Patent Application
20170068080
It is noted that citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
The invention relates to a method for light sheet microscopy and arrangements for carrying out this method.
Light sheet microscopy, which is also referred to as light sheet fluorescence microscopy (LSFM) or single plane illumination microscopy (SPIM), is already known per se as a relatively recent development of fluorescence-microscopic methods and arrangements.
Compared to conventional fluorescence microscopy with the property of optical sectioning, like in confocal laser scanning microscopy or two-photon microscopy, the illumination of a thin layer in the sample, corresponding to the thickness of a light sheet, enables the measurement and display of an optical section during light sheet microscopy, while at the same time suppressing the image background. Here, the negative effects as a result of bleaching or light-induced stress in biological samples are reduced during the examination of same. Since the detection can be undertaken in the wide field, it is moreover possible also to quickly detect relatively large sample regions.
The present invention relates to the subject area of light sheet microscopy, in which the light sheet defines an excitation plane which includes an angle δ≠90° with the optical axis of the detection objective, wherein the sample is scanned by this light sheet.
In this respect, DE 10 2005 027 077 A1 describes a microscope comprising an illumination device, in which a light strip is generated, said light strip illuminating the sample in a plane which is inclined in relation to the detection direction by an angle which differs from 90°. The detection direction is defined by an objective, through which the detection light reaches a surface detector.
The illuminated plane is rotatable about an axis of rotation. In different designs, either the surface detector is movable in synchronous fashion with the rotation of the illumination plane or the spatial situation of the intermediate image plane or the image plane is influenced in such a way that it is kept constant independently of the orientation of the illuminated plane.
If, in the first case, the illuminated plane is rotated about e.g. an axis parallel to the optical axis, the surface detector must likewise be rotated about an axis parallel to the optical axis thereof. If the angle between the illuminated plane and the detection direction is modified, the angle between the detector surface and the propagation direction of the incident detection light must be updated in accordance with this change of angle.
In the second case, the spatial situation of the intermediate image plane or of the image plane is influenced by e.g. synchronizing the rotation of prisms with the rotation of the illumination plane in order always to image the illuminated sample region on the detector surface.
A disadvantage in this case is that it is not possible to meet the Scheimpflug condition, according to which the image plane, objective plane and sharpness plane must intersect along a common straight line during optical imaging, in particular for standard imaging conditions, since the angle between the detector surface and the optical axis becomes very large.
DE 10 2011 000 835 A1 describes a scanning microscope with a reflected-light configuration. Here, an objective generates an elongate illumination focus in an object to be imaged and a scanning device moves the illumination focus over a target region to be illuminated in the object by changing the direction of incidence, with which the illumination light is incident into the entrance pupil of the objective. The location of the incidence of the illumination light into the entrance pupil is decentrally offset in relation to the pupil center. As a result, the illumination focus generates a light sheet which, due to the decentral entrance of the illumination light into the entrance pupil of the objective, is oblique in relation to the optical axis.
The fluorescence radiation originating from the target region illuminated by the light sheet is detected by the objective and guided into a detection optical unit. The detection optical unit comprises a first detection objective, which is disposed downstream of a tube lens system. The tube lens system consists of two tube lenses, of which a first is associated with the objective and the other one is associated with the first detection objective. In cooperation with the objective, the first tube lens generates a first intermediate image. Here, the optical axis of the first detection objective is perpendicular to the optical axis of the objective. Since the target region illuminated by the light sheet is arranged at an angle to the optical axis of the objective, the first intermediate image also lies at an angle to the optical axis of the first detection objective. In cooperation with the second tube lens, the first detection objective images the first intermediate image into a second intermediate image, which is likewise at an angle to the optical axis of the first detection objective.
The detection optical unit furthermore comprises a second detection objective, which images the second intermediate image byway of a further tube lens onto the detection surface of a light detector. Here, the optical axis of the second detection objective is both perpendicular to the second intermediate image and perpendicular to the detection surface. A straightened image of the scanned target region is generated on the detection surface as a result of the cooperation between the two detection objectives.
