Enhanced sample imaging using structured illumination microscopy

Abstract

Methods and apparatus for applying structured illumination microscopy (SIM) to scanning microscopes such as confocal laser scanning microscopes or sample scanning microscopes to improve spatial resolution are disclosed. Specific aspects of the disclosure relate to important advances in the ability to (i) increase the light flux to the sample, thereby increasing the signal-to-noise ratio and / or reducing the exposure time, and (ii) reduce the number of raw images to be processed, thereby reducing the image acquisition time. Both effects significantly improve the overall performance, thereby benefiting users of scanning microscopes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application Serial No. 62 / 827,921, filed on April 2, 2019, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The present invention relates to imaging a sample using a scanning microscope (eg, confocal laser scanning or sample scanning microscope) and structured illumination for improved resolution. Background Art

[0004] Conventional optical microscopes have a spatial resolution that is limited by diffraction. When the back aperture of a microscope objective is completely filled with collimated light, the image in the focus or sample plane is an Airy disk with a radius r according to airy (in nanometers nm):

[0005]

[0006] where λ (in nm) is the illumination wavelength and NA (dimensionless) is the numerical aperture of the microscope objective and provides a measure of its resolving power. Related to this quantity is the resolution of the microscope, which is usually defined as the minimum distance between two objects that allows them to be imaged separately, rather than as a single object. If a sample is imaged by scanning an Airy disk beam across it, the resolution of the resulting image is given by:

[0007]

[0008] where r is the resolution (in nm) in this case, λ and NA are as defined above, n is the refractive index of the ambient medium, and θ is the half-angle of the largest cone of light that can enter or leave the lens. It is obvious from this equation that the spatial resolution can be improved (i.e., the value of "r" can be reduced) by reducing the wavelength of the illumination light or increasing the numerical aperture of the objective. However, this only applies if the linearly polarized beam fills the entire back aperture of the microscope objective. By filling only certain designed components, an interference pattern of the sample surface can be generated and scanned instead of an Airy disk. Importantly, the fringe spacing of the interference pattern is relatively small compared to the diameter of the Airy disk. Such designed illumination patterns and the resulting detection and reconstruction algorithms are known in the art as "structured illumination," a technique that can improve microscope resolution by up to two times.

[0009] Structured illumination microscopy (SIM) systems are commercially available on a variety of fluorescence microscopes. However, these SIM systems use "widefield" illumination, which refers to illuminating a large sample area by sending a collimated light source through a focusing lens before entering the objective. The normal goal of fluorescence imaging is not to collect the entire spectrum, but to simply filter the emitted light at the wavelength of interest and then direct it into a camera. Although widefield microscopy is suitable for a variety of fluorescence imaging applications, there are cases where confocal microscopy is superior. Confocal microscopy utilizes a pinhole to reject out-of-focus light, thereby greatly improving imaging through thick samples. Because the confocal image is constructed pixel by pixel, rather than over a large area (as in widefield imaging), confocal microscopy is ideal for spectral imaging, as the emitted or scattered light can be sent to a spectrometer downstream of the pinhole. Specific uses of confocal microscopy include fluorescence sectioning through thick, inhomogeneous samples, and hyperspectral imaging, such as in the case of imaging Raman microscopy.

[0010] Because confocal microscopes cannot illuminate the entire sample at once as in the case of widefield microscopes, existing SIM systems and their reconstruction algorithms are not directly "drop-in" compatible with confocal microscopes. However, a specific application of SIM to confocal laser scanning microscopy (CLSM) is described in Gao et al. (Optics Letters (2016) Vol. 41 No. 6: 1193-96). According to the publication, the sample is scanned point by point multiple times using a fringe pattern generated by the concentration of light through two "circular pupils" of the microscope objective. More specifically, these fringe patterns are characterized by their directional index m, which refers to the angle of the line connecting the pupils, and the phase shift n of one of the two pupils relative to the other. Using 4 different angles and 4 different relative phases requires the collection of 16 raw images to reconstruct the enhanced image. The existing technology would benefit from improvements in the application of SIM in scanning microscopes such as CLSM, especially in terms of performance efficiency. Summary of the invention

[0011] Various aspects of the present invention are associated with methods and apparatus whereby structured illumination microscopy (SIM) is applied to scanning microscopes, such as confocal laser scanning microscopes or confocal sample scanning microscopes, to improve spatial resolution. Specific aspects relate to discovering important advances in the ability to: (i) increase the light flux to the sample, thereby increasing the signal-to-noise ratio and / or reducing the exposure time, and (ii) reduce the number of raw images to be processed, thereby reducing image acquisition time. Both of these effects significantly improve overall performance, thereby benefiting users of scanning microscopes.

[0012] Depending on the specific method used to image the sample, where SIM is applied to a confocal microscope, interference fringes or a pattern of focused fringes are scanned over the sample area, or more specifically, at discrete points of the sample, such as generating interference fringes or a pattern of focused fringes at regular intervals over the area. The pattern of focused fringes is generated by sending a laser beam to both sides of a microscope objective at a given angle and a phase difference between the two beams. For example, a spatial light modulator can be used to change the angle and phase, and the spatial light modulator is, for example, a known programmable device that can modulate the wavefront of a light beam pixel by pixel. A common type for phase modulation is a liquid crystal on silicon spatial light modulator (LCOS-SLM), which operates in a manner similar to an LCD display, i.e., the liquid crystal changes its refractive index under an applied electric field, thereby causing a phase delay when light interacts with the crystal. The spatial light modulator can use a computer as an auxiliary display so that the color pattern is converted into a phase trace. For example, in the case of an 8-bit color pattern, 128 and 256 can represent phase delays of π and 2π, respectively. If the light is directed only toward the edge of the aperture at the back of the microscope objective, the two beams will interfere constructively and destructively with each other at the sample, producing a sinusoidal pattern with a period of about half the diameter of the Airy disk described above. This forms the basis for the additional resolution in images obtained using SIM compared to conventional confocal microscopy.

[0013] However, the raw image obtained from a single given angle and phase is not sufficient to reconstruct a fully resolved image. Instead, to build an image, a laser generating a beam is scanned across the sample and imaged through the confocal pinhole multiple times, for example at each of a series or predetermined set of discrete angles and phases. That is, the illumination patterns of the two beams corresponding to the illumination area on the back (rear) aperture of the microscope objective are projected at these angles and phases. At a given angle, such as 0 radians, two or more phase images can be collected, for example using a spatial light modulator to delay the phase of one of the beams relative to the other. One image with a phase shift of 0 and other images with the same angle but with non-zero phase shifts such as π / 2, π, 3π / 2 radians relative to each other and images of other focused fringe patterns corresponding to other combinations of angles and phases can be used to synthesize (reconstruct) an entire scanned confocal image of a particular sample area of ​​interest, pixel by pixel. In this way, in a scanning microscope such as a confocal microscope, the use of SIM can advantageously improve the spatial resolution (reduce the value of "r" in the above equation) by a factor greater than about 1.5, such as a factor from about 1.8 to about 2.0.

