阅读说明：本技术 用于经由多视图成像的三维荧光偏振的系统和方法 (System and method for three-dimensional fluorescence polarization via multi-view imaging ) 是由 H.施罗夫 A.库马尔 S.B.梅塔 P.J.拉里维尔 R.奥尔登伯格 Y.吴 T.钱德 于 2018-05-31 设计创作，主要内容包括：公开了用于三维荧光偏振激发的系统和方法,该系统和方法生成三维或更多维度的荧光分子的位置和取向的图。(Systems and methods for three-dimensional fluorescence polarization excitation are disclosed that generate maps of the position and orientation of fluorescent molecules in three or more dimensions.)

1. A fluorescence microscope system, comprising:

a light source for emitting a light beam;

first polarizing optics for converting the light beam into a polarized light beam;

a beam splitter for splitting the polarized beam into a first polarized beam and a second polarized beam;

a first objective lens oriented along a first axis such that the first polarized light beam illuminates the sample along a first angle to produce a first fluorescent emission; and

a second objective lens oriented along a second axis such that the second polarized beam illuminates the sample at a second angle that is non-parallel with respect to the first angle to produce a second fluorescent emission,

wherein the first objective lens is oriented to detect the second fluorescent emission and the second objective lens is oriented to detect the first fluorescent emission.

2. The fluorescence microscope system of claim 1, further comprising:

a first detector in communication with the first objective lens for receiving an image from the second fluorescent emission; and

a second detector in communication with the second objective lens for receiving an image from the first fluorescent emission.

3. The fluorescence microscope system of claim 1, further comprising:

a third objective lens oriented along a third axis in a non-parallel relationship with respect to the first axis of the first objective lens and the second axis of the second objective lens, wherein the third detector detects a third fluorescence emission from the sample.

4. The fluorescence microscope system of claim 2, wherein the third objective lens is in communication with a third detector for receiving images from the third fluorescence emission.

5. The fluorescence microscope system of claim 2, wherein the first detector and the second detector are each in communication with a processor for receiving images of the second fluorescence emissions from the first objective lens and the first fluorescence emissions from the second objective lens.

6. The fluorescence microscope system of claim 4, wherein the third detector is in communication with a processor for receiving an image of a third fluorescence emission from the third objective lens.

7. The fluorescence microscope system of claim 1, further comprising:

second polarizing optics associated with the first objective lens for further altering the polarization of the first polarized beam prior to illuminating the sample; and

second polarizing optics associated with the second objective for further altering the polarization of the second polarized beam prior to illuminating the sample.

8. The fluorescence microscope system of claim 1, wherein the first polarizing optics comprise a wave plate, a polarizer, and a liquid crystal.

9. The fluorescence microscope system of claim 1, further comprising:

a first dichroic mirror in communication with the beam splitter for redirecting the first polarized light beam to the first objective lens; and

a second dichroic mirror in communication with the beam splitter for redirecting the second polarized light beam to the second objective lens.

10. The fluorescence microscope system of claim 1, wherein the first objective lens and the second objective lens illuminate the sample with the first polarized light beam and the second objective lens, respectively, in an alternating sequence.

11. The fluorescence microscope system of claim 1, wherein the first objective lens and the second objective lens detect the first fluorescence emission and the second fluorescence emission, respectively, in an alternating sequence.

12. The fluorescence microscope system of claim 1, wherein the first fluorescent emissions detected by the second objective lens are oriented along a first plane in a non-parallel relationship relative to a second axis of the second objective lens, and wherein the second fluorescent emissions detected by the first objective lens are oriented along a second plane in a non-parallel relationship relative to the first axis of the first objective lens.

13. The fluorescence microscope system of claim 5, wherein the processor generates an orientation distribution of excited dipoles associated with one or more three-dimensional structures based on the first and second fluorescent emissions detected by the first and second objective lenses, respectively.

