[0001] This application claims priority to U.S. Patent Application No. 61/360,352, filed June 30, 2010, and entitled SYSTEM AND METHOD OF PRODUCING

NONDIFFRACTING LIGHT SHEETS BY A MULTIPLICITY OF SPATIALLY OVERLAPPING, MINIMALLY INTERFERING NONDIFFRACTING OPTICAL BEAMS, the entire contents of which are incorporated herein by reference.

FIELD

[0002] This application relates to a system of producing nondiffracting light sheets by a multiplicity of spatially overlapping optical beams, and in particular a selective plane illumination microscopy that utilizes such a system of producing nondiffracting light sheets.

BACKGROUND

[0003] Selective plane illumination microscopy (SPIM) is a technique that uses a thin sheet of light for the illumination of a sample at a detection plane of a detection objective lens that detects a fluorescence signal generated by the sample when illuminated. SPIM provides optical sectioning of the sample by illuminating only those fluorophores that are in the detection plane. The system combines the advantages of wide-field methods (speed, flexibility, and dynamic range) with those of a confocal arrangement (optical sectioning). SPIM also provides quantitative three-dimensional maps of the distribution of a fluorophore within the sample, for example, the expression pattern of GFP-labeled protein, with high spatiotemporal resolution and an excellent signal to noise ratio. However, the standard SPIM technique produces nonuniform axial resolution, which is caused by the diffraction of the laser beam through the sample. As diffraction causes the laser beam to spread and thicken at distances far from its center (beam waist), optical sectioning degrades, thereby forcing a compromise between field of view and axial resolution.

[0004] Other prior art techniques manipulate the phase of the laser beam by a spatial light modulator (SLM) to produce nondiffracting beams, such as Bessel beams, for sample illumination, in order to alleviate the problems mentioned above. However, the concentric rings of multiple nondiffracting beams produce regions of high intensity at points of overlap referred to as interference, thereby degrading both optical sectioning and the quality of the image. Furthermore, scanning a single nondiffracting beam throughout the sample is much slower than producing a multiplicity of minimally interfering nondiffracting beams that illuminate the sample near-simultaneously. Accordingly, there is a need in the art for a SPIM microscope that addresses the problems of the prior art.

SUMMARY

[0005] In an embodiment, a system may include an illumination source that transmits a beam of light. A first optical arrangement transforms the beam of light into a plurality of beams with each of the plurality of beams having a different wavelength. A second optical arrangement transforms the plurality of beams into a plurality of nondiffracting beams with each of the plurality of nondiffracting beams having different wavelengths such that the plurality of nondiffracting beams spatially overlap with at least another one of the plurality of nondiffracting beams with reduced interference.

[0006] In another embodiment, a method for producing nondiffracting sheets of light may include:

transmitting a beam of pulsed light,

transforming the beam of pulsed light into a plurality of beams, each of the plurality of beams having a different wavelength, and

focusing the plurality of beams through an annular aperture for transforming the plurality of beams into a plurality of nondiffracting beams, each of the plurality of nondiffracting beams having different wavelengths such that the plurality of nondiffracting beams overlap with one another with reduced interference.

[0007] In yet another embodiment, a microscope may include a laser source that transmits a pulsed laser beam, a diffraction grating for causing the pulsed laser beam to be split into a plurality of pulsed laser beams with each of the plurality of pulsed laser beams having different wavelengths. A first optic arrangement magnifies the plurality of pulsed laser beams having different wavelengths from the diffraction grating and images the plurality of pulsed laser beams through an aperture for transforming the plurality of pulsed laser beams into a plurality of nondiffracting beams. Each of the plurality of nondiffracting beams has a different wavelength. A second optic arrangement demagnifies the plurality of nondiffracting beams, and then focuses the plurality of nondiffracting beams onto a sample at the detection plane of the second optic arrangement, wherein the plurality of nondiffracting beams spatially overlap at least one other of the plurality of nondiffracting beams, but with reduced interference. [0008] Additional objectives, advantages and novel features will be set forth description which follows or will become apparent to those skilled in the art upon examination of the drawings and detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a simplified illustration of an embodiment of a selective plane illumination microscopy system; and

[0010] FIGS. 2 and 2B are simplified illustrations showing the plurality of nondiffracting beams relative to a detection plane of the microscopy system.