[0014] A specific aspect of the invention relates to the discovery of parameters that allow a significant increase in light throughput in the entire system by using the illumination pattern of two light beams, such that the corresponding illumination area of ​​the microscope objective is non-circular. In the case of a circular area, two light beams with circular profiles generate a sinusoidal pattern on the sample along the displacement axis of the two circles, which displacement axis refers to a line through the centers of the two circles. The key factor in generating the best sinusoidal curve on the sample is the length or distance that the two circles are displaced along their axes. Importantly, the presence (or absence) of light above or below the circles does not affect the quality of the sinusoidal curve generated on the sample, but the displacement distance is still maintained. Therefore, the back aperture can be further illuminated in the area outside the circle, rather than just illuminating with two circles, to increase the amount of light introduced without sacrificing image quality.

[0015] For example, an "illumination enhancement region" may be formed by a slit-shaped illumination region, wherein the maximum slit width is the same as the diameter of a circular "pupil" representing the largest inscribed circle within the slit. Additional light introduced through the microscope objective will also interfere at selected angles and phases to form a sinusoidal focus fringe pattern. In a specific embodiment, an illumination region in the form of a slit or a circular segment of a much larger circle corresponding to the rear circular aperture of the microscope objective provides an illumination enhancement region outside (outside) the largest inscribed circle, which is used to increase the light flux, which can advantageously increase the signal-to-noise ratio and / or reduce the exposure time. This is very important for the overall performance of a confocal microscope or other scanning microscope, since traditionally the light flux is very limited because most of the light generated by the laser is not directly incident on the objective. Advantageously, a change to using one or more (and preferably two) non-circular illumination regions is easily achieved. The corresponding significant (e.g., four-fold) increase in light flux allows image collection with an equal signal-to-noise ratio but with a lower camera exposure time compared to using a reference or baseline circular region represented by the largest inscribed circle within the illumination region. As a result, the overall image acquisition time is reduced, providing a number of benefits to users in the field of scanning microscopy. According to other embodiments, non-circular illumination areas can be configured to provide the same total illumination as reference or baseline circular areas, but with a smaller maximum inscribed circle than these reference or baseline circular areas. This allows the spacing of the areas to be increased along the displacement axis, thereby having the effect of improving image quality.

[0016] Other aspects relate to the discovery that in applying SIM to confocal laser scanning microscopy or sample scanning microscopy, there is no mathematical restriction that requires collecting images with three phases per angle, and in fact, illumination with no more than two phases is sufficient, as shown by 1D and 2D physical optics propagation simulations. The direct impact of this is that the total number of raw images to be collected is reduced, which means that the overall image acquisition time is also reduced. For example, compared to techniques using four phases per angle (e.g., 16 images are collected in total, with four separate angles) or even only three phases per angle (e.g., 9 images are collected in total, with three separate angles), the image acquisition time can be reduced by about 50% or about 33%, respectively. Such improvements in acquisition time are significant not only because time is valuable to the user, but also because the reduction in image acquisition time reduces the system's sensitivity to thermal drift or vibration, which in turn can cause significant aberrations in the image. Image quality is thereby improved. Collecting fewer raw images also reduces the software overhead requirements for their management, tracking and storage. Thus, the benefits of SIM in terms of improved spatial resolution, for example by a factor of 1.8 or more through the use of high numerical aperture (NA) objectives, can be achieved at significantly reduced cost compared to using known methods and apparatus.

[0017] These and other embodiments relate to methods and apparatus for utilizing SIM to improve image resolution, for example when performing hyperspectral imaging, such as in imaging Raman spectroscopy, or other label-based imaging, such as in fluorescence microscopy, as will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A and 1B is the input beam profile, or the illuminated area of ​​the microscope objective back aperture, which is in the form of a circle ( Figure 1A ) and slits, or circular segments with larger diameters ( Figure 1B ).

[0019] Figure 1C The diagram shows the rotation axis with different angles. Figure 1B Input beam profile shown.

[0020] Figure 2 Schematic diagram of a confocal scanning microscope utilizing SIM (confocal SIM) to image a sample with improved resolution.

[0021] Figure 3Plotted are simulated point spread functions for confocal SIM, where illumination at 532 nm wavelength is focused into a 25 μm pinhole through a 100x, 0.7 NA objective lens and a 75 mm focal length lens. The six simulated images are based on using three different angles of the beams (0, π / 3, 2π / 3), and for each angle, the illumination patterns of the two beams are phase-shifted relative to each other by 0 and π.

[0022] Figure 4 Shown are simulated sample images using conventional CLSM, and the same simulated images but using confocal SIM to improve resolution. Figure 3 The six angle and phase combinations shown reconstruct the super-resolved image to obtain an improvement over the illumination pattern swept across the sample.

[0023] The accompanying drawings should be understood to represent embodiments of the invention and / or the principles involved, wherein the same reference numerals are used in the drawings to represent the same or similar elements. As will be clear to those skilled in the art having knowledge of the present disclosure, other methods and devices using SIM will have associated steps and components determined in part according to the specific imaging application. DETAILED DESCRIPTION

[0024] Representative embodiments of the present invention relate to methods for imaging a sample by illuminating multiple points of the sample, such as an area of ​​interest, with multiple focused fringe patterns (or interference fringes), which are generated by light beams illuminating at various angles around a rotation axis and shifted relative to each other at various phases. Therefore, these multiple focused fringe patterns can be designated as "first focused fringe pattern", "second focused fringe pattern", "third focused fringe pattern", etc., where the terms "first", "second", "third", etc. are only used to indicate that these focused fringe patterns are associated with the characteristics of the light beams that interfere to generate them, namely, their respective characteristic rotation axis angles and phase shifts (e.g., angles corresponding to the "first rotation axis", "second rotation axis", "third rotation axis", etc., or offsets of the "first phase", "second phase", "third phase", etc.). These terms are not meant to be interpreted as a specific order in which the focused fringe patterns are generated. These terms should also not be interpreted as necessarily requiring different angle and / or phase values. In fact, according to a preferred embodiment, some of the focused fringe patterns used to illuminate a given sample at multiple points are generated by beams having the same rotation axis angle (having a rotation axis substantially coincident with the rotation axis) as the beams used to generate other focused fringe patterns, but other focused fringe patterns are generated by beams offset by different phases. As described in more detail herein, the rotation axis angle refers to the orientation of separate illumination areas on the back aperture of the microscope that are illuminated by these beams. Similarly, some focused fringe patterns can be generated by beams offset by the same phase as the beams used to generate other focused fringe patterns, but other focused fringe patterns are generated by beams having different rotation axis angles. Therefore, for any two focused fringe patterns used to illuminate a sample, they can differ only in their characteristic rotation axis angles, characteristic phase shifts, or characteristic rotation axis angles and phase shifts.

