Patent ID: 12235426

DETAILED DESCRIPTION OF EMBODIMENTS

FIG.1is a schematic illustration of a microscopic imaging system1according to a first exemplary embodiment. The microscopic imaging system1is configured so that detection light is detected using a detector system12, wherein the detection light is generated by the object4in response to illumination light, which is generated by a light source2and which is directed to the object4. The detection light may include fluorescent light and/or reflected illumination light.

The light source2may include a laser system so that the beam3of illumination light which is emitted from the light source2is a laser beam. However, it is also conceivable (in particular in the third exemplary embodiment which will be described in connection withFIG.6) that the light source2includes a non-laser light source, such as a halogen lamp. By way of example, the laser may be configured as a semiconductor-diode laser, a gas or a solid-state laser. The beam3of illumination light is guided to the object4using an optical system which includes a beam multiplier5, a beam splitter6and focusing optics7. The beam3of illumination light may include one or more wavelengths within the range of 200 nanometers and 1800 nanometers or within a range of between 300 nanometers and 1500 nanometers. More specifically, it may have a wavelength that is suitable for exciting commonly used fluorophores, such as DAPI, FITC, or several species of fluorescent proteins, which typically operate with excitation wavelengths ranging from the near ultraviolet, through the visible range, to the near infrared.

The beam multiplier5receives the beam3of illumination light which is generated by the light source2, to generate a plurality of beamlets8and9of illumination light. The configuration of the beam multiplier5and the beamlets8and9will be described in detail further below with reference to the second and third exemplary embodiments described in connection withFIGS.4and6. For simplicity of illustration, inFIG.1, only two beamlets8and9are shown. However, it is conceivable that the beam multiplier generates more than two beamlets, such as more than 5 beamlets or more than 10 beamlets. The number of beamlets may be less than 500 or less than 130.

The beamlets8and9, which are emitted by the beam multiplier5are incident on the beam splitter6. The beam splitter6may be configured as a wavelength-sensitive beam splitter, such as a dichroic beam splitter, which transmits different wavelengths than it reflects. Additionally or alternatively, the beam splitter6may be configured as a cube beam splitter. The beam splitter6, which is shown inFIG.1, is configured so that the portion of illumination light which is reflected, is suppressed compared to the portion of illumination light which is transmitted unreflected. Further, the portion of unreflected detection light is suppressed compared to the portion the detection light which is reflected. In an alternative embodiment, the beam splitter6is configured so that the portion of illumination light, which is transmitted unreflected, is suppressed compared to the portion of illumination light which is reflected and the portion of detection light which is reflected is suppressed compared to the portion of detection light which is transmitted unreflected.

The beamlets8and9of illumination light, which exit from the beam splitter6are incident on focusing optics7, which are configured to focus each of the beamlets8and9onto a line focus LF1, LF2within the object4. InFIG.1, the axes of the line foci LF1and LF2are oriented perpendicular to the paper plane. The number of line foci LF1and LF2is equal to the number of beamlets8and9. In alternative embodiments, it is also conceivable that the number of line foci is greater or smaller than the number of beamlets8and9.

The focusing optics7are rotationally symmetric or substantially rotationally symmetric. The focusing optics7define an optical axis A1, along which the illumination light is incident on the object4.

It is to be understood that configurations are conceivable in which the focusing optics7substantially deviate from a rotational symmetry. By way of example, the focusing optics7may include one or more plane-symmetric optical elements, such as a cylinder lens. For plane-symmetric optical elements, the optical axis lies within the symmetry plane. Furthermore, the invention is not limited to focusing optics7having only one optical axis. By way of example, the focusing optics7may include an array of optical elements, which define a plurality of optical axes along which the illumination light is incident on the object.

As is illustrated in more detail inFIG.2, the optical system is configured so that the line foci LF1and LF2, when seen relative to the optical axis A1, are mutually displaced from each other in an axial direction and also mutually displaced from each other in a lateral direction (i.e. measured perpendicular to the optical axis A1). In the schematic illustration ofFIG.2, the longitudinal axes of the line foci LF1and LF2extend perpendicular to the paper plane and parallel to each other. It is to be understood that the line foci LF1and LF2do not need to extend parallel relative to each other. It is further to be understood that the line foci LF1and LF2do not necessarily represent perfect lines having a constant width and/or a constant intensity in a direction along the line focus' axis. Rather, each of the line foci LF1and LF2may have an elliptical or substantially elliptical beam profile having a variable width along its longitudinal axis. Specifically, when seen along the line focus' longitudinal axis, the intensity profile may have an intensity maximum, wherein the intensity profile tapers off on either side of the intensity maximum.

