achievable scanning speed, since the scanning components need to be physically displaced in order to perform 3D scanning.
Rapid acousto-optic deflectors (AOD) have been proposed as an alternative to the conventional mechano-optic solutions.
Kaplan et al. (“Acousto-optic lens with very fast focus scanning”, OPTICS LETTERS/Vol. 26, No. 14/July 15, (2001)) proposed an acousto-optic lens made up of two AODs with counter propagating acoustic waves locked in phase, to achieve purely focal plane shift along the z axis without lateral moving of the beam. Changing the focus of the acousto-optic lens is achieved by changing the sweep rate of the acoustic frequencies through the acousto-optic devices.
In U.S. Pat. No. 7,227,127 the above principle is made use of in order to provide 3D scanning. The focus of the beam can be moved in space in a diamond like volume by using four acousto-optic deflectors, two for both lateral directions (x and y). Lateral scanning is a result of the acoustic frequency differences of the two AODs diffracting in the same lateral direction, whereas depth focusing (i.e. focus shift along the z axis) is achieved by changing the sweep rate of the acoustic frequencies in the same AODs. Thus the focus point can be quasi independently adjusted in the x-z and y-z planes, where z is the longitudinal direction corresponding to the optical axis of the device. This also leads to strong astigmatism, when the acoustic frequency sweep rates in the x and y deflecting units are not perfectly matched.
However there are various problems associated with the state of the art AOD 3D scanning technology such as spatial and temporal dispersion, especially when applied in combination with multi-photon scanning technologies.
Prior art AOD systems eliminate spatial dispersion to a more or less extent by applying various dispersion compensating elements between the AODs or by special arrangement of the pair of AODs as discussed in WO2010/076579. Nevertheless, prior art optical arrangements were unable to reduce the angular dispersion of the AODs to a sufficient extent, mainly because of its spatial non-uniformity.
A known way of collimating an optical beam with angular dispersion is to place a prism 10 in the optical path as illustrated in
It is an object of the invention to overcome the problems associated with the prior art laser scanning microscopes and in general any optical system comprising acousto-optic deflectors which introduces angular dispersion into a deflected optical beam.
In particular, it is an object of the invention to provide an angular dispersion compensator system, which reduces the wavelength dependence of the deflection angle of a deflected optical beam.
The inventors have realised that the angular dispersion compensating property of a prism can be made use of in an optical system wherein the angular dispersion of an electromagnetic beam is dependent on the distance from the optical axis of the optical system by providing a compensator having two surfaces that together work like prisms with varying and matching incidence and opening angles. The inventors have also realised that in a laser scanning microscope wherein acousto-optic deflectors (AODs) are used for deflecting the scanning laser beam, a first lens group can be used to focus the deflected beams into a focal plane corresponding to one of the surfaces of such a compensator, whereby the beams of different angular dispersion are separated spatially allowing for compensating for the angular dispersion independently for each beam by the appropriately shaped compensator.
The above objects are achieved by the compensator system according to claim 1 and the method according to claim 5.
Further advantageous embodiments of the invention are defined in the attached dependent claims.
Further details of the invention will be apparent from the accompanying figures and exemplary embodiments.
The compensator system 20 comprises a first lens group 22 and a second lens group 24 which are arranged spaced apart from each other downstream of the AOD 14 along a common optical axis Z corresponding to the optical axis of the objective 16. In
A compensator element 26 is arranged between the first lens group 22 and the second lens group 24 such that a first surface 26a of the compensator element 26 lies substantially in the focal plane FP of the first lens group 22 while a second surface 26b of the compensator element 26 faces the second lens group 24.
By way of illustration two deflected beams 18 and 18′ are depicted in
The first lens group 22 focuses the first beam 18 to a first point P lying substantially in the focal plane FP of the first lens group 22 at a first distance r from the optical axis Z (and the focus point F of the first lens group 22). The second beam 18′ is focused to a second point P′ lying substantially in the focal plane FP of the first lens group 22 which is at a second distance r′ from the optical axis Z. The distance r, r′ from the optical axis Z depends on the deflection angle θdef, θdef′ of the beam 18, 18′ exiting the second AOD 14, hence the beams 18, 18′ are spatially separated in accordance with their different deflection angle θdef and θdef′. As will be clear later on, the different deflection angle θdef, θdef′ results in different angular dispersion, hence the beams 18, 18′ are also spatially spread with regard to their different angular dispersion, whereby the angular dispersion of the focused beams 18, 18′ varies with the distance r, r′ from the optical axis Z. Thus the first lens group 22 effectively serves to spatially separate the beams 18, 18′ of different angular dispersion.