[0025] The terms "light" and "light beam", particularly in the case of light emitted through and focused by a microscope objective and providing an illumination area at the rear aperture of the objective, refer to electromagnetic radiation of any wavelength, but preferably having a wavelength λ ranging from the ultraviolet (UV) portion of the electromagnetic spectrum to the mid-infrared portion of the electromagnetic spectrum, for example in the range of about 200 nm to about 11 μm. Typically, visible light having a wavelength of, for example, about 380 nm to about 750 nm will be used. Unless otherwise indicated, all angles are expressed in radians, where 2π radians = 360°.

[0026] The method described herein utilizes structured illumination microscopy (SIM), such as in the case of confocal SIM, when specifically applied to confocal laser scanning microscopy (CLSM). According to such methods, multiple points of the sample of interest are not illuminated by a laser beam occupying the entire back aperture of the microscope objective as in the case of a conventional confocal microscope, but by two or more, but preferably only two beams (e.g., collimated laser beams) emitted by the microscope objective and focused by the microscope objective. Irradiation of a sinusoidal focused fringe pattern generated by the interference. Specifically, these beams only fill a portion of the back aperture of the objective, such as the designed illumination area. The light beams are focused by the microscope objective and emitted to these areas on the sample, for example, at opposite ends of the back aperture diameter. A given focused fringe pattern can be scanned or swept over the sample area and used to obtain illumination data, for example, at discrete sample intervals corresponding to a given sample frequency, such as several samples per micrometer. The illumination data can be, for example, an intensity value obtained from a single element detector or a spectrum obtained from a spectrometer.

[0027] For example, Figure 1A and 1B Two illustrations of corresponding input beam profiles 10, 20 are provided, with corresponding, respective illumination areas 12, 22 of the beams projected onto the back aperture 14 of the microscope objective. The left figure shows these beams as white areas (in Figure 1A In the case of Figure 1B In the right image, these dark backgrounds are removed to show more detail. Figure 1A and 1B In the embodiment described above, the light beams are emitted from opposite ends of the diameter of the rear aperture 14 of the microscope, which diameter coincides with the displacement axis 16, along which the light beams are spaced or separated. Thus, the light beams illuminate only "illumination areas" the sum of which is less than the aperture area. In general, image quality can be improved by configuring these illumination areas 12, 22 so that they are positioned near the edges of the rear aperture 14, or coincide with at least a portion of these edges, to obtain the maximum possible separation distance. In particular, the quality of the sinusoids generated on the specimen by the interference of the light beams providing these illumination areas 12, 22 is a function of the distance between them, with the greater the distance, the higher the quality.

[0028] exist Figure 1A In the embodiment of FIG. 4 , these illumination areas 12 are in the form of a circle or pupil and are spaced apart along a displacement axis 16 , which also includes a center 18 of the rear aperture 14 . Figure 1B Different input beam profiles 20 are depicted, where the separated illumination area 22 is non-circular (compared to Figure 1AThese areas include a maximum inscribed circle 25, i.e. corresponding to the largest circle that can be inscribed within these respective illumination areas 22, and in the illustrated embodiment, is equal in area to Figure 1A 12. Thus, the maximum inscribed circle 25 can have a corresponding center 27 that is co-linear with the center 18 of the back aperture 14 along the displacement axis 16. In the case of a maximum inscribed circle that does not have such a co-linear center, the term "maximum inscribed circle" as it applies to the corresponding illumination area may, but does not necessarily, refer to a maximum inscribed circle that has such a co-linear center (i.e., co-linear with the center 18 of the back aperture 14) in some embodiments.

[0029] As from Figure 1B It is obvious from the non-circular illumination areas 22 shown that these illumination areas also include illumination enhancement areas 29, which refer to any illumination areas outside the maximum inscribed circle 25, which are along the direction of the displacement axis 16, that is, in Figure 1B No closer in the horizontal direction shown than any portion of the maximum inscribed circle 25. Light in such an illumination enhancement region 29 that meets these requirements is advantageously used to increase light throughput without sacrificing image quality (resolution). In contrast, light between two chords 23 of a circular segment or slit-shaped illumination region 22, while increasing light throughput, will still reduce resolution by shifting the designed illumination pattern in the direction of a conventional confocal microscope in which the entire back aperture is illuminated. In view of this description, it will be appreciated that an illumination region 22 that is a circular segment (i.e., the region defined by the edge of the circle and the chords) provides a maximum illumination enhancement region for any given diameter of the maximum inscribed circle 25, which in turn defines a given displacement distance and therefore a given degree of resolution. According to Figure 1B In the preferred embodiment, the chords 23 of the slit or circular segment shaped illumination area 22 are parallel and separated by a displacement distance d, in such a way that the displacement distance d affects the light flux and resolution in the manner described above. Also in this preferred embodiment, the slit shaped illumination area 22 coincides with the opposite side portions or arcs 21 of the circular back aperture 14 of the microscope objective, so that these areas define a circular segment that includes (i) an arc 21 that substantially coincides with the arc of the circular back aperture 14 or these opposite side portions 21, and (ii) a chord 23 that also defines a chord of the circular back aperture 14. Furthermore, in this embodiment it can be seen that the diameter 28 of the largest inscribed circle 25 corresponds to the slit width at its widest point, which in this case is along the displacement axis 16.

[0030] In general, the use of any non-circular illumination region that provides an illumination enhancement region can improve the trade-offs involved in applying SIM to confocal microscopy. This is the trade-off of (i) increasing the displacement distance, improving the fineness of the generated fringe pattern, and thus improving the resolution of the generated image, at the expense of (ii) reducing the light flux, reducing the signal-to-noise ratio and / or increasing the exposure time. That is, the illumination enhancement region advantageously compensates for (ii) without affecting (i), or alternatively allows for improvement in (i) without affecting (ii). Advantageously, the illumination enhancement region can account for a significant proportion of the total illumination region and thereby increase the baseline amount of light flux from the largest inscribed circle by, for example, at least about 1.5 (e.g., from about 1.5 to about 6.0) or at least about 2.0 (e.g., from about 2.0 to about 5.0) times, which multiple represents the ratio of the total illumination region to the area of ​​the largest inscribed circle. For example, in the case of a slit-shaped illumination region, this ratio increases as the ratio of the diameter of the largest inscribed circle (in this case, the slit width through the displacement axis) to the diameter of the back aperture decreases, i.e., as the slit becomes smaller relative to the back aperture. For example, a slit-shaped illumination area with a slit width of 3 / 8, 1 / 4, or 1 / 8 of the back aperture width provides light from an illumination enhancement area representing 59%, 68%, or 78% of the total illumination area, respectively, resulting in an increase factor of 2.4, 3.1, or 4.5 relative to the baseline amount, respectively. In the specific case of such circular segments or slit-shaped illumination areas, the ratio of the slit width to the back aperture diameter is typically about 0.02 to about 0.4, typically about 0.05 to about 0.3, and often about 0.1 to about 0.25.