[0011] Corresponding reference characters indicate corresponding elements among the view of the drawings. The headings used in the figures should not be interpreted to limit the scope of the claims.

DETAILED DESCRIPTION

[0012] Selective plane illumination microscopy (SPIM) is a technique that provides fast, 3-dimensional biological imaging with minimal photo-bleaching and photo-damage to a sample. However, the axial resolution of the image produced by conventional SPIM microscopes suffers increasing degradation at distances from the illumination beam waist due to diffractive spreading of the illumination source. Other techniques manipulate the phase of the laser beam by a spatial light modulator to produce non-diffracting beams, such as Bessel beams, for sample illumination; however, Bessel beams produce regions of high intensity outside the detection plane, caused by concentric rings ("ringing") or interference, thereby degrading the quality of the image.

[0013] In physics, interference is the addition (superposition) of two or more waves that produces a new wave pattern. Interference usually refers to the interaction of waves that are correlated or coherent with each other, either because these waves come from the same source or because they have the same or nearly the same frequency. The principle of superposition of waves states that the resultant displacement at a point is equal to the vector sum of the displacements of different waves at that point. For example, if a crest of a wave meets a crest of another wave at the same point then the crests interfere constructively and the resultant wave amplitude is increased. However, if a crest of a wave meets a trough of another wave then the waves interfere destructively, and the resultant wave amplitude is decreased.

[0014] Both destructive and constructive interference produce unwanted concentric ringing that degrades the quality of the image detected by a Bessel-beam based SPIM microscope as discussed above. In particular, constructive interference focuses energy into planes other than the detection plane of the microscope, thereby producing peaks in the rings, while destructive interference generates intensity nulls, which produces troughs in the rings. Accordingly, both constructive and destructive interference above and below the detection plane of the microscope create difficulties in measuring the fluorescence signal generated by the illumination of a sample under detection by the microscope. As used herein, the term "interference" will refer to both constructive and destructive interference as described above.

[0015] As such, embodiments of a system and method that produces nondiffracting sheets of light using a multiplicity of spatially overlapping optical beams as disclosed herein include particular properties and characteristics that address issues related to reducing interference. The system and method as described herein overcomes these deficiencies by producing beams of light having different wavelengths that are transformed into nondiffracting beams that also have different wavelengths, which cause the nondiffracting beams to spatially overlap (causing a sheet) with minimal interference between the nondiffracting beams. Furthermore, the interference due to any individual nondiffracting beam, is reduced if the illumination source is a femtosecond laser beam, which produces multiphoton excitation of the sample and eliminates regions of high intensity that would otherwise exist due to the mechanism of linear absorption for generating fluorescence in a biological specimen under observation.

[0016] Referring to the drawings, one embodiment of the system and method for producing nondiffracting sheets of light using the multiplicity of spatially overlapping optical beams is embodied in a microscope is illustrated and generally indicated as 10 in FIG. 1. In an embodiment, the microscope 10 may be a SPIM microscope that includes a laser device 12, such as a femtosecond laser device that generates a pulsed laser beam 36, which is reflected off a diffraction grating 14. In a particular embodiment, the diffraction grating 14 may have the following characteristics: 830.8 grooves/mm, 19.7 degree blaze, input angle = 49.8 degrees and an output angle = 0 degrees. In a particular embodiment, the pulsed laser beam 36 may have an input beam diameter = 10 mm. The output angle of the diffraction grating 14 should be normal to the face of the diffraction grating 14 such that the groove distance, input angle, wavelength lambda, and the order "m" to satisfy the following equation m(lambda) = d sine theta i.