14. A fluorescence microscope system, comprising:

a laser source for emitting a laser beam;

first polarizing optics for converting the laser beam into a polarized beam;

a beam splitter for splitting the polarized beam into a first polarized beam and a second polarized beam;

a first objective lens oriented along a first axis such that the first polarized light beam illuminates the sample along a first angle to produce a first fluorescent emission from the first plurality of excited dipoles; and

a second objective lens oriented along a second axis such that the second polarized beam illuminates the sample at a second angle that is non-parallel with respect to the first angle to produce a second fluorescent emission from a second plurality of excited dipoles,

wherein the first objective lens is oriented to detect the first plurality of excited dipoles oriented at an angle perpendicular to the first axis and the second objective lens is oriented to detect the second plurality of excited dipoles oriented at an angle perpendicular to the second axis.

15. The fluorescence microscope system of claim 14, wherein the first objective lens is oriented to not detect the first plurality of excited dipoles oriented at an angle parallel to the first axis, and the second objective lens is oriented to not detect the second plurality of excited dipoles oriented at an angle parallel to the second axis.

16. A method for determining the position and orientation of an excited dipole generated by fluorescent emissions from a sample, comprising:

generating a laser beam;

converting the laser beam into a polarized beam;

separating the polarized beam into a first polarized beam and a second polarized beam;

illuminating the sample through a first objective lens oriented along a first angle to produce a first fluorescent emission; and

generating a second fluorescent emission by illuminating the sample with a second objective lens oriented at a second angle that is non-parallel with respect to the first angle,

detecting a first plurality of excited dipoles in the second fluorescent emission through the second objective lens oriented along a plane in non-parallel relationship with the first axis, and detecting a second plurality of excited dipoles in the first fluorescent emission through the first objective lens oriented along a second plane in non-parallel relationship with the second axis.

17. The method of claim 16, wherein the polarization state of the laser beam is arbitrarily changed.

18. The method of claim 17, further comprising:

for each polarization state, a series of images of the first and second fluorescent emissions are collected by one or more detectors.

19. The method of claim 17, further comprising:

processing a series of images of the collected first and second fluorescent emissions to determine a location and orientation of each of the first and second pluralities of excited dipoles.

20. The method of claim 17, wherein a reconstruction algorithm is applied in processing the collected series of images of the first and second fluorescence emissions.

Technical Field

The present disclosure relates to systems and methods for three-dimensional fluorescence polarization excitation, and in particular to fluorescence microscopy that generates maps of the position and orientation of fluorescent molecules in three or more dimensions.

Background

Most fluorophores, including fluorescent proteins, absorb and emit light as dipoles (dipoles). This creates the opportunity to reveal not only the position of the fluorophore and its associated molecular assembly, but also their orientation. Polarized light microscopes that are equipped to excite and/or detect polarized fluorescence have taken advantage of this opportunity. The orientation and dynamics of the molecular assembly determine the directionality of cellular function or disease. For example, directed cell migration during wound healing or metastasis relies on the flow of a patterned actin network that generates a net force towards the direction of migration.

Molecular orientation is revealed by using polarized light for dipole excitation or polarization analysis of dipole emission, or both. However, current microscopes illuminate and image a sample from a single view direction. Accordingly, the polarization of the excitation light and the emitted fluorescence light is mainly defined in the plane perpendicular to the illumination/view direction, as shown in fig. 2A and 2B. Dipoles parallel to the direction of the illumination/view cannot be excited nor effectively detected, thus making it difficult or even impossible to determine the complete orientation distribution of the fluorophore in combination with the three-dimensional structure.

It is based upon these observations that the various aspects of the present disclosure have been conceived and developed.

Drawings

FIG. 1 is a simplified diagram illustrating one embodiment of a fluorescence microscope system according to one aspect of the present disclosure;

fig. 2A shows a schematic diagram of a conventional epi-fluorescence microscope according to an aspect of the present disclosure, and fig. 2B shows a schematic diagram of a conventional dual view selective plane illumination microscope system according to an aspect of the present disclosure;

fig. 3A and 3B illustrate measurement schemes for obtaining three-dimensional dipole orientation using epi-detection of fluorescence intensity produced by polarization-resolved excitation, according to an aspect of the present disclosure;

FIGS. 4A and 4B illustrate another measurement scheme for obtaining three-dimensional dipole orientation using orthogonal detection of fluorescence intensity produced by polarization-resolved excitation, according to an aspect of the present disclosure; and

fig. 5A is an image showing maximum intensity projection images corresponding to an imaging volume obtained with different polarizations of illumination light according to an aspect of the present disclosure, and fig. 5B shows a resulting reconstruction according to an aspect of the present disclosure.