[0031] It will be appreciated from this disclosure that many possible non-circular illumination areas may advantageously allow for the luminous flux from the illumination enhancement areas described herein. For example, a lens shape (soccer ball cross-section), a polygon, or a rounded polygon may be used. In some cases, such as in the case of a lens-shaped illumination area, all illumination areas outside of its largest inscribed circle may meet the characteristics of an "illumination enhancement area" as described above. In other cases, the shape of a non-circular illumination area may provide an illumination area outside of its largest inscribed circle, which would not meet the characteristics of an "illumination enhancement area" as defined above. Regardless of the specific shape, preferably (i) at least about 85%, at least about 95%, or all of the illumination area outside the maximum inscribed circle is "illumination enhancement area", (ii) the illumination enhancement area occupies at least about 50% (e.g., about 50% to about 95%), at least about 75% (e.g., about 50% to about 90%), or at least about 80% (e.g., about 80% to about 90%) of the entire illumination area, (iii) the maximum inscribed circle has a center that is substantially coincident with the displacement axis and is therefore substantially collinear with the center of the back aperture, and / or (iv) the entire illumination area occupies less than about 25% (e.g., about 1% to about 25%) or less than about 15% (e.g., about 2% to about 15%) of the area of ​​the back aperture.

[0032] like Figure 1B As shown, Figure 1C Likewise depicted is a slit-shaped illumination area 22 spaced along a displacement axis 16 coinciding with the diameter of the back aperture 14 of the microscope objective. Figure 1C A rotation axis 32 perpendicular to the displacement axis 16 is further illustrated. According to this embodiment, the rotation axis 32 extends between the separated illumination areas 22 and does not intersect, and coincides with the symmetry axis, about which the illumination areas 22 are mirror images of each other. As shown, representative angles of the rotation axis 32 are 0 (0°), π / 3 (60°), and 2π / 3 (120°), and the interference light beams emitted at these different angles produce different focused fringe patterns, i.e., generated from different directions. By scanning or sweeping across the sample surface having a focused fringe pattern, in which the characteristic angles of the rotation axis are different, this allows the image resolution to be enhanced along different directions. Therefore, as Figure 1C As shown, at different angles of rotation axis of 0, π / 3 and 2π / 3, image resolution enhancement in the entire space can be achieved. Therefore, in some embodiments, a sample can be scanned or swept with multiple focused fringe patterns, thereby illuminating points of the sample for the first, second and third time with focused fringe patterns having different first, second and third rotation axes (e.g., 0, π / 3 and 2π / 3). However, for imaging applications in which resolution enhancement along a given direction is not required, it can be considered sufficient to illuminate the sample with a focused fringe pattern having two different rotation axes or even only one rotation axis. Although in Figure 1C , the vertical direction specifically corresponds to an angle of 0 for the rotation axis 32, but it is understood that this is an arbitrary direction and the primary consideration is the ability to enhance image resolution along a desired direction or a combination of different directions using a combination of different focused fringe patterns in which the separated illumination areas have different rotation axes.

[0033] Figure 2A confocal scanning microscope (e.g., a confocal laser scanning microscope) 200 and its operation for imaging a sample with improved resolution using structured illumination microscopy (SIM) is depicted. A laser 201 provides a horizontally polarized and collimated beam 207, for example, with a beam diameter from about 2 mm to about 20 mm, such as from about 5 mm to about 15 mm, via a polarization-maintaining fiber 203 and a fiber collimator 205. The beam is directed to a spatial light modulator (SLM) 209, which can be an electrically programmable device that modulates light according to a fixed spatial (e.g., pixel) pattern. Specifically, the programming information of the address of each pixel changes its optical properties, thereby providing appropriate modulation of the input or readout light. In this case, the SLM 209 is used to modify the phase of the light, particularly by displaying a phase grating 211 that causes diffraction. The combined SLM 209 and phase grating 211 projects a light pattern according to the desired illumination area and rotation axis direction as described above after the modulated light beams 213', 213" leave the SLM, through additional equipment and onto the back aperture 14 of the microscope objective 223. Such additional equipment may include (i) a beam compressor 215, such as a variable beam compressor, which is configured to reduce the beam spacing to within the diameter of the back aperture 14; (ii) an edge filter 217 for distinguishing between excitation wavelengths and emission wavelengths, (iii) a laser scanning module 219 with a galvanometer for steering and moving the light beam, and (iii) a dichroic mirror 221 with appropriate reflection / transmission properties. The objective 223 can use air with a refractive index of 1 as the ambient medium. Otherwise, the objective 223 can operate with a medium with a higher refractive index (e.g., from about 1.2 to about 1.6), such as oil in the case of an oil immersion objective, thereby increasing the resolution capability of the microscope.

[0034] SLM 209 may display the corresponding Figure 1B2-D pattern (image) of the non-circular illumination area 22 shown. In some embodiments, blurring can be used to reduce diffraction occurring at the edges of the displayed pattern (e.g., the edges of the circular segment pattern) or to "soften" these edges. For example, blurring can be achieved by performing a convolution of an image with a Gaussian kernel of a certain standard deviation, where a larger standard deviation results in a larger blur of the resulting image in the direction. Blurring of the image before it is displayed on the SLM 209 or other phase modulation device can be achieved by applying a software algorithm called a Gaussian blur filter 208. The SLM 209 can be, for example, a phase-only SLM or an amplitude-phase SLM combined in other ways, with the additional ability to modulate amplitude. The phase-only SLM displays a phase grating that causes diffraction of the emitted light. However, by selecting locations where the phase grating pattern is displayed and not displayed, amplitude modulation can be achieved. For example, if a slit-shaped pattern needs to be generated, the SLM can be configured to display a grating pattern in that shape, while not displaying the grating elsewhere. In this case, the light diffracted from the grating will have an amplitude distribution in the shape of a slit, and the 0th order diffracted beam will have a negative image of the slit. Since the SLM is also able to modulate the phase on top of the grating pattern, a phase delay (e.g., a phase delay of π) can be added to one of the two slits.

