Patent Description:
Most fluorophores, including fluorescent proteins, absorb and emit light as dipoles. This creates the opportunity to reveal not only the position, but also the orientation of fluorophores and of the molecular assemblies to which they are bound. Polarized light microscopes that are equipped to excite and/or detect polarized fluorescence already exploit this opportunity. The orientation and kinetics of molecular assemblies determine directionality of cellular function or disease. For example, directional cell migration during wound healing or metastasis relies on the flow of a patterned actin network which generates net force towards the direction of migration.

The molecular orientation is revealed by using either polarized light for dipole excitation or polarization analysis of the dipole emission, or both. However, current microscopes illuminate and image the sample from a single viewing direction. Accordingly, the polarization of the excitation light and the emitted fluorescence is primarily defined in the plane perpendicular to the illumination/viewing direction as illustrated in <FIG>. Dipoles parallel to the illumination/viewing direction can neither be excited nor detected efficiently, thus making it difficult or even impossible to determine the complete orientation distribution of fluorophores bound to three-dimensional structures.

It is with these observations in mind, among others, that various aspects of the present disclosure were conceived and developed. Background art is represented by <CIT>.

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

As described herein, systems and methods for extending fluorescence polarization imaging so that the dipole moment of a fluorescent dye emitted by a sample may be excited regardless of the three-dimensional orientation of the dipole. In one aspect, the dipole is excited from multiple directions, thereby ensuring that the excitation of the sample occurs along multiple orientations even if the dipole is unfavorably oriented along the axial (propagation) axis of the detection objective. In one embodiment, a dual-view inverted selective-plane illumination microscope (diSPIM) is used to illuminate the sample and detect the resulting polarized fluorescence emissions emitted by the sample from two different directions that are non-parallel relation relative to each other. In one embodiment, polarization-resolved excitation of the sample and epi-detection of the emitted polarized fluorescence captures the three-dimensional orientation of the excitation dipole 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 in alternating sequence of excitation and non-parallel detection of the fluorescence emitted by the sample captures substantially most of the projection of the three-dimensional orientation of the excitation dipole in the axial or meridional plane of the respective detection objective. In one embodiment, the system includes a processor in operative communication with one or more detectors for capturing data related to the position and three-dimensional orientation of each excitation dipole detected in the polarized fluorescence emissions emitted by the sample detected by one or more objective lenses. The processor is operable for computing the three-dimensional orientation and position of the excitation dipole in each voxel detected in the fluorescence emission emitted by the sample being illuminated. In some embodiments, the system captures a plurality of images with different excitation polarization such that the processor may determine the position and three-dimensional orientation of each excitation dipole detected by one or more detectors. Referring to the drawings, embodiments of a system for determining the three-dimensional dipole orientation and position of each voxel of an illuminated sample are illustrated and generally indicated as <NUM> in <FIG>.

Referring to <FIG>, one embodiment of a fluorescence microscopy system, designated <NUM>, is illustrated. In one aspect, the fluorescence microscopy system <NUM> is operable to determine the position and orientation of excitation dipoles in fluorescence emissions emitted from a sample <NUM> being illuminated.

In some embodiments, the fluorescence microscopy system <NUM> includes a light source <NUM> for emitting a light beam <NUM> that is polarized by a first polarization optics <NUM> for polarizing the light beam <NUM> into polarized light <NUM>. In some embodiments, the first polarization optics <NUM> may include a wave plate, a polarizer, and/or one or more liquid crystals to polarize the light beam <NUM>. In some embodiments, the light source <NUM> may be a laser for emitting a laser light beam; however, in other embodiments, the light source <NUM> may be other sources of light, such as lamps that emit a light beam capable of being polarized.

In some embodiments, the polarized light <NUM> may be split by a beam splitter <NUM> into split polarized light 103A and 103B. In some embodiments, the split polarized light 103A is redirected by a first dichroic mirror <NUM> through a first objective lens <NUM> which may include a second polarization optics <NUM> for further polarizing the split polarized light 103A into split polarized light 103C for illumination of sample <NUM> along a first axis <NUM>. As shown, the split polarized light 103B is redirected by a dichroic mirror <NUM> through a second objective lens <NUM> which may include a third polarization optics <NUM> for further polarizing the split polarized light 103B into split polarized light 103D for illumination of sample <NUM> along a second axis <NUM> that is in orthogonal relation relative to the first axis <NUM>.

As the sample <NUM> is illuminated, those polarized fluorescence emissions <NUM> emitted by the sample <NUM> substantially along a plane orthogonal to the first axis <NUM> are detected by the first objective lens <NUM>, while those polarized fluorescence emissions <NUM> emitted by the sample along a plane substantially parallel to the first axis <NUM> are not detected by the first object lens <NUM>. In addition, those polarized fluorescence emissions <NUM> emitted by the sample <NUM> substantially along a plane orthogonal to the second axis <NUM> are detected by the second objective lens <NUM>, while those polarized fluorescence emissions <NUM> emitted by the sample <NUM> along a plane substantially parallel to the second axis <NUM> are not detected by the second objective lens <NUM>. In this arrangement, the orthogonal relationship between the first objective <NUM> and the second objective <NUM> allows the fluorescence microscopy system <NUM> to detect the excitation dipoles regardless of their axis of orientation. In other embodiments, the first and second objectives <NUM> and <NUM> may be oriented at a non-parallel angle relative to each other.