Specifically, as is illustrated inFIG.2, the line focus LF1and the line focus LF2, when measured along the optical axis A1, are displaced from each other by an axial separation distance Δz (measured along the optical axis A1) and by a lateral separation distance

Δx (measured along a direction d, which is perpendicular relative to the optical axis A1). The lateral separation distance Δx is selected so that for each of the line foci LF1and LF2, confocal detection of detection light27,28which is emitted from the respective line focus LF1and LF2in response to the illumination light can be separately performed by using the optical system and the detector system12(shown inFIG.1), which is configured to detect the detection light.

In other words, a sufficiently large lateral separation distance Δx allows, for each of the line foci LF1and LF2, confocal detection of the detection light27,28emitted from the respective line focus LF1, LF2without crosstalk from the neighboring line focus.

By way of example, the optical system may be configured so that the lateral displacement distance Δx between the line foci may be larger than 0.5 times or larger than 2 times the diffraction-limited width of the line foci LF1and LF2measured along the lateral direction d. The lateral displacement distance Δx may be less than 100 times or less than 1000 times the diffraction-limited width. The line foci LF 1 and LF2lie within a common plane P, wherein a normal vector n1to the plane P is angled relative to the optical axis A1. By way of example, an angle alformed by the normal vector n1and the optical axis A1may be greater than 0.01 degrees or greater than 0.03 degrees. The angle a1may be less than 6 degrees or less than 4.5 degrees. If the imaging system generates more than two line foci, each of the line foci may lie within the common plane P.

It has been shown by the inventor that a different configuration (which is not covered by the claims) in which the line foci are located in an object plane which is perpendicular to the optical axis and in which the object is tilted relative to the object plane, results in significant aberrations which severely degrade the resolution of the microscopic imaging system. Specifically, these aberrations are dominated by coma and astigmatism. The inventor has further shown that such aberrations do not occur if a configuration is used in which the line foci LF1and LF2are axially and laterally displaced relative from each other, as has described above in connection withFIG.2.

Returning toFIG.1, the optical system of the imaging system1is further configured to image each of the line foci LF1and LF2on a respective light receiving portion SP1and SP2on a light receiving surface10of the light-sensitive detector system12, on which a plurality of light-sensitive detector elements of the light-sensitive detector system12are arranged, as will be described in detail below in connection withFIG.3. Therefore, the light receiving portions SP1and SP2are substantially located at positions, which are optically conjugate to the locations of the line foci LF1and LF2within the object4. The light receiving portions SP1and SP2are non-overlapping.

As can be seen fromFIG.1, the optically conjugate positions of the light receiving portions SP1and SP2are achieved by tilting the light receiving surface10of the light-sensitive detector system12with respect to the optical axis A2along which the detecting light is incident on the light-sensitive detector system12so that a surface normal {right arrow over (n)}2to the light receiving surface10and the optical axis A2forms an angle a2which depends on the angle al (shown inFIG.2) formed by the normal vector {right arrow over (n)}1and the optical axis A1. Therefore, the detector system12is configured to compensate for the axial displacement of the line foci LF1and LF2within the object4. By way of example, the angle a2may be greater than 1 degrees or greater than 3 degrees. The angle a2may be less than 60 degrees or less than 45 degrees. It is to be noted that, depending on the configuration of the optical system (in particular depending on the configuration of the beam splitter6), the optical axes A1and A2may be parallel relative to each other, may be angled 90 degrees or may be angled at an angle which is different from 90 degrees.

Additionally or alternatively, it is conceivable that the optical system is configured to at least partially compensate for for the axial displacement of the line foci LF1and LF2within the object4. By way of example, the optical system may include an array of refractive optical elements having different values of refractive optical power. For each of the line foci LF1and LF2, the detection light27,28which is emitted from the respective line focus LF1, LF2, may traverse one of the refractive optical elements so that detection light from different line foci LF1, LF2, traverse different refractive elements.

