Phase mask for structured lighting

Abstract

An embodiment of a phase mask is described, comprising a light-blocking layer disposed on a substrate, the light-blocking layer having several light-transmitting regions each configured as a first pattern, the first pattern including two segments having different phase configurations, and the light-blocking layer including at least three angular orientations of the first pattern.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority from U.S. Patent Application No. 62 / 978,351, filed on February 19, 2019, which is hereby incorporated by reference in its entirety for all purposes.

[0002] The present invention generally relates to a phase mask configured to generate an interference fringe pattern for structured illumination microscopy.

Background Art

[0003] It is generally understood that for various fluorescence microscopes, structured illumination microscopy (SIM) systems are commercially available. However, these SIM systems typically use “wide - field” illumination. As used herein, the term “wide - field illumination” generally refers to the illumination of a large sample area by sending collimated light from a light source through a focusing lens before it enters an objective lens element. The normal purpose of fluorescence imaging is not to collect the entire spectrum, but simply to filter the emitted light of the target wavelength and direct it to a camera. Typically, wide - field microscopy is suitable for various fluorescence imaging applications, although confocal microscopy may be superior in some cases. Confocal microscopes utilize a pinhole (sometimes also referred to as an aperture) to exclude out - of - focus light, thereby significantly improving imaging and being particularly useful for imaging through thick samples. Confocal images are constructed pixel - by - pixel rather than over a wide area (as in wide - field imaging), so confocal microscopy is well - suited for spectral imaging because it can send emitted or scattered light to a spectrometer downstream of the pinhole. Specific applications of confocal microscopy include fluorescence sectioning with thick and heterogeneous samples and hyperspectral imaging such as in the case of Raman microscopy imaging.

[0004] An example of the application of SIM in confocal microscopy is described in U.S. Patent Application No. 16 / 837,512, filed April 1, 2020, entitled "Enhanced Sample Imaging Using Structured Illumination Spectroscopy," which is incorporated herein by reference in its entirety for all purposes. For example, Application '512 describes scanning a sample multiple times point by point using an interference fringe pattern, which takes advantage of the fact that what is typically called interference fringes (for example, a pattern of alternating bands of light and dark at uniform intervals resulting from the in-phase or out-of-phase nature of light) can have finer periodicity than a focused beam. Application '512 describes generating the interference fringe pattern using what is called a Spatial Light Modulator.

[0005] Those skilled in the art understand that embodiments of SLMs are well known and capable of spatially modulating both the intensity and phase of the illumination beam, which is important for confocal-structured illumination combined microscopy applications. However, while SLMs are excellent devices for generating intensity and phase in arbitrary patterns, they are generally inferior devices for use in commercial products. For example, embodiments of SLMs are generally prohibitively expensive, inefficiently utilize optical power, and require various complex optical and electronic overheads.

[0006] Therefore, there is a need for a device that can spatially modulate the intensity and phase of the illumination beam and does not suffer from the drawbacks of SLM. [Overview of the project]

[0007] Systems, methods, and products addressing these and other needs are described herein with respect to exemplary, non-exclusive implementations. Various alternatives, modifications, and equivalents are possible.

[0008] An embodiment of a phase mask is described, comprising a light-blocking layer disposed on a substrate, wherein the light-blocking layer has several light-transmitting regions, each configured as a first pattern. The first pattern includes two segments having different phase configurations, and the light-blocking layer includes at least three angular orientations of the first pattern.

[0009] In some implementations, the substrate is constructed of optically transparent glass, which may include BK7 glass. The optically transparent glass may also include an anti-reflective coating, and the light-blocking layer may include a chromium layer disposed on the substrate. Furthermore, light-transmitting apertures may be distributed radially on the substrate, and in some cases, six instances of the first pattern are distributed around it, with two instances of the first pattern for each angular orientation.