In one arrangement, the polarized fluorescence emissions <NUM> detected by the first objective lens <NUM> may be redirected by the first dichroic mirror <NUM> through a first tube lens <NUM> for detection by a first detector <NUM>. In a further arrangement, the fluorescence emissions <NUM> detected by the second objective lens <NUM> may be redirected by the second dichroic mirror <NUM> through a second tube lens <NUM> for detection by a second detector <NUM>.

In some embodiments, a third objective <NUM> may be positioned below a plane <NUM> of the sample <NUM> and oriented along a third axis <NUM> that forms a <NUM> degree angle relative to the first and second axes <NUM> and <NUM>, respectively. The third objective lens <NUM> functions to detect fluorescence emissions <NUM> emitted below the plane <NUM> of the sample <NUM> and at an angle perpendicular to the plane <NUM> of the sample <NUM>. In some embodiments, the fluorescence emissions <NUM> detected by the third objective <NUM> may be imaged through a third tube lens <NUM> for detection by a third detector <NUM>. In some embodiments, the third objective <NUM> may be oriented in a non-parallel angle relative to the first and second axes <NUM> and <NUM>, respectively.

In some embodiments, the first detector <NUM>, second detector <NUM> and third detector <NUM> are in operative communication with one or more processors <NUM> that utilize one or more algorithms for computing the position and three-dimensional orientation of the excitation dipole based on the images of the fluorescence emissions <NUM> captured from the first detector <NUM>, second detector <NUM> and third detector <NUM>, respectively.

In some embodiments, the first and second objective lenses <NUM> and <NUM> may be, for example, a Nikon <NUM> NA, Nikon <NUM> NA, Nikon <NUM> NA, Special Optics <NUM> NA lenses, although other types or kinds of objective lenses are contemplated.

In some embodiments, the fluorescence microscopy system <NUM> may operate in either in an epi-detection mode of operation (<FIG>) or an orthogonal detection mode of operation (<FIG>). In the epi-detection mode of operation shown in <FIG>, a single objective lens, e.g., first objective <NUM> focuses the polarized light rays in a direction along an axis A to illuminate a sample <NUM> and then the same objective detects the resulting fluorescence emissions emitted by the sample. In this mode of detection, the polarized fluorescence emissions emitted by the sample along a plane perpendicular to the axis of the first objective <NUM> are detected by the first objective <NUM>.

As shown in <FIG>, in the epi-detection mode of operation the fluorescence microscopy system <NUM> the same objective lens, e.g., first objective <NUM>, illuminates the sample <NUM> and then detects the polarized fluorescence emissions of those emission dipoles oriented along a plane <NUM> that is in non-parallel relation relative to the axis <NUM> of first objective <NUM>. In this arrangement, the second objective <NUM> is inactive when the first objective <NUM> illuminates the sample <NUM> and detects the resulting polarized fluorescence emissions. In alternating fashion, once the first objective <NUM> completes the sequence of illumination and detection, the second objective <NUM> then illuminates the sample <NUM> and then detects the polarized florescence emissions of those excitation dipoles not oriented in parallel relation to the second objective <NUM>. In other words, the first objective <NUM> detects the polarized fluorescence emissions of those excitation dipoles not detectable by the second objective <NUM> and vice-versa.

In the orthogonal detection mode of operation shown in <FIG>, a two-objective arrangement alternately illuminates and detects the resulting polarized fluorescence emissions along a plane perpendicular to the axis of the respective detection objective. Specifically, the first objective <NUM> illuminates the sample <NUM> and then a second objective <NUM> detects those excitation dipoles oriented along a plane that is in non-parallel relation relative to the axis of the second objective <NUM>. In alternating fashion, the second objective <NUM> then illuminates the sample <NUM> and the first objective <NUM> detects those excitation dipoles oriented along a plane that is in non-parallel relation relative to the axis of the first objective <NUM>. In this manner, the first and second objectives <NUM> and <NUM> are capable of detecting those excitation dipoles oriented along axes that are not directly parallel to the axis of the respective objective such that the first and second objectives <NUM> and <NUM> are collectively capable of detecting excitation dipoles aligned along any particular orientation.