As mentioned before, the first surface 26a of the compensator element 26 lies substantially in the focal plane FP of the first lens group 22, hence the beams 18, 18′ are practically focused onto the first surface 26a of the compensator element 26. It is clear that the deviation between the actual point of focus P, P′ and the first surface 26a increases with the increase of the deflection angle θdef, θdef′, however, in spite of this deviation the later field of view can be considerably ameliorated with the compensator system 20 according to the invention as will be clear from the following discussion.
Since the beams 18, 18′ of different angular dispersion (and deflection angel θdef, θdef) are practically focused onto different points P, P′ of the first surface 26a of the compensator element 26 it is possible to design the compensator element 26 such as to act locally as a prism for compensating the angular dispersion of the beam 18, 18′ focused onto a given local surface region. If the point P of focus is enlarged as illustrated in
The second lens group 24 is arranged so as to substantially parallelise the different wavelength components 18a, 18b, 18c of each deflected beam 18, 18′ exiting the compensator element 26. The compensator system composed of the first lens group 22, the compensator element 26 and the second lens group 24 is designed to have an angular magnification M, hence it transforms the deflection angles θdef, θdef′ to M*θdef, M*θdef′, thus maintaining the angular variation, of the beams 18, 18′ deflected at different acoustic frequencies f1, f2 and f1′, f2′ respectively. The second lens group 24 creates the image at the objective's 16 entrance pupil (not shown). The magnification of the system is engineered to create the same size image of the acousto-optic deflector aperture as that of the objective back aperture.
In the following part of the description a calculation method is proposed for designing the compensator system 20, in particular for calculating the two surfaces 26a, 26b of the compensator element 26.
The deflection angle θ1 in the first acousto-optic deflector 13 is governed by the Bragg equation in isotropic and the Dixon equations in anisotropic interactions.
The Bragg relation [equation (1)] expresses a linear dependence of the deflection angle's sine on the optical wavelength:
Here λ is the optical wavelength in the acousto-optic crystal, θ1 is the angle closed by the incident and diffracted optical waves, f1 is the acoustic frequency and va is the acoustic velocity in the material. The angular dispersion is the derivative of the deflection angle θ1 with respect to the optical wavelength λ:
In the anisotropic case the Dixon equations indicate that the deflection angle θ1 depends on the propagation directions of the incident optical beam (not depicted) and acoustic waves relative to the crystal's axes, expressed with the angle γ1 between the incident optical beam's wave vector and the crystallographic [100] axis and the angle αc, closed by the acoustic wave vector and the [1-10] axis:
Equation (3) is valid for acoustically rotated configuration (α0≠0) and when the interaction plane coincides with the plane determined by the [001] and the [110] (or [1-10]) crystallographic axes.
The angular dispersion in this case is also the derivative of the function of (3) with respect to the wavelength λ. The wavelength dependence of the deflection angle θ1 outside the first AOD 13 at different acoustic frequencies f1 is shown in
As mentioned above, in advanced acousto-optic deflection systems the deflection in a given direction (perpendicular to the optical axis Z of the optical system) is realized with two consecutive AODs 13, 14 to also provide lens effect with controllable focus. This arrangement can also compensate for the angular dispersion in the beams 18, 18′ deflected by both AODs 13, 14. When using two AODs 13, 14 of similar material and interaction configuration, the angular dispersion is completely cancelled in a given deflection direction. This direction is conveniently chosen to coincide with the optical axis Z for the subsequent optics (practically the objective 16) which leads the deflected beams 18, 18′ to the target. The angular dispersion is only partially cancelled in deflection directions not parallel to this axis z, moreover, the remaining dispersion is increasing when the deflection angle θdef, θdef′ departs from the axis z.