[0035] Specifically, the SLM 209 can be used to (eg, program) modulate the phase shift of a light beam 213' projected onto and emitted through the objective lens 223 relative to another light beam 213". The modulated light beams 213', 213" are used to provide an illumination area, such as Figures 1A to 1C As described with respect to these figures, and with further reference to Figure 2, the light beams 213', 213" are focused by the objective lens 223 and interfere with the sample 225 to generate focused fringe patterns with different rotation axis angles and / or phase shifts between the light beams, as described above. Each focused fringe pattern is used to scan multiple points of the sample 225 and collect illumination data corresponding to the original image. This can be done, for example, using a detector 250 to detect the emitted light downstream of the focusing lens 229 and the confocal pinhole 231. The detector 250 can be a single element detector that provides intensity values, so that the image data can be collected in the form of a 2-D array. Otherwise, the detector 250 can be a multi-element detector, such as a spectrometer that provides spectral data, so that the image data can be collected in the form of a 3-D data cube, where the third dimension is the spectral dimension. The image reconstruction algorithm is the same in each case, although in the case of spectral data It usually takes longer. For Raman spectroscopy imaging, the detector 250 can be a commercially available spectrometer, such as a DXR2 spectrometer. The confocal microscope 200 can be operated as a laser scanning device, according to which the sample is maintained in a fixed position and a given image is established pixel by pixel by scanning a focused fringe pattern for the image on the sample, or its associated illumination data is obtained. Alternatively, the confocal microscope 200 can be operated as a sample scanning device to obtain the pixel information, according to which the laser is maintained in a fixed position and the sample is moved, for example in two or possibly three dimensions using a motorized stage 227 (e.g., an XYZ motorized stage). The processor 260 (e.g., within a personal computer PC) can be used to coordinate the operation of the Gaussian blur filter 208, the SLM 209, the laser scanning module 219, the motorized stage 227 and / or the detector 250, and can be operably linked thereto (e.g., electrically or wirelessly), and can contain an instruction program for collecting and processing illumination data and for executing an image reconstruction algorithm.

[0036] By using different focused fringe patterns with different rotation axis angles of the beams and / or different phase shifts between the two beams, illumination data for multiple "raw" images can be obtained, where each raw image corresponds to each focused fringe pattern used to illuminate (scan or sweep) the sample at multiple points. The raw image data can be used to reconstruct a super-resolution composite image with higher resolution than that obtained using a conventional confocal microscope. Therefore, a representative method for imaging a sample can include illuminating multiple points of the sample with a first focused fringe pattern and at least one different focused fringe pattern, such as illuminating an area of ​​interest on the sample, wherein each of these focused fringe patterns is generated at each of the multiple points by interference of a beam (e.g., from a collimated laser source). These beams can be emitted through a microscope objective, such as at the back or opposite ends of the diameter of the rear aperture. The beam irradiates less than the entire aperture to provide "structured illumination". For the first focused fringe pattern, the beam is directed to a separate illumination area of ​​the microscope objective, which can be regarded as a "first separated illumination area" for generating the pattern. The first separated illumination area can have a rotation axis as described above, which can be considered as a "first rotation axis", and the light beams can be phase-shifted relative to each other by a "first phase". As described above, the corresponding modifiers "second", "third", "fourth", "fifth" and "sixth" can be used as a convenient way to refer to the separated illumination areas, rotation axes and phases of the corresponding "second", "third", "fourth", "fifth" and "sixth" focused fringe patterns, and these modifiers do not require any specific order for sample imaging, and these modifiers do not require different separated illumination areas, rotation axes and phases. Possible values ​​for the rotation axis angle and phase shift include 0. Any of these higher-order modifiers does not necessarily require all lower-order modifiers. Thus, for example, a sample can be imaged with the "first", "second", "fifth" and "sixth" focused fringe patterns described herein without having to be imaged with the "third" and "fourth" focused fringe patterns described herein.

[0037] In addition to illuminating a plurality of points of the sample with a first focus fringe pattern, a representative method further comprises illuminating the points with at least one different focus fringe pattern, wherein the light beams (and thus the light projected onto the back aperture at the separated illumination regions) are phase-shifted differently relative to each other relative to the first phase. For the at least one different focus fringe pattern having a different phase shift relative to the first focus fringe pattern, the rotation axis angle may be the same as or different from the rotation axis angle of the first focus fringe pattern. That is, for the at least one different focus fringe pattern, either (i) the same separated illumination region of the microscope objective is used, which has the same rotation axis that is not angled relative to the first rotation axis (i.e., substantially coincident), or (ii) different separated illumination regions of the microscope objective are used, which have another rotation axis that is angled relative to the first rotation axis, thereby resulting in different directions in which the light beams are focused on the sample. According to a specific embodiment (belonging to the above case (i)), the at least one different focus fringe pattern may be a second focus fringe pattern having a different phase shift relative to the first focus fringe pattern. According to other specific embodiments, the at least one different focus fringe pattern may be two or more, such as two, three, four or five different focus fringe patterns. Therefore, the sample is illuminated using a total of at least two different focus fringe patterns (a first focus fringe pattern and at least one different focus fringe pattern), such as a total of three, four, five or six different focus fringe patterns, which have different characteristic rotation axis angles (for a given focus fringe pattern, the angle of the rotation axis of the separated illumination area) or their characteristic phase shifts (for a given focus fringe pattern, the phase of the light beam offset projected onto the separated illumination area), or both.

[0038] Performing this illumination (scan or sweep) multiple times allows the acquisition or acquisition of corresponding illumination data for each of a plurality of raw images, each of the plurality of raw images corresponding to each of a plurality of focused fringe patterns, namely a first focused fringe pattern and at least one different focused fringe pattern. As described above, the illumination data may be obtained, for example, from a single-element detector (for collecting data in the form of a 2-D array) or a spectrometer (for collecting data in the form of a 3-D data cube). Regardless of the specific detector, a super-resolved synthetic image may then be reconstructed from the illumination data for each of the plurality of raw images according to the following image reconstruction algorithm:

[0039]

[0040] Among them I syn is the synthetic image, n imagesis the number of original images, corresponding to the number of focus fringe patterns used. For each of these focus fringe patterns, namely the first focus fringe pattern and at least one different focus fringe pattern, i is the index corresponding to its characteristic rotation axis angle, and j is the index corresponding to its characteristic phase shift. The indices i, j in the above equations can have respective X and Y unique numbers (for example, the rotation axis angle index i can have 3 numbers: 1, 2, and 3; and the phase shift index j can have 2 numbers: 1 and 2), corresponding to respective X and Y unique values ​​of the rotation axis angle and phase shift. For example, a set of X·Y focus fringe patterns with X unique rotation axis angles and Y unique phase shifts can be utilized, such as in the case of 6 focus fringe patterns with 3 unique rotation angles and 2 unique phase shifts. The term is the illumination data for each of the multiple raw images, with corresponding rotation axis angles and phase shift indices i, j. A is a factor representing the contrast of the focused fringe pattern, which may be the amplitude of the sine wave of the interference pattern generated on the sample, and phase is the value of the phase shift (in radians).