Corresponding reference characters indicate corresponding elements throughout the several views of the drawings. The headings used in the figures do not limit the scope of the claims.

Detailed Description

As described herein, systems and methods for extending fluorescence polarization imaging so that the dipole moment of a fluorescent dye emitted by a sample can be excited regardless of the three-dimensional orientation of the dipoles. In one aspect, the dipoles are excited from multiple directions, ensuring that excitation of the sample occurs along multiple orientations even if the dipoles are disadvantageously oriented along the axial (propagation) axis of the detection objective. In one embodiment, a dual-view inverted selective-plane illumination microscope (bispim) is used to illuminate a sample and detect the resulting polarized fluorescence emissions of the sample emitted from two different directions in a non-parallel relationship to each other. In one embodiment, polarization-resolved excitation of the sample and epi-detection of the emitted polarized fluorescence capture the three-dimensional orientation of the excited dipoles along the focal plane of the same excitation/detection objective used to excite the sample and detect the emitted fluorescence. In one embodiment, polarization-resolved excitation of the sample and non-parallel detection of fluorescence emitted by the sample in an alternating excitation sequence captures a substantial portion of the projection of the three-dimensional orientation of the excited dipoles onto the axial or meridional plane of the respective detection target. In one embodiment, the system includes a processor in operative communication with one or more detectors for capturing data regarding the position and three-dimensional orientation of each excited dipole detected in polarized fluorescence emissions emitted by the sample detected by the one or more objective lenses. The processor is operable to calculate the three-dimensional orientation and position of the excited dipole in each voxel detected in the fluorescent emissions emitted by the illuminated sample. In some embodiments, the system captures multiple images with different induced polarizations so that the processor can determine the position and three-dimensional orientation of each excited dipole detected by the one or more detectors. Referring to the drawings, an embodiment of a system for determining the three-dimensional dipole orientation and position for each voxel of an illuminated sample is shown in fig. 1-4 and is generally indicated at 100.

Referring to FIG. 1, one embodiment of a fluorescence microscope system is shown generally at 100. In one aspect, the fluorescence microscope system 100 is operable to determine the position and orientation of excited dipoles in fluorescent emissions emitted from the illuminated sample 114.

In some embodiments, the fluorescence microscope system 100 includes a light source 102, the light source 102 for emitting the light beam 101 polarized by first polarizing optics 129, the first polarizing optics 129 for polarizing the light beam 101 into polarized light 103. In some embodiments, the first polarizing optics 129 may include a wave plate, a polarizer, and/or one or more liquid crystals to polarize the light beam 101. In some embodiments, the light source 102 may be a laser for emitting a laser beam; however, in other embodiments, the light source 102 may be other sources of light, such as a lamp that emits a beam of light that is capable of polarization.

In some embodiments, polarized light 103 may be split by beam splitter 106 into separate polarized light 103A and 103B. In some embodiments, a first dichroic mirror (dichroic mirror)134 redirects the separated polarized light 103A through a first objective lens 108, which first objective lens 108 may include second polarizing optics 130 for further polarizing the separated polarized light 103A into separated polarized light 103C for illuminating the sample 114 along the first axis 300. As shown, dichroic mirror 136 redirects the separated polarized light 103B through second objective lens 110, which second objective lens 110 may include third polarizing optics 132 for further polarizing the separated polarized light 103B into separated polarized light 103D to illuminate sample 114 along a second axis 302 in an orthogonal relationship with respect to first axis 300.

When the sample 114 is illuminated, those polarized fluorescent emissions 105 of the sample 114 that are emitted substantially along a plane orthogonal to the first axis 300 are detected by the first objective lens 108, while those polarized fluorescent emissions 105 of the sample 114 that are emitted along a plane substantially parallel to the first axis 300 are not detected by the first objective lens 108. In addition, those polarized fluorescent emissions 105 emitted by the sample 114 substantially along a plane orthogonal to the second axis 302 are detected by the second objective lens 110, while those polarized fluorescent emissions 105 emitted by the sample 114 along a plane substantially parallel to the second axis 302 are not detected by the second objective lens 110. In this arrangement, the orthogonal relationship between the first objective lens 108 and the second objective lens 110 allows the fluorescence microscope system 100 to detect excited dipoles regardless of their orientation axes. In other embodiments, the first objective lens 108 and the second objective lens 110 may be oriented at non-parallel angles with respect to each other.