As is further schematically indicated inFIG.1, the object4is moved along a scanning direction {right arrow over (s)} relative to the foci LF1and LF2for scanning the line foci LF1and LF2through the object4. The scanning movement is performed using a scanning stage (not shown inFIG.1) on which the object4is mounted and which is configured to displace the object4relative to the foci LF1and LF2. The scanning direction g is oriented perpendicular or substantially perpendicular to the longitudinal axis of each of the line foci LF1and LF2. It is, however, also conceivable that the scanning direction {right arrow over (s)} is angled relative to the axes of the line foci LF1and LF2at an angle which is significantly less than 90 degrees. The scanning direction {right arrow over (s)} is also angled relative to the common plane P, within which the line foci LF1and LF2extend. As will be discussed in detail further in connection withFIG.3, the scanning movement allows acquisition of three-dimensional confocal image data from the volume of the object4.

FIG.3schematically illustrates the arrangement of light-sensitive detector elements20on the light receiving surface10of the detector system12(shown inFIG.1). The light-sensitive detector elements20have a light-sensitive surface and are pixels. Each of the light-sensitive detector elements is configured to separately detect a respective portion of the detection light emitted from the object. For each of the foci LF1and LF2, an array21,22of the light sensitive detector elements20is provided, wherein each of the arrays21and22represents a pixel block formed from a plurality of pixel lines and represents one of the light receiving portions SP1and SP2. Each of the arrays21and22may be configured as a TDI (time delay integration) block. In a portion of the light receiving surface10, which is located between the arrays21and22, no further detector elements are arranged so that a non-photosensitive surface region23is formed. By way of example, a width g of the non-photosensitive surface region23(i.e. a separation distance between the light-sensitive surfaces of the detector elements of the neighboring arrays21and22), measured perpendicular to a longitudinal axis L of the arrays21and22may be greater than 4 or greater than 12 times the pixel size (measured along an edge of a square-shaped pixel). Additionally or alternatively, for each pair of neighboring light sensitive surfaces, which is in one of the arrays21and22, a displacement h between the light-sensitive surfaces of the pair is less than 0.25 times, or less than 0.1 times, or less than 0.05 times the separation distance g. The width g may be less than 15 times or less than 45 times or less than 60 times or less than 100 times or less than 200 times the displacement h.

Each of the arrays21and22is dimensioned so that it acts as a confocal spatial filter, which acts in a similar manner as a pinhole in conventional scanning confocal microscopes. Therefore, for each of the line foci LF1, LF2, the respective array21,22allows confocal filtering of the detection light for filtering out detection light emitted from the respective line focus and for suppressing light which is emitted from axial and/lateral positions which are different from the respective line focus. It is, however, also conceivable that each of the arrays21,22have an extent which is larger than required for performing the confocal filtering and that the confocal filtering is provided by using only detection signals from a portion of the light-sensitive detector elements20of the arrays21,22. Further, it is also conceivable that for performing the confocal filtering, a conventional image sensor is used and for each of the line foci LF1, and LF2, the detector signals of a portion of the light-sensitive pixels of the image sensor is used so that the pixels provide the required confocal filtering. This provides the advantage of using a readily available off-the-shelf component.

Furthermore, the plurality of light-sensitive detector elements20within each of the arrays21and22allow spatially resolved light detection within each of the line foci LF1, and LF2in a direction along the longitudinal axis of the line foci LF1, and LF2and/or in a width direction of the line foci LF1, and LF2.

Compared to conventional two-dimensional image sensors, the light-sensitive detector system12of the present embodiment prevents detecting pixel signals which do not provide useful information, since the light receiving surface10only has light sensitive detector elements at focus positions of the detection light, i.e. at the light receiving portions SP1and SP2, which are optically conjugate to the locations of the foci LF1and LF2in the object4. Further, the detector system12of the present embodiment is configured so that in the non-photosensitive region23, at least a portion of the read out electronics (logic and connective circuitry, in particular charge to voltage converters CVC) is arranged. Thereby, more circuitry can be arranged on the light-sensitive detector system12so that a higher read-out-speed can be obtained. The circuitry in the non-photosensitive region also allows providing an increased fill factor in the arrays21and22of detector elements so that the light collection efficiency within the foci LF1and LF2can be improved.