There are basically two configurations that can be used in this setup: the second AOD 14 of the pair is set for the same input and output polarization as the first AOD 13. In this case the polarization of the light must be rotated by 90° with a half wave plate between the two AODs 13, 14 in order to have the correct entrance polarization in the second AOD 14. Moreover, to fulfil the criterion of keeping a point on the same place during focusing, the direction of the acoustic wave in the second AOD 14 must be opposite to that in the first.
In the second AOD 14 the incident beam 18, 18′ is already dispersed, so the incidence angle is wavelength dependent and also depends on the acoustic frequency f1 of the first AOD 13—γ12(λ,f1). If the second AOD 14 uses the same configuration as the first AOD 13, then equation (3) can be used with the new incidence angles.
The new incidence angles can be calculated from equation (3) by adding a fix angle δ representing the orientation of the second AOD 14 with respect to the beam deflected by the first AOD's 13 crystal at a given acoustic frequency f1 taken as the reference—it is usually the centre frequency of the acoustic band.
γ12(λ,f1)=θ(λ,f1)−δ (5)
In the second configuration the second AOD 14 operates with 90° rotated incident and output polarization as the first AOD 13. Therefore the output polarization of the first AOD 13 must not be rotated (the half wave plate can be omitted). Moreover the acoustic wave direction should be the same in the second AOD 14 as in the first AOD 13, it should not be rotated. The deflection angle θ2 after the second AOD 14 can be calculated by solving equation (6).
The angle θ2 can be expressed analytically too, as one of the radicals of the fourth grade equation, but it is complicated. Hence we propose to solve the equation numerically:
Otherwise the operation of the two setups using different incident polarizations and acousto-optic cell rotations are comparable, and can be described using almost the same physical picture.
If the optical system is a microscope the deflection angle θ2 is transformed into a focal point position on the target by an optical chain. The last element of this chain is the microscope objective 16. Conveniently, the position coordinates are defined as the x, y and z distances from an origin. This origin is the point where the objective's 16 optical axis Z intersects the principal focal plane. The deflected beam direction that coincides with this optical axis Z needs to be selected.
The second AOD 14 also has a central acoustic frequency, usually the center of the working acoustic band. In both setup variations particular acoustic frequencies f1 and f2 can be selected in the two consecutive AODs 13, 14, such that the beam 18, 18′ deflected twice does not contain angular dispersion after the second AOD 14, because the second AOD 14 exactly cancels out the dispersion introduced by the first AOD 13. This occurs when the frequencies f1 and f2 are nearly equal
The beam without angular dispersion is conveniently set to coincide with the optical axis Z of the optical system subsequent to the second AOD 14.
The angular dispersion in the beams 18, 18′ diffracted at other acoustic frequencies f1 and f2 in the first and second AOD 13, 14 respectively, is not zero, and is depending on the frequency difference:
Formally the optical axis z can be found where the right side of equation (7) is equal to zero. With δ=0 and identical AODs 13, 14 this occurs at f1=f2. With increasing frequency difference f1−f2, both the angle θ2, and the angular dispersion increases, following the prediction of equation (6).
The visible deflection angles θdef, θdef′ outside the second AOD 14 can be calculated using its wavelength dependent refractive index, which is slightly different in the two cases because of the different polarization states.
θdef(λ,f1,f2)=a sin [n(λ)·(θ2(λ,f1,f2)−θ2(λc,fc1,fc2))] (8)
Here λc, fc1, fc2 are the central optical wavelength and acoustic frequencies, respectively, that determine the optical axis Z after the AODs 13, 14.
By generalization of the angular dispersion compensation method based on a prism 10 it is possible to design the optimized compensator system 20 that compensates for the above described spatially variable angular dispersion. This compensator system 20 consists of spherical and aspheric lenses and fulfils the following criteria:
As discussed above the compensator system 20 comprises three main parts: the first lens group 22 focusing the deflected beams 18, 18′ into the focal plane FP of the first lens group 22, the compensator element 26 compensating for the angular dispersion of the deflected beams 18, 18′ and the third lens group 24 parallelizing the spectral components 18a, 18b, 18c and creating the image at the objective's 16 entrance pupil.
The compensator element 26 can be fabricated as a glass piece with a first surface 26a of radius R1 and a second surface 26b of radius R2.