As shown in <FIG>, in the orthogonal detection mode of operation the fluorescence microscopy system <NUM> utilizes the two-objective arrangement discussed above to alternately illuminate and detect the resulting fluorescence emissions of those excitation dipoles oriented along a plane that is in non-parallel relation relative to the axis of the respective detection objective. Referring to <FIG>, in a first sequence of operation the first objective <NUM> illuminates the sample <NUM> along the axis <NUM> of the first objective <NUM> which generates fluorescence emissions emitted from the sample <NUM>. The second objective <NUM> then detects the fluorescence emissions from those excitation dipoles oriented along a plane that is in non-parallel relation relative to the axis <NUM> of the second objective <NUM>. Referring to <FIG>, in a second sequence of operation the second objective <NUM> illuminates the sample <NUM> along the axis <NUM> of the second objective <NUM> which generates fluorescence emissions emitted from the sample <NUM>. The first objective <NUM> then detects the fluorescence emissions from those excitation dipoles oriented along a plane that is in non-parallel relation relative to the axis <NUM> of the first objective <NUM>. The alternating sequence of illumination and detection allow the fluorescence microscopy system <NUM> to detect the fluorescence emissions emitted by the excitation dipoles despite the orientation of each respective excitation dipole since the excitation dipoles are oriented along a plane that is in non-parallel relation relative to either the first objective <NUM> or the second objective <NUM>.

In some embodiments, the processor <NUM> generates a position and orientation for each excitation dipole based on the images of the first fluorescence emission, second fluorescence emission and third fluorescence emission detected by the first objective <NUM>, second objective <NUM> and third objective <NUM>, respectively. As such, the processor <NUM> generates an orientation distribution of the detected excitation dipoles bound to one or more three-dimensional structures in at least a three-dimensional orientation.

In addition, the polarization state of the laser light beam <NUM> may be changed arbitrarily by the first polarization optics <NUM>, second polarization optics <NUM> and/or third polarization optics <NUM>. The detector <NUM> may collect images of the first, second and/or third fluorescence emissions <NUM> for each polarization state.

In some embodiments, a beam splitter <NUM> is not required and each of the first, second and third objective lenses <NUM>, <NUM> and <NUM> may have a dedicated light source <NUM> that is not necessarily independent of the other two light sources <NUM>.

A prototype microscopy system was constructed so that the method could be reduced to practice. The prototype microscopy system included a diSPIM with asymmetric objectives (<NUM> NA, Nikon; <NUM> NA, Special Optics, corresponding to first and second objectives <NUM> and <NUM> shown in <FIG>) equipped with polarization optics to produce polarized excitation through each objective. To demonstrate the method, actin filaments were imaged that were immunostained with Alexa Fluor <NUM> phalloidin. This label binds strongly to actin filaments, producing a strongly polarized fluorescence that depends on the relative orientation of the bound filament and polarized excitation. Eight volumetric stacks were obtained, first by exciting the sample with polarized illumination introduced by <NUM> of varying orientation (<NUM>°, <NUM>°, <NUM>°, and <NUM>°, examples in <FIG>, LEFT), collecting fluorescence through <NUM>; and then by repeating this process with polarized illumination through <NUM> (also of orientation <NUM>°, <NUM>°, <NUM>°, and <NUM>°, collecting fluorescence through <NUM>).

Next, a reconstruction was performed on a processor utilizing an algorithm that predicts average orientation in each voxel. The algorithm uses a model of the excitation and radiation processes to predict the relationship between the average dipole orientation and the intensities measured by the instrument. To recover the average dipole orientation from the measured intensities the object was expanded onto spherical harmonic functions and solved the linearized reconstruction problem in angular frequency space. As shown in <FIG>, example raw data (Alexa Fluor Phalloidin labeling actin in fixed U2OS cells) showing maximum intensity projection images corresponding to imaging volumes obtained with different polarizations of illumination light (introduced through <NUM> NA objective lens (first objective lens <NUM>), and collected through a <NUM> NA objective lens (second objective lens <NUM>). Orientation of the input illumination is indicated in the inset to each image. This data, plus an additional set of <NUM> volumes (excited with different orientations through the second objective lens <NUM>, collected through the first objective lens <NUM>, form the input data to the reconstruction algorithm. <FIG> shows the result of reconstruction. Example projection from data, showing orientations (brown glyphs) inside each voxel. In one aspect, the reconstruction allows us to derive orientation from multi-view fluorescence images captured with different orientations of the illumination.

Claim 1:
A fluorescence microscopy system (<NUM>) comprising:a laser source (<NUM>) for emitting a laser light beam (<NUM>);a first polarization optics (<NUM>) for converting the laser light beam (<NUM>) into a polarized light beam (<NUM>);a beam splitter (<NUM>) for splitting the polarized light beam (<NUM>) into a first polarized light beam (103A) and a second polarized light beam (103B);a first objective (<NUM>) oriented along a first axis (<NUM>) such that the first polarized light beam (103A) illuminates a sample (<NUM>) along a first angle to produce a first fluorescence emission (<NUM>) from a first plurality of excitation dipoles; and a second objective (<NUM>) oriented along a second axis (<NUM>) such that the second polarized light beam (103B) illuminates the sample (<NUM>) at a second angle that is not parallel relative to the first angle to produce a second fluorescence emission (<NUM>) from a second plurality of excitation dipoles,wherein the first objective (<NUM>) is oriented to detect the first plurality of excitation dipoles oriented at an angle perpendicular to the first axis (<NUM>) and the second objective (<NUM>) is oriented to detect the second plurality of excitation dipoles oriented at an angle perpendicular to the second axis (<NUM>).