FIG.4is a schematic illustration of a microscopic imaging system1a according to a second exemplary embodiment. Components of the imaging system1a, which are similar or corresponding to components of the first exemplary embodiment illustrated inFIGS.1to3, bear the same numerals but are succeeded by the suffix letter “a” to avoid confusion.

In the imaging system1a of the second exemplary embodiment, the beam multiplication system5aincludes a diffractive optical element13awhich is in the path of the illumination light between the light source2aand the beam separator6a. The reflective optical element13amay include a grating, which may be configured to function as a reflective grating or as a transmissive grating. By way of example, the grating may include an electrically addressable and/or an optically addressable grating. Examples for electrically and/or optically addressable gratings are spatial light modulators. The spatial light modulator may use liquid-crystal technology. The spatial light modulator may include an array of pixels, wherein each of the pixels generates a phase delay to a portion of the beam3aof illumination light, which traverses or impinges on the respective pixel. The phase delay may be adjustable by means of a signal provided to the spatial light modulator. The signal may be an electrical and/or an optical signal. The diffractive plane defined by the diffractive optical element13amay be a pupil plane of the focusing optics7aor of the optics, which focuses the illumination light, which exits from the diffractive optical element, into the foci (point foci or line foci) within the object.

Additionally or alternatively, the diffractive optical element13amay include an optical element which imparts non-adjustable phase delays to the beam3aof illumination light which is incident on the diffractive optical element13a. By way of example, the diffractive optical element13aincludes a plate made of glass, quartz and/or a polymer, such as polycarbonate. The plate may have a surface structure, such as a corrugated surface structure, which is configured to generate a desired variation in optical path length across the beam profile, which generates the plurality of beamlets. The corrugated surface structure may be generated using an etching and/or embossing process.

As will be described hereinafter, the beamlets8a,9a, which are generated using the diffractive optical element13aare configured so that the focusing optics7a, which include an objective lens, focus these beamlets into the line foci LF1and LF2with their mutual axial and lateral displacement as described above. For simplicity of description, the following description of a design process for the diffractive optical element13arelates to a transmissive grating. However, the principles can be adapted to design reflective gratings.

The diffractive optical element13ais configured so that the illumination light, which is transmitted through the beam multiplication system5aincludes a plurality of beamlets8a,9aso that each of the beamlets diverges from or converges toward a real or virtual focus. The real or virtual foci are displaced from each other. The diffractive optical element13ais configured so that the displacement of the real or virtual foci causes the lateral and axial displacement of the line foci in the object4a. Specifically, in the imaging system1aaccording to the second exemplary embodiment, the beamlets8aand9ain a region between the beam multiplication system5aand the beam splitter6ahave different divergences which relate to virtual foci (not shown inFIG.4) from which the beamlets8a,9a, diverge and which are displaced from each other. The focusing optics7aare configured to image each of these virtual foci into one of the line foci LF1and LF2within the object. Therefore, with respect to the focusing optics7a, the virtual foci on the one hand and the line foci LF1and LF2on the other hand are located at optically conjugate positions.

As can further be seen fromFIG.4, the beam multiplication system5aincludes adaptation optics24a, which are disposed in the illumination light between the diffractive optical element13aand the beam splitter6a. The adaptation optics24afunction as a beam expander which adapts the cross-sections of each of the beamlets8a,9ato the entrance pupil of the focusing optics7a. Adapting the beam diameters to the entrance pupil of the focusing optics7a, allows generation of line foci LF1and LF2in the object4a, which have a comparatively small width, since the full numerical aperture of the focusing optics7ais used. Thereby, an optimal lateral and axial resolution of the confocal scanning microscope can be obtained.

In the exemplary embodiment, which is shown inFIG.4, the adaptation optics24aare configured as an afocal Keplerian telescope having two relay lenses14aand17a, the principal planes of which being displaced from each other by the sum of their focal lengths. In alternative embodiments, other configurations of the adaptation optics are contemplated, which are not configured as a Keplerian telescope or which are not configured to be afocal.