[0010] Furthermore, the two segments may be configured as circular segments, and in some implementations, the circular segments may have a first side with an arc shape and a second side with a substantially linear shape. The three angular orientations of the first pattern may include angles of 0, π / 3, and 2π / 3. In addition, the phase configuration of the first segment may include a phase delay of zero, the phase configuration of the second segment may include a phase delay of π, and the second segment may have a longer optical path length than the first segment. In some instances, this is achieved by having the second segment coated with a material.

[0011] Furthermore, one embodiment of a confocal microscope is described, comprising a light source configured to generate a light beam and a phase mask. The phase mask has a light-blocking layer disposed on a substrate, the light-blocking layer having several light-transmitting regions, each configured as a first pattern. The first pattern comprises two segments having different phase configurations from each other, and the light-blocking layer comprises at least three angular orientations of the first pattern. The confocal microscope also includes a device operably coupled to the phase mask to move the phase mask to position the light-transmitting apertures in the path of the light beam.

[0012] In some implementations, the light-transmitting aperture comprises six instances of a first pattern, with each angular orientation having two instances of the first pattern. In some cases, the three angular orientations may include angles of 0, π / 3, and 2π / 3, and may include a phase configuration for a first segment with a zero phase delay and a phase configuration for a second segment with a π phase delay. The phase configuration may include a second segment having a longer optical path length than the first segment, which may be achieved by using a material coating within the second segment.

[0013] In addition, one embodiment of a confocal microscope is described, comprising a light source configured to generate a light beam, a detector configured to generate a signal in response to light from a sample, and a phase mask. The phase mask has a light-blocking layer disposed on a substrate, the light-blocking layer having several light-transmitting regions, each configured as a first pattern. The first pattern comprises two segments having different phase configurations from each other, and the light-blocking layer comprises at least three angular orientations of the first pattern. The confocal microscope also includes a device operably coupled to the phase mask, which moves the phase mask to position the light-transmitting aperture in the optical path from the sample.

[0014] In some implementations, light from the sample is generated from the interaction between the light beam and the sample.

[0015] The embodiments and implementations described above are not necessarily exclusive or inclusive of one another, and can be combined in any way that is non-contradictory and otherwise possible, whether presented in relation to the same or different embodiments or implementations. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and / or implementations. Furthermore, any one or more functions, steps, operations, or techniques described elsewhere in this specification can be combined with any one or more functions, steps, operations, or techniques described in the summary of the invention in alternative implementations. Therefore, the embodiments and implementations described above are illustrative, not limiting. [Brief explanation of the drawing]

[0016] The above and further features will be more clearly understood from the following detailed explanation when read in conjunction with the attached drawings. In the drawings, similar reference numbers indicate similar structures, elements, or method steps, and the number at the left of the reference number indicates the drawing number in which the reference element first appears (for example, element 110 first appears in Figure 1). However, all of these rules are intended to be typical or illustrative, not limiting.

[0017] [Figure 1] This is a functional block diagram of one embodiment of a confocal microscope that communicates with a computer. [Figure 2] Figure 1 is a simplified schematic representation of one embodiment of a confocal microscope having a phase mask. [Figure 3] This is a simplified schematic representation of a top view of one embodiment of the phase mask shown in Figure 2, illustrating multiple patterns of light-blocking layers on the substrate. [Figure 4] Figure 3 is a simplified schematic representation of a side view of one embodiment of the phase mask, showing the base and fasteners that hold the substrate. [Figure 5] Figure 3 is a simplified schematic representation comparing images acquired using the phase mask with images acquired using a standard confocal configuration. [Figure 6] Figure 3 is a simplified schematic representation comparing images acquired using the phase mask with images acquired using a standard confocal configuration.

[0018] Similar reference numbers refer to corresponding parts across several figures in the drawing. [Modes for carrying out the invention]

[0019] As will be described in more detail below, embodiments of the invention described include a phase mask configured to spatially modulate the intensity and phase of an illumination beam. More specifically, the phase mask is configured for SIM using a confocal microscope corresponding to Raman spectroscopy and / or fluorescence spectroscopy.