[0041] Given that the second term in the above sum is weighted by the cosine (phase) factor, certain phase shifts, especially those with relatively low values ​​of this factor, have a significant impact on the composite image I syn That is, in some embodiments, the marginal improvement in resolution of the composite image resulting from additional illumination data corresponding to a focused fringe pattern with a characteristic phase shift such that the cosine (phase) is relatively low does not justify the additional acquisition and processing time associated with collecting that illumination data. In some embodiments, none of the focused fringe patterns have a characteristic phase shift, or otherwise for any of the first focused fringe pattern and at least one different focused fringe pattern, the separated illumination regions have no offset phase and the cosine is 0. For example, such embodiments exclude the use of additional illumination data corresponding to a focused fringe pattern with a characteristic phase shift of π / 2 or 3π / 2. syn In certain more specific embodiments, the possibility of using a wider range of focus fringe patterns may be excluded. For example, according to such embodiments, none of the focus fringe patterns has a characteristic phase shift, or otherwise for any of the first focus fringe pattern and at least one different focus fringe pattern, the separated illumination areas do not have an offset phase with an absolute value of a cosine less than 0.5. Such embodiments exclude the possibility of using a wider range of focus fringe patterns when generating I syn A focused fringe pattern is used having a characteristic phase shift in the range of, for example, π / 3 to 2π / 3 or 5π / 3 to 5π / 3.

[0042] As described above, illuminating the sample with a first focused fringe pattern having a characteristic rotation axis angle and phase shift, in addition to a second (or third or fourth) focused fringe pattern having the same rotation axis angle but a different phase shift, may be sufficient to provide image resolution enhancement along the rotation axis angle direction. Thus, in a specific embodiment, at least one different focused fringe pattern may be a second focused fringe pattern for which the beams are offset by a second phase different from the first phase, but for which the second separated illumination areas are identical to those of the first focused fringe pattern, such that the separated illumination areas have a second rotation axis that is not angled relative to (substantially coincident with) the first rotation axis. In a preferred embodiment, the second phase differs from the first phase by π, such as when the first phase shift (or the phase by which the beams generating the first focused fringe pattern are offset relative to each other) is zero (0) and the second phase shift (or the phase by which the beams generating the second focused fringe pattern are offset relative to each other) is pi (π).

[0043] Likewise, if it is desired to enhance the image resolution along another direction, a third focus fringe pattern and a fourth focus fringe pattern may be used, which have another characteristic rotation axis angle but different characteristic phase shifts. According to a representative imaging method, at least one different focus fringe pattern may therefore include using not only the second focus fringe pattern, but also the third focus fringe pattern and / or the fourth focus fringe pattern, and preferably using both the third focus fringe pattern and the fourth focus fringe pattern. For the third focus fringe pattern, the light beam may be offset by a third phase substantially the same as the first phase, but the third separated illumination area may have a third rotation axis that is angled relative to both the first rotation axis and the second rotation axis. For the fourth focus fringe pattern, the light beam may be offset by a fourth phase substantially the same as the second phase, but the fourth separated illumination area may have a fourth rotation axis that is not angled relative to (substantially coincident with) the third rotation axis. In a preferred embodiment, the fourth phase differs from the third phase by π, such as when the third phase shift (or the phase at which the light beams generating the third focus fringe pattern are offset relative to each other) is zero (0) and the fourth phase shift (or the phase at which the light beams generating the fourth focus fringe pattern are offset relative to each other) is pi (π). In other preferred embodiments, the third rotational axis, or both the third rotational axis and the fourth rotational axis, are at an angle of about π / 6 to about π / 2, such as about π / 4 to about π / 2, relative to the first rotational axis, or otherwise relative to both the first rotational axis and the second rotational axis. For example, the first rotational axis and / or the second rotational axis may have a reference rotational axis angle of zero (0), and the third rotational axis and / or the fourth rotational axis may be at an angle of π / 3 relative to the first rotational axis and / or the second rotational axis.

[0044] If it is necessary to enhance the image resolution along another direction, a fifth focus fringe pattern and a sixth focus fringe pattern may be used, which have another characteristic rotation axis angle but a different characteristic phase shift. According to a representative imaging method, at least one different focus fringe pattern may therefore include not only the second focus fringe pattern, the third focus fringe pattern, and the fourth focus fringe pattern, but also the fifth focus fringe pattern and / or the sixth focus fringe pattern, and preferably the fifth focus fringe pattern and the sixth focus fringe pattern. For the fifth focus fringe pattern, the light beam may be offset by a fifth phase that is substantially the same as the first phase and the third phase, but the fifth separated illumination area may have a fifth rotation axis that is angled relative to all of the first rotation axis, the second rotation axis, the third rotation axis, and the fourth rotation axis. For the sixth focus fringe pattern, the light beam may be offset by a sixth phase that is substantially the same as the second phase and the fourth phase, but the sixth separated illumination area may have a sixth rotation axis that is not angled relative to (substantially coincides with) the fifth rotation axis. In a preferred embodiment, the sixth phase differs from the fifth phase by π, such as when the fifth phase shift (or the phase by which the beams generating the fifth focused fringe pattern are offset relative to each other) is zero (0) and the sixth phase shift (or the phase by which the beams generating the sixth focused fringe pattern are offset relative to each other) is pi (π). In other preferred embodiments, the fifth rotation axis or both the fifth rotation axis and the sixth rotation axis are at an angle of about π / 2 to about 5π / 6, such as about π / 2 to about 3π / 4, relative to the first rotation axis or otherwise relative to both the first rotation axis and the second rotation axis. For example, the first rotation axis and / or the second rotation axis may have a reference rotation axis angle of zero (0), and the fifth rotation axis and / or the sixth rotation axis may be at an angle of 2π / 3 relative to the first rotation axis and / or the second rotation axis.