In one arrangement, polarized fluorescent emissions 105 detected by first objective lens 108 may be redirected by first dichroic mirror 134 through first tube lens 122 for detection by first detector 116. In a further arrangement, fluorescent emissions 105 detected by second objective lens 110 may be redirected by second dichroic mirror 136 through second barrel lens 124 for detection by second detector 118.

In some embodiments, third objective 112 may be located below plane 304 of sample 114 and oriented along a third axis 306, third axis 306 forming an angle of 135 degrees with respect to first axis 300 and second axis 302, respectively. Third objective lens 112 is used to detect fluorescent emission 105 that is emitted below plane 304 of sample 114 and at an angle that is perpendicular to plane 304 of sample 114. In some embodiments, fluorescent emissions 105 detected by third objective 112 may be imaged through third tube lens 126 to be detected by third detector 120. In some embodiments, third objective 112 may be oriented at non-parallel angles with respect to first axis 300 and second axis 302, respectively.

In some embodiments, the first detector 116, the second detector 118, and the third detector 120 are in operable communication with one or more processors 128, the one or more processors 128 utilizing one or more algorithms to calculate the position and three-dimensional orientation of the excited dipoles based on images of the fluorescent emissions 105 captured from the first detector 116, the second detector 118, and the third detector 120, respectively.

In some embodiments, the first objective lens 108 and the second objective lens 110 may be Nikon 0.8NA, Nikon 1.1NA, specialty optics 0.71NA lenses, for example, although other types or kinds of objective lenses are also contemplated.

In some embodiments, the fluorescence microscope system 100 can operate in an epi-detection mode of operation (fig. 2A) or an orthogonal detection mode of operation (fig. 2B). In the epi-detection mode of operation shown in fig. 2A, a single objective lens, such as first objective lens 108, focuses polarized light rays in a direction along axis a to illuminate sample 114, and then the same objective lens detects the resulting fluorescent emission emitted by the sample. In this detection mode, the first objective lens 108 detects polarized fluorescence emissions emitted by the sample along a plane perpendicular to the axis of the first objective lens 108.

As shown in fig. 3A and 3B, in an epi-detection mode of operation of fluorescence microscope system 100, the same objective lens, e.g., first objective lens 108, illuminates sample 114 and then detects polarized fluorescence emissions of those emitting dipoles oriented along a plane 304 in a non-parallel relationship with respect to axis 300 of first objective lens 108. In this arrangement, the second objective lens 110 is inactive when the first objective lens 108 illuminates the sample 114 and detects the resulting polarized fluorescent emissions. In an alternating manner, once the first objective lens 108 completes the sequence of illumination and detection, the second objective lens 110 then illuminates the sample 114 and then detects polarized fluorescence emissions of those excited dipoles that are not oriented in a parallel relationship with the second objective lens 110. In other words, the first objective lens 108 detects those polarized fluorescence emissions of the excited dipoles that the second objective lens 110 cannot detect, and vice versa.

In the quadrature detection mode of operation shown in FIG. 2B, the two objective lens arrangement alternately illuminates and detects the resulting polarized fluorescence emission along a plane perpendicular to the axis of the respective detection objective lens. Specifically, the first objective lens 108 illuminates the sample 114, and then the second objective lens 110 detects those excited dipoles that are oriented along a plane in a non-parallel relationship with respect to the axis of the second objective lens 110. In an alternating manner, the second objective lens 110 then illuminates the sample 114, and the first objective lens 108 detects those excited dipoles that are oriented along a plane in a non-parallel relationship with respect to the axis of the first objective lens 108. In this manner, first objective lens 108 and second objective lens 110 are able to detect those excited dipoles that are oriented along axes that are not directly parallel to the axes of the respective objective lenses, such that first objective lens 108 and second objective lens 110 are collectively able to detect excited dipoles aligned in any particular orientation.