As explained before, the first lens group 22 focuses the deflected beams 18, 18′ into its focal plane FP, where the deflected beams 18, 18′ are spatially separated, and the wavelength components 18a, 18b, 18c within each deflected beam 18 are separated too, but with at least about one order of magnitude less amount, typically by one-two orders of magnitude less amount. The first lens group 22 of focal length foc1 transforms the deflection angle θdef, θdef′ into spatial distance r, r′ measured from the optical axis Z in the focal plane FP:
r(λ,f1,f2)=θdef(λ,f1,f2)·foc1 (9)
The angular dispersion into spatial spread of the spectral components 18a, 18b, 18c can be described as follows:
Δr(λ1,λ2,f1,f2)=(θdef(λ1,f1,f2)−θdef(λ2,f1,f2))·foc1 (10)
The angular separation of the spectral components 18a, 18b, 18c increases with increasing distance r from the optical axis Z. The main angle, which can be associated with the frequency components is r(λ)/foc1
To compensate effectively for the angular dispersion a prism 10 is the most forehand solution. The amount of angular dispersion, which a prism 10 can compensate for, can be matched to a given dispersion value by properly selecting the incidence angle α1 and the prism opening angle αP. The output angle after the prism is given by:
The wavelength dependent incidence angle α1 can be determined from the tilt angle β of the prism 10 (i.e. the angle between the optical axis Z and the angle of incidence) and the wavelength dependent angle θdef of propagation:
α1(λ)=θdef(λ,f1,f2)−β (12)
and the total deflection angle of the prism 10:
The zero angular dispersion requirement after the prism can be expressed as:
To compensate for an angular dispersion that increases with the distance r from the optical axis Z both the tilt angle β and the prism opening angle αP should be varied: the two functions β(r) and αP(r) can be computed.
From these relations the nominal radiuses R1 and R2 of the first surface 26a and of the second surface 26b that together work like prisms with varying and matching tilt angles (or incidence angles) and opening angles, can be calculated.
In the first approach, where these surfaces 26a, 26b are assumed to be spherical surfaces with perfect symmetry around the above defined optical axis, the radius R1 of the first surface 26a can be expressed as:
R
1
=r/sin [β(r)] (15)
and the radius R2 of the second surface 26b can be expressed as:
The optimal surfaces 26a, 26b are however not spherical surfaces, but they can be expressed as aspheric and conic surfaces with the main radii given by equations (15) and (16):
The conic and aspheric parameters (indicated in
The second lens group 24 of the setup typically comprises a single lens which accounts for imaging the deflected beams 18, 18′ onto the objective's 16 back aperture and for filling the objective aperture at each deflection angle M*θdef, M*θdef′. Its focal length and position are also optimized. For example an Olympus scan lens of 210 mm focal length may be used at this place, when the compensator system 20 is used in combination with a LumPlan 20x Olympus objective. As is clear from the above equations the parameters of the custom surfaces 26a, 26b of the compensator element 26 can be optimized for a variety of, commercially available lenses preselected for the second lens group 24 of the angular compensator system 20. Optimization and analysis must be performed for each combination to give the highest field of view within a given angular resolution range.
When compared with a conventional setup which would use a simple telescopic lens system instead of the inventive compensator system 20, we have found that using the inventive compensator system 20 it was possible to obtain 700 μm lateral field of view whereas using a conventional telescopic lens system only 420 μm lateral field of view was obtained with the same focused spot size and dispersion at the edges of this area.
It is clear that the same equations can be used to calculate the surface 26a, 26b of the compensator element 26 in a direction perpendicular to x, say y, if a second pair of AODs are used to deflect the beams 18, 18′ in a lateral direction (y) perpendicular to the deflection direction (x) occasioned by the first pair of AODs 13, 14.
An arrangement similar to that depicted in
The above-described embodiments are intended only as illustrating examples and are not to be considered as limiting the invention. Various modifications will be apparent to a person skilled in the art without departing from the scope of protection determined by the attached claims.
Number | Date | Country | Kind |
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P1100727 | Dec 2011 | HU | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/HU2012/000003 | 1/5/2012 | WO | 00 | 10/13/2014 |