A disadvantage here is the comprehensive technical, and hence cost-intensive, outlay required for the imaging since a second light microscope structure is required for observing the intermediate image generated by a microscopic relay system.
Furthermore, the detection efficiency is low since the collection NA is limited due to the required obliqueness of various optical units, in particular of the second detection objective with a high NA, and the transmission of the overall system is low due to the multiplicity of required optical units. Also, the flexibility of the use of the system is greatly restricted since the objective properties are dependent on the overall structure and the objective is therefore not interchangeable in respect of different practical applications. Moreover, the observed sample plane is not orientable in an arbitrary manner in relation to the optical axis since the excitation geometry is predetermined by the arrangement of the detection objectives.
What is more, the described technical solutions require mechanical movements and are therefore susceptible to wear-and-tear and faults in both of the cited publications.
Proceeding therefrom, the object of the invention lies in developing a method for light sheet microscopy, which no longer has the illustrated disadvantages. Another object underlying the invention is that of specifying at least one arrangement suitable for carrying out the new method.
For a method of the type set forth at the outset, this object is achieved by the following method steps:
The sample volume to be imaged is either the entire volume of a sample to be examined or a spatial part to be examined of the overall volume of the sample.
Within the meaning of the invention, the nominal depth of field defined by the design of the respectively employed objective is defined to be the depth of field Sobj. Byway of example, the following applies for a high-aperture aplanatic objective:
where α is the aperture angle of the objective, n is the refractive index of an immersion liquid and NA is a numerical aperture in accordance with α=arcsin(NA/n). A depth of field Sobj of Δz=Sobj=0.83 μm is obtained for a water immersion objective with an NA of 1.2. In the case of standard objectives, as can be used for the method according to the invention, the depth of field Sobj lies in the range from 0.5 μm to 170 μm.
Depth of field Seff is understood to mean an effective depth of field obtained over the course of the detection beam path, which is greater than the depth of field Sobj. According to the invention Seff≧T applies.
The direction of the optical axis of the objective corresponds to the z-coordinate direction of a Cartesian coordinate system. The spatial alignment of the light sheet is defined within the meaning of the invention by the propagation direction of the illumination light formed to be the light sheet. The light sheet is the region of the illumination beam path provided for illuminating the sample volume, in which region the illumination light does not exceed a spatial extent of e.g. 10 μm measured in the detection direction or in the direction of the optical axis of the objective and in which it is therefore suitable for detecting or measuring a sample volume according to the principle of light sheet microscopy.
The light sheet can be generated in different procedures; it can either be projected into the sample volume in the form of a widened collimated laser beam, which e.g. is focused in only one direction by means of a cylindrical lens, or be generated with a punctiform laser focus moving in a scanning fashion. It is also conceivable to generate the light sheet by means of light forming elements such as e.g. SLMs (spatial light modulators).
Depending on the sample volume to be scanned, it is possible to vary
Advantageously, the sample volume is scanned using the reflected-light method. The axially non-discriminating detection, provided according to the invention, advantageously permits, in combination with the light sheet illumination with a known tilt, sample regions to be illuminated and imaged, which sample regions not only lie in the focal plane of the detection objective, but also lie, as seen in the axial direction, at different depths in the sample upstream or downstream of the focal plane.
In alternative embodiments of the method according to the invention, two- or three-dimensional images of the scanned sample volume are generated in a manner dependent on the selection of the optical means for imaging the detection light on the reception plane of the area sensor and the algorithm for the signal processing. Embodiments in which two- or three-dimensional images of the scanned sample volume are generated according to the extended depth of field procedure or light-field technology, for example with the inclusion of diffractive optical elements (DOE) into the detection beam path, are advantageous.
Illuminating the sample through the objective, in transmitted light or else laterally in accordance with the selective plane illumination microscopy (SPIM) method, lies within the scope of the method according to the invention.