Since the adaptation optics24aare disposed in the beamlets8a,9aof the illumination light between the diffractive optical element13aand the beam splitter6a, the adaptation optics24aare located out of the path of the detection light. Thereby, it is prevented that the intensity of the detected light on the light receiving surface10aof the light-sensitive detector12ais reduced by the adaptation optics24a. Thereby, an improved low light sensitivity can be obtained. However, it should be noted that for many applications, a sufficient low light sensitivity can be obtained if the adaptation optics24aare traversed by the illumination light as well as by the detection light.

The diffractive optical element13ais configured to impart a phase W (x,y) on the incident beam3of illumination light, wherein (x,y) are the coordinate values in a plane defined by the diffractive optical element13a, which, in the exemplary embodiment, is oriented perpendicular to a beam axis of the beam3aof illumination light.

The plane can be divided into a plurality of zones, wherein to each of the zones, a discrete index j is assigned, which is denoted herein as the zone index.

Points with coordinates (x,y) are in zone j if the following holds true:
jλ≤K(x,y)<(j+1)λ,
where λ is the beam wavelength, and K(x,y) is a function which is denoted herein as the zone function. As an illustrative example, in a conventional grating, the zone function is a linear function of x. Within each zone j, a variable t that takes values 0≤t<1 is defined by:

t=K⁡(x,y)λ-floor⁢⁢(K⁡(x,y)λ),
where floor(x) is defined as the largest integer which is smaller than x. The phase profile W(x,y), which is added to the incoming beam3aof illumination light, is defined by a function ƒ(t), which is denoted herein as the profile function, according to:

W⁡(x,y)=f⁡(t)=f⁡(K⁡(x,y)λ-floor⁢⁢(K⁡(x,y)λ)).
The profile function ƒ(t), therefore, depends on the shape of the grating within each zone. The complex valued transmission function for the diffractive optical element13a, which is defined as

T⁡(x,y)=exp⁡(2⁢π⁢iW⁡(x,y)λ)
can be written as a sum over diffraction orders with index m:
T(x,y)=Σm∞=−∞Cmexp(2πimK(x,y)),
wherein:

Cm=∫01⁢dt⁢⁢exp⁡(-2⁢π⁢i⁢m⁢t)⁢exp(2⁢π⁢⁢if⁡(t)λ).
The diffraction efficiency of order m is ηm=|Cm|2.

It follows from the foregoing that the profile function ƒ(t) can be designed to give a desired distribution of light intensity over the diffraction orders m, and the zone function K(x,y) can be designed to give a desired phase to the different contributing orders m.

In order to obtain the axial and lateral displacement of the line foci LF1and LF2, as described above, the following zone function K(x,y) can be chosen:
K(x,y)=ax+b(x2+y2).

As can be seen from the following equations, a and b are coefficients that determine the lateral and axial displacement between the line foci LF1and LF2. Referring toFIG.4, if F14ais the focal length of relay lens14a, then, the lateral displacement Δx1between the focal lines generated in the region15abetween lenses14aand17aand close to the location of the focal plane of relay lens14ais given by:
Δx1=aF14aλ,
and the axial displacement Δz1between the focal lines is given by:
Δz1=2bF14a2λ.

The lateral magnification between the line foci in the region15aand the foci in the object4ais M15a4a=F17a/F7b, with F17abeing the focal length of relay lens17aand F7abeing the focal length of the focusing optics7a, which may include or may be formed by an objective lens. This leads to the following expressions for the lateral displacement Δx (seeFIG.2) and the axial displacement Δz between the line foci LF1and LF2in the object4a:
Δx=M15a4aΔx1
Δz=nM15a4a2Δz1,
where n is the refractive index in the object4a, which is typically close to the refractive index of water for biological samples. The magnification step from the object4ato the sensor12acan be handled in exactly the same way as described above. The lateral magnification is M4a12a=F18a/F7awith F18abeing the focal length of the tube lens18a, giving a lateral displacement Δx2and an axial displacement Δz2of the line foci on the light receiving portions SP1and SP2on the light receiving surface10aof the light-sensitive detector12a:

Δ⁢x2=M4⁢a⊥2⁢a⁢Δ⁢x⁢⁢Δ⁢z2=M4⁢a⁢1⁢2⁢a2n⁢Δ⁢z.
The angle α2(illustrated for the first exemplary embodiment inFIG.1) formed between the normal vector to the light receiving surface10aand the optical axis along which the detection light is incident on the light receiving surface10amust satisfy the condition:

Δ⁢z2Δ⁢x2=tan⁢⁢α2,
and Δr (shown inFIG.3for the first exemplary embodiment), which is the displacement between adjacent arrays21,22of light-sensitive detector elements20of the light receiving surface10a, must satisfy the following condition:

Δ⁢x2cos⁢⁢α2=p⁢Δ⁢r
with p being an integer.