As shown in fig. 4A and B, in the orthogonal detection mode of operation, fluorescence microscope system 100 utilizes the two-objective arrangement discussed above to alternately illuminate and detect the resulting fluorescent emissions of those excited dipoles oriented along a plane in a non-parallel relationship with respect to the axis of the respective detection objective. Referring to fig. 4A, in a first sequence of operations, the first objective lens 108 illuminates the sample 114 along an axis 300 of the first objective lens 108, which generates fluorescent emissions emitted from the sample 114. The second objective lens 110 then detects fluorescent emissions from those excited dipoles oriented along a plane in a non-parallel relationship with respect to the axis 302 of the second objective lens 110. Referring to fig. 4B, in a second operational sequence, the second objective lens 110 illuminates the sample 114 along an axis 302 of the second objective lens 110, which generates fluorescent emissions emitted from the sample 114. The first objective lens 108 then detects the fluorescent emissions from those excited dipoles that are oriented along a plane in a non-parallel relationship with respect to the axis 300 of the first objective lens 108. The alternating sequence of illumination and detection allows the fluorescence microscope system 100 to detect the fluorescent emissions emitted by the excited dipoles regardless of the orientation of each respective excited dipole, as the excited dipoles are oriented along planes that are in a non-parallel relationship with respect to the first objective lens 108 or the second objective lens 110.

In some embodiments, processor 128 generates a position and orientation for each excited dipole based on images of the first, second, and third fluorescent emissions detected by first, second, and third objective lenses 108, 110, and 112, respectively. As such, the processor 128 generates an orientation distribution of the detected excited dipoles that are bound to the one or more three-dimensional structures with at least a three-dimensional orientation.

In addition, the polarization state of the laser beam 101 may be arbitrarily changed by the first polarization optics 129, the second polarization optics 130, and/or the third polarization optics 132. For each polarization state, the detector 128 may collect images of the first, second, and/or third fluorescent emissions 105.

In some embodiments, beam splitter 106 is not required, and each of first, second, and third objective lenses 108, 110, and 112 may have a dedicated light source 101 that is not necessarily independent of the other two light sources 102.

Testing

A prototype microscope system was constructed so that the method could be simplified for practice. The prototype microscope system included a disprim (1.1NA, Nikon; 0.71NA, special optics, corresponding to the first objective lens 110 and the second objective lens 108 shown in fig. 1) with asymmetric objective lenses equipped with polarizing optics to produce polarized excitation through each objective lens. To demonstrate this approach, actin filaments immunostained with AlexaFluor 488 phalloidin (pharioidin) were imaged. The label binds strongly to actin filaments, producing strongly polarized fluorescence depending on the relative orientation of the bound filaments and the polarization excitation. The eight volume stacks were obtained by first exciting the sample by polarized illumination introduced with different orientations 108(0 °, 45 °, 90 ° and 135 °, example in fig. 5, left side), collecting the fluorescence by 110, and then repeating this process by polarized illumination with 110 (also orientations of 0 °, 45 °, 90 ° and 135 °, collecting the fluorescence by 108).

Next, reconstruction is performed on the processor using an algorithm that predicts the average orientation in each voxel. The algorithm uses a model of the excitation and radiation process to predict the relationship between the average dipole orientation and the intensity measured by the instrument. To recover the average dipole orientation from the measured intensities, the object is extended to spherical harmonics and the linearized reconstruction problem is solved in angular frequency space. As shown in fig. 5A, example raw data (Alexa Fluor phalloidin labeled actin in fixed U2OS cells) shows maximum intensity projection images corresponding to the imaging volume obtained with differently polarized illumination light (introduced through the 0.71NA objective lens (first objective lens 108)) and collected through the 1.1NA objective lens (second objective lens 110). The orientation of the input illumination is indicated in the inset of each image. This data plus an additional set of 4 volumes (acquired by the first objective lens 108, excited at different orientations by the second objective lens 110) collected by the first objective lens 108 form the input data to the reconstruction algorithm, the result of which is shown in fig. 5B. An example projection from the data shows the orientation inside each voxel (brown glyph). In one aspect, the reconstruction allows us to derive the orientation from multi-view fluorescence images captured with illumination at different orientations.

It should be understood from the foregoing that, while particular embodiments have been illustrated and described, various modifications can be made thereto without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teachings of the present invention as defined by the appended claims.