[0020] Figure 1 provides a simplified exemplary embodiment of a user 101 who can interact with a computer 110 and a microscope 120. Embodiments of the confocal microscope 120 may include a variety of commercially available microscopes. For example, the confocal microscope 120 may include a DXR confocal-enabled Raman microscope available from Thermo Fisher Scientific. Figure 1 also shows a network connection between the computer 110 and the confocal microscope 120, but it will be understood that Figure 1 is intended to be illustrative and may include additional or fewer network connections. Furthermore, network connections between elements may include "direct" wired or wireless data transmission (e.g., represented by a lightning bolt) and "indirect" communication via other devices (e.g., switches, routers, controllers, computers, etc.), and therefore the embodiment in Figure 1 should not be considered limiting.

[0021] Computer 110 may include any type of computing platform, such as a workstation, personal computer, tablet, smartphone, one or more servers, compute cluster (local or remote), or other current or future computer or computer cluster. A computer typically includes known components such as one or more processors, an operating system, system memory, memory storage devices, input / output controllers, input / output devices, and display devices. More than one implementation of computer 110 may be used to perform various operations in different embodiments, and it will also be understood that the representation of computer 110 in Figure 1 should not be considered limiting.

[0022] In some embodiments, computer 110 may employ a computer program product that includes a computer-usable medium having stored therein control logic (e.g., a computer software program including program code). When executed by a processor, the control logic causes the processor to perform some or all of the functions described herein. In other embodiments, some of the functions are implemented primarily in hardware, such as using a hardware state machine. Implementations of hardware state machines to perform the functions described herein will be apparent to those skilled in the art. Also, in the same or other embodiments, computer 110 may employ an Internet client that includes a dedicated software application that enables access to remote information via a network. The network may include one or more of many types of networks well known to those skilled in the art. For example, the network may include a local or wide area network that may employ what is commonly referred to as the TCP / IP protocol suite for communication. The network may include the worldwide interconnected computer network system commonly referred to as the Internet, or may include various intranet architectures. Those skilled in the art will also understand that some users within a network environment may prefer to employ what is commonly referred to as a "firewall" (which is sometimes also referred to as a packet filter or border protection device) to control information traffic entering and leaving a hardware and / or software system. For example, a firewall may include hardware or software elements, or a combination thereof, and is typically designed to enforce security policies set by a user, such as a network administrator.

[0023] As described herein, embodiments of the invention described include a phase mask for SIM in a confocal microscope. In the embodiments described, the phase mask is particularly useful for Raman spectroscopy and / or fluorescence spectroscopy using SIM. For example, as described above, the phase mask has a substantially higher level of efficiency and lower cost than a SLM and is also easy to implement (e.g., the phase mask does not require the additional optical components and software required by a SLM to operate effectively).

[0024] FIG. 2 provides a simplified, exemplary embodiment of a confocal microscope 120 that includes a phase mask 200. The confocal microscope 120 includes elements typical of a commercially available confocal microscope, such as a light source 215 that generates a light beam 217. The light source 215 can include any type of light source used in confocal microscopy, including, but not limited to, lasers, light emitting diodes (LEDs), broadband, or other types of light sources known to those skilled in the art. Embodiments of the confocal microscope 120 can also include a beam splitter 225 that selectively reflects light in a particular wavelength range to the objective lens 227 and the sample 205 and is transmissive within a particular wavelength range such that light passes through and reaches the lens 229 and through an aperture 223 (e.g., a “pinhole” type aperture) to the detector 235. The detector 235 can include any type of detector typical of a commercially available confocal microscope, such as a CCD, a photomultiplier tube, or other types of detectors known to those skilled in the art. Those skilled in the relevant art will also understand that FIG. 2 is provided for illustrative purposes and that other elements and / or configurations of the confocal microscope 120 are considered to be within the scope of the invention described. For example, before reaching the detector 235, the light can first pass through a spectrometer for spectrally dispersing the light.