The zone function K(x,y) can be improved by adding higher order terms which depend on the coordinates x and y in order to compensate for the spherical aberration that occurs when beams of high NA are focused by the focusing optics7ato form the line foci LF1and LF2within the object4a, or in order to compensate spherical aberration that occurs by other causes, such as lens manufacturing errors. Objective lenses with high numerical aperture are known to comprise multiple lens elements, assembled in such a way that errors in the curvature and/or the thickness of the lens elements easily give rise to spherical aberration

In a further alternative embodiment, the diffractive optical element13is configured so that the focusing optics7a, focuses the beamlets8a,9ainto a plurality of line foci within the object4. The inventors have shown that this can, for example, be achieved by adapting the phase profile W (x,y), which has been described above, by adding a phase profile Wast(y) of two-fold rotational symmetry within the diffractive plane so that the total phase profile Wtot(x,y), which is added to the incoming beam3aof illumination light (having plane wavefronts), is expressed as:
Wtot(x, y)=W(x, y)+Wast(y),
which transforms the point foci into line foci within the object. Specifically, the phase profile Wtot(x,y), which has a two-fold rotational symmetry, causes each of the beamlets to be astigmatic. The above polynomial Wtot(x,y) represents a polynomial representation, which is an uniquely determined polynomial representation and which has its origin on an optical axis of the beam multiplication system5aand/or the focusing optics7aand in the diffraction plane of the diffraction optical element13a. Wast(y) is an univariate monomial or an univariate polynomial and is an additive component of Wtot(x,y).

The inventor has further shown that different expressions of the phase profile Wast(y) can be used to generate line foci of different levels of homogeneity with regards to line width and intensity.

By way of example, according to an exemplary embodiment, the phase profile Wast(y) is expressed by the following formula:

Wast⁡(y)=12⁢c(yF⁢⁢NA)2,
with c being a constant so that Wast(y) is an univariate term of degree 2. The value c/NA is a measure for the length of the line focus, since it corresponds substantially to the length of the line focus. F is the object-side focal length of the focusing optics7a(i.e. the focal length for the section of the illumination light, which converges toward the line foci within the object) and NA is the numerical aperture at the object4. The inventor has further shown that using the above phase profile Wast(y), the line intensity of each of the line foci within the object4can be described by:

If⁡(x1,y1)=A(1-(NA⁢⁢y1c)2)⁢sinc(2⁢π⁢1-(NA⁢⁢y1c)2⁢x1⁢NAλ)2,
with λ being the wavelength, A being a constant, and sinc (x) being defined as sinc(x)=sin (x)/x. x1is a coordinate perpendicular to a longitudinal axis of the line focus and y1is a coordinate in a direction along the longitudinal axis of the line focus.

From this expression, it follows that, as seen along the longitudinal axis of the line focus, the peak intensity varies as

(1-(NA⁢⁢y1c)2)
and the line width varies as

1/1-(NA⁢⁢y1c)2.

The inventor has further shown that it is possible to adapt the phase profile Wast(y) of the diffractive optical element13aso that the variation of the intensity and the line width is reduced. By way of example, the inventor has shown that using a phase profile Wast(y), which has the form

Wast⁡(y)=34⁢w⁢⁢NA[(yF·NA)2+1p⁢(yF·NA)4]
(i.e. an univariate polynomial, which consists of terms of degree 2 and 4) results in less variation of peak intensity and line width. The value w is a measure for the length of the line focus, since 2w is substantially equal to the length of the line focus. It is conceivable that Wast(y) includes univariate terms of other degrees than 2 and 4 in order to further reduce intensity variation and/or line width variation of the line foci.