[0045] Thus, a representative sample imaging method described herein includes obtaining illumination data for each of a plurality of raw images, wherein each raw image corresponds to a given focused fringe pattern in a set of focused fringe patterns. Each focused fringe pattern is generated by the interference of a pair of light beams emitted through a microscope objective to illuminate a plurality of points of a sample, such as by scanning a pattern around a fixed sample to perform laser scanning microscopy, or otherwise by moving the sample around a fixed pattern to perform sample scanning microscopy. The set of focused fringe patterns may include X·Y members, the members being a combination of or a matrix defining (i) X unique angles of the orientation of the axis of rotation of the light beam and (ii) Y unique phases of the light beam offset. Advantageously, as described herein, by judiciously selecting corresponding Y unique values ​​of the phase shift, enhanced image resolution is achieved with reduced time and data processing by limiting or eliminating the collection of illumination data that provides little or no additional benefit in conventional methods. For example, if the cosine of the values ​​of phase shift indices 1 and 2 is significant (e.g., where phase shift index 1 has a value of 0 and phase shift index 2 has a value of π), it may be desirable to select a phase difference corresponding to the difference in the values ​​of phase shift indices 1 and 2 of π, but not if the cosine of these values ​​is less significant (e.g., where phase shift index 1 has a value of π / 2 and phase shift index 2 has a value of 3π / 2). Thus, it can be appreciated that in some cases, simply selecting the difference in the phase shift indices without selecting further values ​​may not be sufficient. However, by appropriately selecting the index values, a sample can be imaged according to the methods described herein with no more than two unique values ​​of the phase shift, and / or no more than six, or even no more than four, focused fringe patterns in total.

[0046] In representative embodiments, for example, a sample may be imaged with two, four, or six of the focused fringe patterns 1 to 6 characterized in Table 1 below with respect to their rotation axis angle index i and phase shift index j.

[0047] Table 1

[0048] Focus on stripe pattern Rotation axis angle index i Phase shift index j 1 1 1 2 1 2 3 2 1 4 2 2 5 3 1 6 3 2

[0049] For example, the sample can be imaged with only focused fringe patterns 1 to 2, only focused fringe patterns 1 to 4, or only focused fringe patterns 1 to 6. The value of the rotation axis angle index i can be, for example, 0, π / 3, and 2π / 3, and the value of the phase shift index j can be, for example, 0 and π. In this case, for each rotation axis index i and phase shift index j in Table 1, the above-mentioned specific focused fringe patterns 1 to 6 can be more specifically characterized by the values ​​of the rotation axis angle and the phase shift, which are provided in Table 2 below.

[0050] Table 2

[0051] Focus on stripe pattern Rotation axis angle value Phase shift value 1 0 0 2 0 π 3 π / 3 0 4 π / 3 π 5 2π / 3 0 6 2π / 3 π

[0052] Further embodiments of the invention relate to an apparatus for performing Raman spectroscopy or fluorescence microscopy according to any of the imaging methods described herein. Such an apparatus may include a component such as a programmable spatial light modulator (SLM) for modulating the phase of a beam projected onto the back aperture of a microscope objective, as described herein, and interfering to generate a focused fringe pattern on a sample. Preferably, the SLM displays a phase grating that causes the beam to pass through separate illumination areas of the microscope objective, which are non-circular. The apparatus, or more specifically, the software of a processor used in the apparatus may further perform Gaussian blurring (according to a software algorithm known as a Gaussian blur filter) to reduce or eliminate diffraction along the edges of these non-circular separate illumination areas.

[0053] In general, aspects of the invention relate to advantages in sample imaging applications obtained by directing a laser beam into separate illumination regions of a microscope's back aperture that are, in some embodiments, non-circular (e.g., in the shape of a slit or a circle segment). Two non-circular illumination regions can increase light throughput for a given displacement (distance) through the back aperture compared to using a circular illumination region. For example, along a Figure 1A and 1B The horizontal displacement axis 16 shown, the distance between the two circles ( Figure 1A ) and the distance between the two non-circular slits ( Figure 1B ) are the same. However, with two non-circular slits (circular segments in this particular embodiment), substantially more light enters the microscope objective in the vertical direction. For example, the light intensity of the back aperture can be increased by at least four times to achieve this goal. Alternatively, for a given light flux, two non-circular illumination areas can increase the displacement compared to using a circular illumination area, thereby providing a high-quality (finer) sinusoidal curve generated by interference. By judiciously selecting the focused fringe pattern profile for sample scanning, other aspects are associated with advantages resulting from the efficiency of illumination data collection and processing. Those skilled in the art, using the knowledge gained from this disclosure, will recognize that various changes can be made to the disclosed imaging methods and apparatus to achieve these and other advantages without departing from the scope of the invention. Therefore, it should be understood that the features described herein are susceptible to change or replacement. The specific embodiments illustrated and described herein are for illustrative purposes only and are not limitations of the invention set forth in the appended claims.

[0054] The following examples are presented as representative of the present invention. These examples should not be construed as limiting the scope of the invention, as these and other equivalent embodiments will be apparent in view of this disclosure and the appended claims.

[0055] Example 1

[0056] Simulation study

[0057] Imaging of real samples with "ground truth images" was simulated using confocal SIM according to the improved imaging method described in this paper and compared with traditional CLSM. The simulation results were obtained using a light propagation library available online as "PROPER". This suite of programs for simulating the propagation of light through optical systems using Fourier transform algorithms (Fresnel, angular spectrum method) has been thoroughly validated as a physical optics propagation tool and is commonly used by astronomers to model the performance of telescopes. The library was used to simulate, in particular, the confocal and confocal SIM point spread functions of a 532nm laser through a 100x, 0.7NA objective, focused to a 25μm confocal pinhole through a 75mm focal length lens. Figure 3 Plotted for three different angles (0, π / 3, 2π / 3) of the light beam, and for each angle, the light beam is offset relative to each other by the phase of 0 and π, the simulated point spread function of the confocal SIM example. Then, by taking the convolution of the point spread function and the ground truth image, the collected result images are simulated using these patterns. In the case of the original confocal SIM image with an angle of 0 and a phase shift of π, the image itself is of little use. Only when the raw image data of all angles and phases are collected and used to reconstruct the super-resolution image according to the image reconstruction algorithm described herein, the improvement relative to the traditional CLSM is obvious.

[0058] Figure 4 A collection of simulated sample images using conventional CLSM and the same simulated images but using confocal SIM to improve resolution are shown. Figure 3 The six angle and phase combinations shown reconstruct super-resolved images achieve improvements to the illumination pattern swept across the sample. The image reconstruction algorithm is as described above. Therefore, the simulation work demonstrates the successful simulation of the confocal SIM image using the above improvements on the simulated microscope resolution standard. In other studies, it was demonstrated in both 1D and 2D simulations that only 2 different illumination phase shifts are required at a given rotation axis angle.

[0059] Example 2

[0060] Experimental Demonstration

[0061] according to Figure 2A prototype microscope for performing confocal SIM was built using the configuration shown. The dichroic mirror provides the ability to observe the laser spot on the sample using a microscope camera. The camera was used to acquire images of the spot generated by the SIM pattern from the SLM to demonstrate that the same focused fringe pattern as in the physical optics propagation simulation study described in Example 1 was generated. This was based on a side-by-side comparison with simulation results, taking into account the circular border around the image obtained in this experimental demonstration to help indicate the approximate alignment with the confocal pinhole. In the simulation study, the simulated SIM laser patterns already took into account the application of a confocal mask, which is why the outer shape of these patterns is circular, as shown in Figure 1. Figure 3 shown.