[0025] In the embodiment shown in Figure 2, the phase mask 200 is positioned in the path of the light beam 217 to pattern the excitation light delivered to the sample 205. However, it will also be understood that the phase mask 200 can be positioned in the path of light 219 to pattern the light from the sample 205 to the detector 235 (e.g., light emitted, scattered, etc., as a result of the interaction between the light beam 217 and the sample 205).

[0026] Figure 3 provides an exemplary embodiment of a top view of the phase mask 200. Embodiments of the phase mask 200 include a substrate 307 constructed from a light-transmitting material having known optical properties. For example, the substrate 307 may include a 60 mm × 60 mm area constructed from optical glass of the type used in lenses and other optical components, such as what is called "crown glass," which has good optical and mechanical properties and is resistant to chemical and environmental damage. One particular type of crown glass useful for the phase mask 200 includes glass containing borosilicate additives, such as BK7 glass available from Schott AG.

[0027] As shown in Figure 3, the substrate 307 includes a light-blocking region 305, which may include any type of configuration that can block the transmission of light through the substrate 307. One embodiment includes a configuration comprising the deposition of a chromium material layer on the surface of the substrate 307 (which may be, for example, the top or bottom surface). Also, although Figure 3 shows the light-blocking region 305 as a substantially circular ring, it will be understood that the ring configuration is illustrative and other configurations may be available (for example, a substantial portion of the surface area of ​​the substrate 307 on one side may include the light-blocking region 305, or the light-blocking region 305 may be configured as a linear strip).

[0028] Furthermore, Figure 3 shows multiple light-transmitting regions within the light-blocking region 305, indicated as pattern 310. As stated above, the ring configuration of the light-blocking region 305 and the configuration of each pattern 310, as shown in Figure 3, are illustrative and should not be considered limiting. In some embodiments, pattern 310 may include a linear strip-shaped light-blocking region 310, or it may include a substantial portion of the surface area of ​​the substrate 307, such that the configuration of pattern 310 is substantially linear. For example, in a ring or linear configuration, pattern 310 may include a pattern-covered region of about 4 mm × 4 mm. In a linear embodiment, this may include a linear configuration light-blocking region 305 consisting of about 4 mm × 24 mm.

[0029] Figure 3 also shows fasteners 320, which may include a nut / bolt configuration or any other fastener configuration known to those skilled in the art. Figure 4 shows a side view of the phase mask 200, which includes a substrate 307 having a substantially flat configuration and substantially consistent thickness, held in place by fasteners 320 operably coupled to a base 405. In some embodiments, the base 405 utilizes a clamping mechanism for operably connecting the phase mask 200 to a translational device, such as a motor (e.g., a stepper motor), which rotates the phase mask 200 about an axis around the fasteners 320. In embodiments in which the phase mask 200 includes a pattern 310 in a linear configuration, the translational device is constructed and configured to provide linear motion to the phase mask 200. It will also be understood that the translational device may include other types of elements known in the related art, such as piezoelectrics, and others.

[0030] Furthermore, Figure 3 shows six instances of pattern 310 at various angles to the optical path of the light beam 217 (for example, when an instance of pattern 310 is positioned in the optical path). Each instance of pattern 310 is located in a light-blocking region 305 indicated by position indicator 303. The light-blocking region 305 also includes a substantially circular pattern 313 at a first position indicated by position indicator 303, allowing substantially all of the light beam 217 to pass through the substrate 307, and a region without a light-transmitting pattern at a second position indicated by position indicator 303, blocking substantially all of the light beam 217 from passing through the substrate 307.