Implementing the stimulated emission depletion (STED) principle in the method according to the invention is also conceivable. Here, the thickness of the light sheet contributing to the imaging is reduced by virtue of a double light sheet being superimposed onto the excitation light sheet in such a way that the zero position of the double light sheet substantially lies at the position of the excitation light sheet. The emission stimulated at the wavelength of the double light sheet is not taken into account when evaluating the signal, as a result of which the longitudinal resolution is increased. Furthermore, all methods known per se, in which the resolution is increased by linear or nonlinear means, are combinable by the method according to the invention. Methods from localization microscopy, such as photo activated localization microscopy PALM, stochastic optical reconstruction microscopy (STORM) or super-resolution optical fluctuation imaging (SOFI) for obtaining images in 2D and 3D, are particularly suitable in this respect. Moreover, multi-photon methods for generating the signal light can be used, such as e.g. 2- or 3-photon microscopy, SHG, THG microscopy or CARS excitation.
The object underlying the invention is furthermore achieved by an arrangement for carrying out the method according to the invention described above. This arrangement comprises:
Byway of example, the objective has a nominal depth of field Sobj in the range from 0.5 μm to 170 μm, and the optical device disposed downstream of the objective is embodied to increase the depth of field Sobj by a factor a≧5 to the effective depth of field Seff, where Seff≧T always applies.
The arrangement according to the invention is preferably equipped with optical means
A common objective is preferably available for illumination and detection, and provision is made for shining the illumination light into the entrance pupil of the objective at a location offset parallel to the optical axis thereof. In this case, a device is present, which is embodied to
When operating this embodiment of the arrangement according to the invention, the angle δ or the inclined position of the light sheet is predetermined with the decentral shining of illumination light into the entrance pupil of the illumination and detection objective. The displacement of the entrance location of the illumination light along the circumference of the partial circle in the pupil plane brings about a rotation of the light sheet about the optical axis of the objective. Changing the direction of shining in illumination light or the angle of shining in illumination light relative to the normal of the pupil, which extends parallel to the optical axis within the meaning of the invention, leads to a displacement of the light sheet in the case of an elliptic Gaussian light sheet and influences the lateral extent of a light sheet which arises by scanning an elongate point spread function in the illumination.
By way of example, the optical device for increasing the depth of field Sobj comprises an axicon, a phase mask or a microlens array. Here, the axicon or the phase mask are used in conjunction with the generation of two- or three-dimensional images according to the extended depth of field procedure, while the microlens array is used in conjunction with the generation of two- or three-dimensional images according to the light-field technology.
By way of example, cubic, logarithmic, exponential, fractional power phase masks and rational phase masks can be used within the meaning of the invention as phase masks, as can all further phase masks which can be used for lengthening the detection PSF. Furthermore, it is also possible to use pupil masks, which influence the amplitude of the light to be detected. Thus, pupil masks which have increased absorption in certain regions and therefore bring about apodization or lead to stopping down of the objective NA come into question. Here, it is also possible to advantageously use a mixture of absorbing and phase-influencing regions of a mask.
The reception area of the area sensor is rectangular, aligned at least substantially at right angles to the detection beam path and fixedly arranged in the detection beam path in this position, to be precise independently of a change in position or inclination, or rotation, of the light sheet. In contrast to the prior art, there is no need to update the area sensor or the reception area following a movement of the light sheet. As a result, mechanically moved components are dispensed with and the arrangement is less susceptible to errors. Byway of example, a CCD sensor can be provided as area sensor.
Furthermore, there is no need for a microscopic relay system for generating an intermediate image and consequently there is also no need for a second light microscope structure for observing such an intermediate image such that, compared to the prior art, the solution according to the invention requires substantially less technical outlay and it is less cost intensive as a result. Moreover, the detection efficiency is higher since the NA is not restricted by the oblique position of a plurality of optical units, in particular of high-aperture objectives. Due to the smaller number of required optical units, the transmission of the overall system is higher than in the comparable prior art.