In this equation, the parameterp (which is a measure for a ratio of the coefficient of the term of degree 2 to the term of degree 4) is within a range of between −3 and −10, or in a range of between −4 to −8. The inventor has shown that a particular advantageous configuration is provided ifp has a value of −6.

The inventor has further shown that, additionally or alternatively, less fluctuation in intensity and line width can be obtained, if the ratio (R=C1/C2) of the coefficient of the second-degree term

(C1=3⁢w4⁢F2⁢NA)
to the coefficient of the fourth-degree term

(C2=3⁢w4⁢F4⁢NA3)
has a value, which is between −1 and −11 times the radius of the beam cross-section of the illumination light at a position, where the illumination light traverses the diffractive optical element. The radius may correspond to a half of the FWHM (full width at half maximum). Even lesser fluctuations can be obtained if the ratio is between −3 and −9 times or between −5 and −7 times the radius of the beam cross-section.

Therefore, it has been shown by the inventor that by using a phase profile Wast(y), which includes or which represents or substantially represents a univariate term or a univariate polynomial of a degree equal or greater than 4, a more homogeneous intensity and line width along the longitudinal axis of the line foci can be obtained. This allows achieving an improved, homogeneous resolution, as well as an improved, homogeneous, dynamic range of image intensities along the longitudinal axis. However, it has also been shown that—depending on the application—a sufficient performance of the imaging system can be obtained using a phase profile Wast(y), which represents or substantially represents an univariate term of a degree of 2.

In a further alternative embodiment, in which point foci or line foci are generated within the object, the diffractive optical element13ais configured to have the following zone function:
K(x,y)=ax+b√{square root over (n2−(x2+y2)/F2)},
wherein n is the refractive index of the sample, F is the object-side focal length of the focusing optics7a(i.e. the focal length for the section of the illumination light, which converges toward the foci within the object), and x and y are coordinates in the diffractive plane of the diffractive optical element13a.

It has been shown by the inventor that the above zone function generates a plurality of foci and also provides a correction for the spherical aberration caused by the focusing optics7aso that the focusing optics7acan be configured to have a comparatively high numerical aperture.

Specifically, it has been shown by the inventor that the above zone function allows correction of the spherical aberrations for a focusing optics7a, which is configured to have a numerical aperture at the object, which is greater than or equal to 0.25, or greater than or equal to 0.5. The spherical aberration may be at least partially caused by the focusing optics7a, in particular by an objective lens of the focusing opics. In other words, the diffractive optical element13ais configured to at least partially compensate a spherical aberration of the focusing optics7a, wherein the spherical aberration affects a shape and/or a position of at least a portion of the foci.

Further, the spherical aberration affecting the shape and/or the line width of two or more foci may be different for each of the foci. This makes it difficult to compensate the spherical aberration for all foci using a single corrector, which is configured to correct one value of spherical aberration. However, the inventor has shown that using one or more diffractive optical elements, in particular by using the above zone function, allows compensation of different amounts of spherical aberrations for each of the foci.

Furthermore, the inventor has also shown that the above zone function for correcting the spherical aberration can be combined with a phase profile Wast(y) (in particular an univariate phase function) of two-fold rotationally symmetry. Specifically, the phase profile Wast(y) can be added to the phase profile W (x,y):

W⁡(x,y)=f⁡(t)=f(K⁡(x,y)λ-floor⁢⁢(K⁡(x,y)λ)),
with K(x,y) being the zone function K(x,y)=ax+b√{square root over (n2−(x230y2)/F2)} to get a Wtot(x,y), which generates line foci within the object. Wast(y) may be an univariate term or an univariate polynomial of degree 2 or higher or of degree 4 or higher.

The present disclosure can also be applied to conventional image sensors, comprising a 2D array of adjacent pixels instead of a set of spatially separate line sensor elements, but still tilted at an angle ∝2. In that case the second constraint on the lateral displacement Δr being matched to the distance between adjacent arrays21,22(shown inFIG.3) of light-sensitive detector elements20can be dropped, providing one more degree of freedom in the design of the diffractive optical element. This allows providing a cost-efficient design for a scanning confocal microscope.