[0031] In some embodiments, the phase mask 200 includes three angular orientations, with two instances of the pattern 310 for each angle: one instance of the pattern 310 including the deposited layer segment 315 and the substrate segment 317, and a second instance of the pattern 310 where two substrate segments 317 appear. The pattern 310 may also include two segments configured as "slit" shaped elements (e.g., a slit includes an elongated opening), which may sometimes be referred to as "circular segments." Furthermore, in some embodiments, the diameter of the circular segments of the pattern 310 is matched to the rear aperture diameter of the objective lens 227. For example, in the Olympus 100x0.9NA, the diameter is >= 3.24 mm, and in the Olympus 100x0.8NA with a longer operating distance, the diameter is >= 2.88 mm. Furthermore, in the embodiments described herein, one side of each circular segment may have an arc shape, such as substantially circular (e.g., about 1 / 4 of a circle), and the second side may be substantially straight.

[0032] Importantly, in some instances of pattern 310, there is a difference in optical path length between two segments of pattern 310. In other words, the segments have different optical path lengths that create a phase difference in the light passing through them (for example, a segment with a longer optical path length will produce a phase delay in a portion of the light beam 217 passing through it compared to a segment with a shorter optical path length). In some embodiments, the optical path difference may be created by depositing additional material on the substrate 307 (e.g., the substrate 307 has two substantially flat surfaces with a consistent thickness) in one of the segments of pattern 310 to create a deposited layer segment 315 with a longer optical path in combination with the substrate 307, compared to the substrate segment 317 of pattern 310 which contains only the substrate 307. The deposited material may be the same material as the substrate 307 or other preferred material. Alternatively, or in combination with deposition, the optical path length of one of the segments of pattern 310 may be shortened by removing material from the substrate 307 within the segment.

[0033] For example, the refractive index difference between the BK7 glass used in the substrate 307 and air creates a certain optical path difference between the segments of the pattern 310. Since the refractive index of BK7 glass is 1.52 at an excitation wavelength of 532 nm, light travels more slowly through BK7 than through air. Therefore, the optical path difference can be generated by the controlled deposition of BK7 on one of the segments. Those skilled in the art will know that if n is the refractive index and d is the length, then when d1 = d2 (for example, when light travels the same distance in air as it does through BK7), the optical path difference (OPD) is equal to n1*d - n2*d (which can also be expressed as d = OPD / (n1 - n2)). In this embodiment, at an excitation wavelength of 532 nm, n1 is 1.0003 for air and n2 is 1.52 for BK7 (for example, the value depends on the wavelength). For SIM applications, a π phase difference between segments is highly desirable, which corresponds to an OPD of 266 nm for an excitation wavelength of 532 nm. Solving for d, the desired π phase difference is generated by depositing a BK7 material to a thickness of 512 nm on one of the segments. Therefore, when a portion of the light beam 217 passes through the substrate segment 317 and the deposited layer segment 315 containing a 512 nm BK7 glass coating, the phase difference between the light passing through segments 315 and 317 is π.

[0034] In the embodiment shown in Figure 3, the six instances of pattern 310 include three different angles: 0, π / 3, and 2π / 3. For each angle, pattern 310 includes a substrate segment 317 with a phase delay of 0 and a deposited layer segment 315 with a phase delay of π. In the described embodiment, the six instances of pattern 310 are useful for performing structured illumination by allowing the light beam 217 to pass through each instance of pattern 310 sequentially when each instance is positioned within the optical path of the light beam 217 (for example, rotated within the optical path in a circular configuration as shown in Figure 3, or linearly translated in a linear configuration). For example, an embodiment of a confocal microscope 120 equipped with a phase mask 200 and a computer 110 for image processing can implement SIM to image a sample and obtain a spatial resolution of 150 nm, which is a twofold improvement over the diffraction limit of 300 nm for a typical confocal microscope.

[0035] Figure 5 provides an exemplary example of a comparison between a SIM-Raman image 510 and associated SIM-Raman data 515 acquired using a phase mask 200 and a confocal image 520 and associated confocal data 525 acquired using a standard confocal microscopy configuration, where both the SIM-Raman image 510 and the confocal image 520 have the same field of view of the substrate, including an array of longitudinal and transverse lines separated at a 250 nm pitch. The example in Figure 5 clearly shows that, in visual inspection, the SIM-Raman image 510 has better resolution than the confocal image 520, which is further enhanced by the SIM-Raman data 515, which shows superior intensity discrimination of the data acquired along the data lines 505 compared to the confocal data 525.