A substantial advantage of the invention furthermore consists of spatial images of samples being obtained with objectives which have a comparatively small nominal depth of field Sobj. As a result of this, it is possible to use cost-effective standard objectives or standard imaging conditions. The standard objectives can be interchanged with one another in respect of various practical applications since the objective properties are independent of the overall structure. As a result, the arrangement according to the invention can be used in a substantially more flexible manner.
A PC and means for electronic storage and visually perceivable reproduction of two- or three-dimensional images serve as hardware. The software corresponds to a predetermined algorithm for signal evaluation and for generating the two- or three-dimensional images as a result of the signal evaluation.
In a complementary embodiment, the arrangement according to the invention optionally has a device for changing the location for shining illumination light into the entrance pupil of the illumination and detection objective such that the shining-in can be optionally undertaken decentrally or centrally in relation to the optical axis thereof. As a result, it is possible, when required, to switch from scanning the selected sample volume according to the light sheet microscopy method to scanning according to the conventional laser scanning method. Thus, when the illumination light is shone centrally into the entrance pupil of the objective, the optical image signals are obtained in accordance with confocal laser scanning microscopy.
It is to be understood that the FIGS. and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein.
The present invention will now be described in detail on the basis of exemplary embodiments.
The exemplary embodiment shown in
A laser light source 2, a beam widening optical unit 3 and a scanning device 4 generate an illumination beam path 5, which is deflected toward the objective 1 by means of the splitter surface 6 of a beam splitter 7 and which is shone with lateral offset from the optical axis 8 of the objective 1 into the entrance pupil 24 of the objective 1 (cf.
The illumination light being shone in a decentral manner in relation to the optical axis 8 causes the propagation direction of the light sheet 9 to include an angle 8 with the optical axis 8 which differs from 90°. Here, the size of the angle δ is dependent on the distance between the entrance location 23 of the illumination light into the entrance pupil 24 and the optical axis 8.
A consequence of the light sheet 9 being at an angle relative to the optical axis 8 is that the light sheet 9 also illuminates regions of the sample volume 10 which lie at different depths in front of, and behind, the focal plane 11 in the axial direction, i.e. in the z-direction.
With the scanning movement in the x- and/or y-direction, the scanning device 4 prescribes the current position and alignment of the light sheet 9 within the sample volume 10. Said scanning device 4 is also embodied to rotate the light sheet 9 about an angle about the optical axis 8 or about an axis parallel to the optical axis 8 (cf.
Here, the scanning device 4 is embodied in an exemplary manner to
However, the scope of the invention explicitly also includes each one of these functions being exercised by separate devices embodied therefor.
Reflected, scattered or excited light coming from the illuminated sample regions enters the objective 1 as detection light. The objective 1 forms a detection beam path 12, to which the splitter surface 6 is transparent, as depicted in
In the detection beam path, the axicon 19 is positioned between the two lens groups 15, 16a. The axicon 19 transforms the detection light into a Bessel-shaped light beam with a very long focus and therefore, according to the invention, brings about an increase in the nominal depth of field Sobj of the objective 1 by a multiple to a depth of field Seff, as a result of which the whole sample volume 10 scanned by the light sheet 9 is imaged sharply on the reception plane 18.
A depth-resolved image of each illuminated thin section of the sample volume 10 is established by calculation from the intensity distribution in the detection beam path 12 registered by the area sensor 17 and said image is depicted in a manner that is visually perceptible by an observer. Such restoration methods are known per se. In the case of the so-called wavefront coding by means of phase masks, the data record is initially decoded numerically. This is carried out by a deconvolution with a known point spread function (PSF) determined by the mask, the objective and the microscope system. A projection of the sample volume along the optical axis is obtained after this first step. In a further step, the a priori information about the respectively current illumination geometry is included in the sample restoration in such a way that a unique spatial assignment of each detected event to the scanned sample volume is carried out.