[0062] Using Pelcotec TM CDMS (Critical Dimension Magnification Standard, Ted Pella Company), successfully demonstrated the super resolution of confocal SIM in line scans as well as full 2D images. The CDMS sample has lines spaced 500nm apart, which cannot be resolved by the confocal imaging system. In contrast, the image can be resolved when imaged using confocal SIM. In addition, when imaging at 532nm excitation using a 100x, 0.9NA objective, the confocal resolution calculated according to the equation given in the background technology will be 296nm. This can be experimentally confirmed using a carbon nanotube sample and confirmed on a confocal system by achieving a confocal (non-SIM) resolution of 279nm. With the same system, using the same excitation wavelength, objective and carbon nanotube sample, an improved resolution of 186nm was achieved when using combined confocal SIM imaging.

Claims

1. A method for imaging a sample, the method comprising: illuminating the plurality of points of the sample with a first focused fringe pattern generated at each of the plurality of points by interference of light beams emitted through a microscope objective and irradiated about a first rotation axis; wherein for the first focused fringe pattern, a first separated illumination region of the microscope objective has the first rotation axis, and the light beams are phase-shifted relative to each other by a first phase; The method further comprises illuminating the plurality of points with at least one different focused fringe pattern, wherein the beams are phase-shifted relative to each other by a phase different from the first phase, and wherein (i) the same separate illumination area of ​​the microscope objective has the same rotation axis that is not angled relative to the first rotation axis, or (ii) different separate illumination areas of the microscope objective have another rotation axis that is angled relative to the first rotation axis, wherein the angle of the rotation axis refers to the orientation of the separate illumination areas on a back aperture of the microscope objective, One or both of the separate illumination areas are non-circular.

2. The method of claim 1, further comprising obtaining illumination data for each of a plurality of raw images corresponding to each of the first focused fringe pattern and the at least one different focused fringe pattern from a single-element detector or a spectrometer.

3. The method of claim 2, further comprising reconstructing a super-resolved synthetic image from the illumination data of each of the plurality of original images according to the following equation: Among them I syn is the composite image and n images is the number of each of the plurality of original images, and Wherein for each of the first focus fringe pattern and the at least one different focus fringe pattern: i is the index corresponding to the angle of the rotation axis; j is the index corresponding to the phase shift of the beams relative to each other; is the illumination data of each of the plurality of original images, having an index i corresponding to the rotation axis angle and an index j corresponding to the phase shift; A is a factor representing the contrast of the focused fringe pattern, phase is the phase shift.

4. The method of claim 1 or 2, wherein the separated illumination area comprises a maximum inscribed circle whose center is substantially collinear with the center of the rear aperture of the microscope objective.

5. The method of claim 4, wherein the separate illumination regions each further include an illumination enhancement region outside the maximum inscribed circle.

6. The method of claim 5, wherein the illumination enhancement area occupies at least about 50% of the separated illumination area.

7. The method of claim 5, wherein the illumination area is shaped as a slit that coincides with opposite side portions of a rear circular aperture of the microscope objective.

8. The method according to any one of claims 1 to 3, wherein for the at least one different focused fringe pattern, the light beam is phase modulated using a programmable spatial light modulator.

9. The method of claim 1, wherein for any one of the first focus fringe pattern and the at least one different focus fringe pattern, the separated illumination areas have no offset phase and the phase cosine is zero.

10. The method of claim 9, wherein for any one of the first focus fringe pattern and the at least one different focus fringe pattern, the separated illumination areas have no offset phase having a cosine with an absolute value less than 0.

5.

11. The method of claim 1 , wherein the at least one different focused fringe pattern is a second focused fringe pattern, for which the light beam is offset by a second phase different from the first phase, and the same separated illumination area is a second separated illumination area having a second rotation axis, which is not angled relative to the first rotation axis.

12. The method of claim 11, wherein the at least one different focus fringe pattern further comprises: A third focused fringe pattern for which the light beam is offset by a third phase that is substantially the same as the first phase and for which a third separated illumination area has a third rotation axis that is angled relative to the first rotation axis and the second rotation axis.

13. The method of claim 12, wherein the at least one different focus fringe pattern further comprises: A fourth focused fringe pattern for which the beam is offset by a fourth phase that is substantially the same as the second phase and for which fourth separated illumination regions have a fourth rotation axis that is not angled relative to the third rotation axis. The method of claim 13 , wherein the second phase differs from the first phase by π. The method of claim 14 , wherein the first phase is 0 and the second phase is π.

16. The method of claim 13, wherein the at least one different focus fringe pattern further comprises: a fifth focused fringe pattern for which the beam is offset by a fifth phase that is substantially the same as the first phase and the third phase, and for which a fifth separated illumination region has a fifth rotation axis that is angled relative to the first, second, third, and fourth rotation axes; as well as and a sixth focused fringe pattern, for which the beam is offset by a sixth phase that is substantially the same as the second phase and the fourth phase, and for which a sixth separated illumination area has a sixth rotation axis that is not angled relative to the fifth rotation axis. 17 . The method of claim 16 , wherein the first phase, the third phase, and the fifth phase are 0, and wherein the second phase, the fourth phase, and the sixth phase are π.

18. The method of claim 16, wherein the third rotation axis is at an angle of about π / 6 to about π / 2 relative to the first rotation axis, and wherein the fifth rotation axis is at an angle of about π / 2 to about 5π / 6 relative to the first rotation axis.

19. A method of imaging a sample, the method comprising: obtaining illumination data for each of a plurality of raw images, each raw image corresponding to a given focused fringe pattern in a set of focused fringe patterns, each focused fringe pattern being generated by interference of a pair of light beams emitted through a microscope objective and irradiated about a rotation axis to illuminate a plurality of points of the sample, wherein the set of focused fringe patterns comprises X·Y members, the members being combinations of (i) X phases of the beam offset and (ii) Y angles of the rotation axis orientation of the beam, wherein the set includes no more than two different values ​​of X, wherein the angle of the rotation axis refers to the orientation of a separate illumination area on a back aperture of the microscope objective; and wherein one of the pair of light beams illuminates a non-circular illumination area for the microscope objective for at least one of the focused fringe patterns.

20. An apparatus for performing Raman spectroscopy or fluorescence microscopy, the apparatus comprising a programmable spatial light modulator (SLM) for modulating the phase of the light beam and configured to perform the method according to claim 1.

21. The apparatus of claim 20, wherein the SLM displays a phase grating that causes the light beam to pass through a non-circular split illumination area of ​​a microscope objective.

22. The apparatus of claim 21, configured to perform Gaussian blurring to smooth the non-circular separated illumination areas.

Images