[0017] The plurality of beams 38 having different wavelengths imparted by the diffraction grating 14 is then imaged by a pair of relay lenses 18 and 20, such as lenses 18 and 20 having focal lengths of 300 mm and 100 mm, respectively, through an annular aperture 16 that transforms the plurality of beams 38 having different wavelengths into a plurality of nondiffracting beams 40 with different wavelengths. However, the focal length of the relay lens 18 and 20 may be chosen so that the diameter of the pulsed laser beam 36 is demagnified (or magnified) to fill the annular aperture 16. In an embodiment, the annular aperture 16 may have an inner radius between 2.33 mm to 2.63 mm and an outer radius of 2.66 mm. The value of the outer radius of the annular aperture 16 may be chosen so that, after magnification by the relay lenses 18, 20, 22, and 24, the back focal plane diameter of the first objective lens 26 is filled so as to produce the highest available numerical aperture. The value of the inner radius of the annular aperture 16 determines the length of the nondiffacting beam 40 with smaller values producing a shorter nondiffracting beam 40 while larger values producing a longer nondiffracting beam 40 Based on the characteristics of the first objective lens 26 and the relay lenses 18, 20, 22, and 24 the range of values of the outer radius may be between 2.0-2.66 mm, and the inner radius may be any value less than the range of values for the outer radius. In addition, other embodiments that transform the plurality of beams 38 into plurality of nondiffracting beams 40 may include an axicon, a spatial light modulator (SLM), or a binary phase mask.

[0018] As used herein, the term "nondiffracting beam" means any beam of electromagnetic light that shows little or no diffraction over a significant propagation distance (i.e. the transverse intensity distributions of the beam do not vary over a significant propagation distance), including but not limited to Bessel-like beams (i.e., those beams whose wave amplitude is approximated by a Bessel function).

[0019] The plurality of nondiffracting beams 40 are then reimaged by another pair of relay lenses 22 and 24, for example lenses having focal lengths of 100 mm and 300 mm, respectively, onto the back detection plane of a first objective lens 26, for example an excitation objective lens. In other embodiments, the relay lenses 22 and 24 may have any suitable focal length with the only constraint being that the nondiffracting beams 40 must cover the back focal plane of the first objective lens 26 In an example embodiment, the first objective lens 26 may be a Nikon 0.8 NA 40x water immersion objective lens. The annular aperture 16 may be replaced by an axicon plus additional lenses for increased power efficiency. In one embodiment, this arrangement transforms the energy into a series of nondiffracting beams 40 that overlap each other, but also reduces interference of the beams 40. As shown in FIGS. 1, 2A and 2B, the resulting plurality of nondiffracting beams 40 may illuminate a biological sample 32 and induce a multiphoton fluorescence excitation in the sample 32 along the optical axis and in the vicinity of a detection plane 42 of the first objective lens 26.

[0020] Referring to FIGS. 1 and 2A, the microscope 10 further includes a focusing optic arrangement having a second objective lens 28, for example a detection objective lens, positioned at a 90-degree angle relative to the sample 36 on the detection plane 42. In one embodiment, the second or detection objective lens 28 may be a Nikon 0.8 NA 40x water immersion objective lens. The main constraint on the types of first and second objective lenses 26 and 28 is that these lenses must fit together at 90 degrees angle for a SPIM arrangement such that the 90 degree geometry imposes spatial constraints on the types of objective lenses that may be used. As shown in FIG. 1, a tube lens 34 is positioned along the optic axis of the second objective lens 28 for imaging the fluorescence excitation signal emitted by the sample 32 onto a widefield detector 30. In an embodiment, the tube lens 34 may be a 200 mm Nikon tube lens, however other focal lengths may be used to provide a given magnification. For example, the 200 mm tube lens in the example embodiment provides a magnification of 40x when combined with the first objective lens 26. In one embodiment, the widefield detector 30 may be a charge coupled detector (CCD), for example, an Andor iXon 888).

[0021] The optical overall arrangement of the microscope 10 described above creates a multiplicity of nondiffracting beams of light from an illumination source that overlap spatially with each other but with reduced interference, thereby producing sheets of light that resist diffraction along the optic axis. This arrangement results in better axial resolution of the image than is possible with conventional SPIM microscopes.

[0022] The concept of transforming a pulsed laser beam 36 generated from a laser source 12 that is split into a plurality of beams 38 having different wavelengths and then transformed into a plurality of nondiffracting beams 40 may be applied to other microscopy, such as fluorescence microscopy, where the application of nondiffracting sheets of light to illuminate a sample to generate the resulting excitation profile is desirable.

[0023] 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 this invention as defined in the claims appended hereto.