As can be seen from
In a specific embodiment, the distance of the tube optical unit 13 from the position of the intermediate image 14 is f13=200 mm. Byway of example, the lens group 15 is at a distance of f15=200 mm from the position of the intermediate image 14 and generates an infinite beam path. The axicon 19 is at a distance of 41 mm from the lens group 15. The cone angle of the axicon 19 is ≦2; for example, it is 0.5 in this case. The distance between the axicon 19 and the reception plane 18 varies depending on the embodiment of the arrangement according to the invention; it can be up to 50 cm and is determined by the length of the Bessel beam which is generated by the axicon 19. The reception plane 18 is positioned in the region of the Bessel beam.
The lens groups 15, 16a can both consist of a plurality of lenses and be embodied as individual lenses. In principle, there is no need for a lens group 16a; the scope of the invention also includes an embodiment of the arrangement according to the invention without a lens or lens group 16a. However, a lens group 16a can advantageously be used to set optical parameters such as resolution and lateral magnification.
The difference to the exemplary embodiment according to
The phase mask 20 transforms the detection light into an Airy-shaped light beam and likewise, according to the invention, brings about an increase in the nominal depth of field Sobj of the objective 1 by a multiple to a depth of field Seff, as a result of which the whole sample volume 10 scanned by the light sheet 9 is also imaged sharply on the reception plane 18 in this case.
By applying the extended depth of field (EDoF) technology, a depth-resolved image of each illuminated thin section of the sample volume 10 is established in turn by calculation using the computing unit 26 from the intensity distribution in the detection light beam registered by the area sensor 17 and said depth-resolved image is depicted in a manner visually perceivable by an observer by means of the memory and reproduction unit 27.
The exemplary embodiment according to
The microlens array 21 increases the depth of field Sobj of the objective 1 by a multiple to a depth of field Seff by virtue of the sample volume 10 scanned by the light sheet 9 being imaged as a whole on the reception plane 18. Below, a three-dimensional reconstruction of this volume is undertaken by virtue of a depth-resolved image of the illuminated thin section being established by calculation by means of the light-field technology from the registered intensity distribution of the detection light beam and said depth-resolved image being depicted in a visually perceivable manner.
In the case mentioned here, obtaining and using the a priori information about the respectively current illumination geometry, as described further above on the basis of
Here too, two- or three-dimensional images of the scanned sample volume 10 are obtained by means of a computing unit 26.
In a plan view of the entrance pupil 24 of an objective,
By contrast,
In both cases, this pupil illumination shows, in an exemplary manner, the generation of an inclined elliptic Gaussian light sheet.
As can be seen from
A variation of the distance of the entrance location 23 from the optical axis 8 brings about a change in the angle δ between the light sheet 9 and the optical axis 8. An adaptation of the position of the light sheet 9 to the sample volume 10 which extends in the x- and y-direction is achieved by changing the entrance angle of the illumination light into the pupil plane of the objective 1. A displacement of the focal plane 11 or of the region of the depths of the field Sobj, Seff in the z-direction relative to the sample can be obtained by displacing the sample stage 22 in the z-direction.
In
Moreover, it is possible to identify from
This condition is also satisfied by the case L=T and therefore likewise lies within the scope of the invention.
Within the region L, the spatial extent of the light sheet 9 in the direction of the optical axis 8 does not exceed the value of e.g. 10 μm and it is therefore suitable for detecting or measuring a sample volume 10 according to the principle of light sheet microscopy.
In conjunction with the change in the angle and the displacement of the light sheet 9 or the sample in the x- or y-direction, the depth of field Seff≧T enables the detection and imaging of the entire sample volume 10.
While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2014 102 215.7 | Feb 2014 | DE | national |
The present application claims priority from International Patent Application No. PCT/EP2015/053449 filed on Feb. 19, 2015, which claims priority from German Patent Application No. 10 2014 102 215.7 filed on Feb. 20, 2014, the disclosures of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/053449 | 2/19/2015 | WO | 00 |