[0036] Figure 6 provides a further exemplary example of a comparison between a SIM-Raman image 610 and associated SIM-Raman data 615 acquired using a phase mask 200 and a confocal image 620 and associated confocal data 625 acquired using a standard confocal microscopy configuration, where both the SIM-Raman image 610 and confocal image 620 have the same field of view of the substrate containing the overlapping carbon nanotube array. Again, the example in Figure 6 clearly shows that, in visual inspection, the SIM-Raman image 610 has better resolution than the confocal image 620, which is further enhanced by the SIM-Raman data 615, which shows superior intensity discrimination of the data acquired along the data line 605 compared to the confocal data 625 (for example, the SIM-Raman image 610 clearly resolves two separate carbon nanotubes, while in the confocal image 620, the two together are blurred except for the distal end of the nanotubes).

[0037] While various embodiments and implementations have been described, those skilled in the art should understand that the above are merely illustrative and not limiting, but simply presented as examples. Many other schemes are possible for distributing functionality among the various functional elements of the described embodiments. The functionality of any element may be performed in various ways in alternative embodiments.

Claims

1. It is a phase mask, circuit board and A light-blocking layer disposed on the substrate, having a plurality of light-transmitting regions, each configured as a first pattern including two slit-shaped segments, The first pattern includes a first slit-shaped segment and a second slit-shaped segment having different phase configurations. The phase mask comprises a light-blocking layer including a plurality of light-transmitting regions, each including a light-blocking layer with a different angular orientation, where the angular orientation represents the angle of the first pattern within the mask surface in the observation field.

2. The phase mask according to claim 1, wherein the substrate includes optically transparent glass.

3. The phase mask according to claim 2, wherein the optically transparent glass is provided with an anti-reflective coating.

4. The phase mask according to claim 1, wherein the light-blocking layer includes a chromium layer disposed on the substrate.

5. The phase mask according to claim 1, wherein the plurality of light-transmitting regions are distributed radially on the substrate.

6. The phase mask according to claim 1, wherein the plurality of light-transmitting regions include six instances of the first pattern.

7. The phase mask according to claim 6, wherein the six instances include three sets of two instances of the first pattern having the same angular orientation.

8. The phase mask according to claim 1, wherein each of the two segments includes a circular segment having a first side having an arc shape and a second side having a substantially linear shape.

9. The phase mask according to claim 1, wherein the different angular orientations include relative angles of 0, π / 3, and 2π / 3.

10. The phase mask according to claim 1, wherein the phase configuration of the first segment includes a phase delay of zero, and the phase configuration of the second segment includes a phase delay of π.

11. The phase mask according to claim 10, wherein the second segment includes an optical path length longer than that of the first segment, and the second segment is provided with a material coating.

12. It is a confocal microscope, A light source configured to generate a light beam, The phase mask according to claim 1, A confocal microscope comprising: a device operably coupled to the phase mask and configured to move the phase mask to position the light-transmitting region within the path of the light beam.

13. The confocal microscope according to claim 12, wherein the plurality of light-transmitting regions include six instances of the first pattern.

14. The confocal microscope according to claim 13, wherein the six instances include two instances of the first pattern in each angular orientation.

15. The confocal microscope according to claim 12, wherein the phase configuration of the first segment includes a phase delay of zero, and the phase configuration of the second segment includes a phase delay of π.

16. The confocal microscope according to claim 15, wherein the second segment includes an optical path length longer than that of the first segment.

17. It is a confocal microscope, A light source configured to generate a light beam, A detector configured to generate a signal in response to light from a sample, The phase mask according to claim 1, A confocal microscope comprising: a device operably coupled to the phase mask and configured to move the phase mask to position the light-transmitting region within the path of light from the sample.

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