As is explained in the following, a profile function ƒ(t) can be selected that distributes the power over a limited set of orders m=m1, . . . , m2, where m1and m2are positive or negative integers, such that substantially all the power of the beam3aof illumination light which is incident on the diffractive optical element13ais concentrated into this set of diffraction orders, preferably such that each of the diffraction orders receive a substantially equal amount of power. This will give focal lines of approximately equal light intensity.

Specifically, the inventors have found that a suitable profile function can be determined by parameterizing the profile function ƒ(t) and finding the optimum parameters using a numerical search algorithm. For a profile function providing a symmetric distribution of power over the orders, i.e. ηm=η−m, it follows from the expression for Cmthat ƒ(t)=ƒ(−t). A suitable parameterization for ƒ(t) is therefore the following finite cosine series:

f⁡(t)=∑j=1K⁢cj⁢cos⁡(2⁢π⁢⁢jt).
An optimum distribution of the power over the 2N+1 diffraction orders is obtained when ηi=ni′, where

ηi′={12⁢N+1,i≤N0⁢,⁢otherwise.
The cost function Z is defined as

Z⁡(C0,…⁢,CK)=∑m=-MK⁢(ηm-ηm′)2,
where an integer M>N is used. Now, the minimum of Z can be found for parameters c0, . . . , cKusing a generic numerical search algorithm. As an initial condition, all ciare set to zero, except c0=1.

FIG.5is a histogram of the power of illumination light (y-axis) in arbitrary units versus diffraction order (x-axis) for a profile function ƒ(t) which was determined using the above-described method. As can be seen fromFIG.5, an optimal power distribution can be obtained for 11 lines, providing a suitable solution using the parameters N=5, K=14, M=15. The resulting profile function ƒ(t) concentrates 87% of the power of the beam3aof illumination light which is incident on the diffractive optical element13a(shown inFIG.4) into the 11 diffraction orders. For each of these 11 diffraction orders, the power of the respective diffraction order varies from an average power value of these 11 diffraction orders by less than 5%.

FIG.6is a schematic illustration of a microscopic imaging system1baccording to a third exemplary embodiment. Components of the imaging system1b, which are similar or corresponding to components of the first and second exemplary embodiments illustrated inFIGS.1to5, bear the same numerals but are succeeded by the suffix letter “b” to avoid confusion.

The beam multiplication system5bof the microscopic imaging system1bincludes an array19bof refractive optical units25b,26b, each of which receiving a separate portion of the beam3bof illumination light provided by the source2b. Each of the refractive optical units25b,26bis configured to focus the respective portion of the beam3bof illumination light, thereby forming one of the beamlets8b,9b. Each of the refractive optical units25b,26bis configured as a cylindrical lens. For each of the refractive optical units25b,26b, an optical axis of the respective refractive optical unit25b,26bis inclined relative to a beam axis of the beam3bof illumination light which is incident on the array19b.

Each of the beamlets8b,9b, which exits the respective refractive optical unit25b,26bis focused by the respective refractive optical unit25b,26bto form a line focus in a region between the array19band the adaptation optics24bso that the line foci are displaced relative from each from each other by an axial distance Δz3and a lateral distance Δx3. Since the imaging system1bdoes not rely on a refractive optical element, it is possible use a halogen lamp as light source2b. The foci in the region between the array19bof refractive optical units25band26band the adaptation optics24bare located at positions which are optically conjugate to the positions of the line foci LF1and LF2within the object4b.

Although the microscopic imaging systems described in the exemplary embodiments above are configured as a scanning confocal microscopy systems, it is to be understood that the present invention is not limited to such systems. Specifically, it is conceivable that the teaching of the present disclosure is used for microscopy systems which provide no scanning functionality and/or do not rely on confocal detection. By way of example, the microscopic imaging system may be configured to inspect the reflectivity and/or fluorescent light emission at two-selected locations within the object which are located at different depths so that no scanning functionality is required. Further, if there are low requirements for the axial resolution, it is conceivable to detect the detection light without using confocal filtering of the detection light.

The above embodiments as described are only illustrative, and not intended to limit the technique approaches of the present invention. Although the present invention is described in details referring to the preferable embodiments, those skilled in the art will understand that the technique approaches of the present invention can be modified or equally displaced without departing from the protective scope of the claims of the present invention. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. Any reference signs in the claims should not be construed as limiting the scope.