Phase mask for structured illumination

An embodiment of a phase mask includes a light blocking layer disposed on a substrate, where the light blocking layer has a number of optically transmissive regions each configured as a first pattern. The first pattern includes two segments that have different phase configurations from each other, and the light blocking layer includes at least three angular orientations of the first pattern.

FIELD OF THE INVENTION

The present invention is generally directed to a phase mask configured to generate fringe patterns for Structured Illumination Microscopy.

BACKGROUND

It is generally appreciated that Structured Illumination Microscopy (SIM) systems are available commercially on a variety of fluorescence microscopes. However, these SIM systems typically use “wide field” illumination. The term, “wide field illumination, as used herein, generally refers to the illumination of a large sample area by sending collimated light from a source through a focusing lens before entering an objective lens element. The normal intent of fluorescence imaging is not to collect an entire spectrum, but rather to simply filter the emission light for the wavelength of interest and then direct it into a camera. Typically, wide field microscopy is suitable in a variety of fluorescence imaging applications, but there are also cases in which a confocal microscope is superior. Confocal microscopes utilize a pinhole (also sometimes referred to as an aperture) to reject light that is out of focus, thereby vastly improving imaging and is particularly useful for imaging through thick samples. Since a confocal image is constructed pixel-by-pixel, rather than over a large area (as in wide field imaging), confocal microscopy is well adapted for spectroscopic imaging because the emission or scattered light can be sent into a spectrograph downstream of the pinhole. Specific uses for confocal microscopy include fluorescence sectioning through thick, non-homogenous samples, as well as hyperspectral imaging, such as in the case of imaging Raman microscopy.

An example of an application of SIM in a confocal microscope is described in U.S. application Ser. No. 16/837,512, titled “Enhanced Sample Imaging Using Structured Illumination Spectroscopy”, filed Apr. 1, 2020, which is hereby incorporated by reference herein in its entirety for all purposes. For example, the '512 application describes scanning a sample point-by-point, multiple times using fringe patterns that take advantage of the fact that what is typically referred to as an interference fringe (e.g. a pattern of evenly spaced alternating bright and dark bands due to light being in our out of phase) can have a finer periodicity than a focused beam. The '512 application describes using what is referred to as a spatial light modulator (SLM) to generate the fringe patterns.

Those of ordinary skill in the art appreciate that embodiments of SLM's are well known and are capable of modulating both the intensity and phase of an illumination beam spatially, which is important for combined confocal-structured illumination microscopy applications. However, while an SLM is an excellent device for generating any arbitrary pattern of intensity and phase, it is generally a poor device to use in a commercialized product. For example, embodiments of SLM are generally prohibitively expensive, inefficiently utilize light power, and require a variety of complex optical and electronic overhead.

Therefore, a need exists for a device that is capable of spatially modulating the intensity and phase of an illumination beam, and does not suffer from the drawbacks of an SLM.

SUMMARY

Systems, methods, and products to address these and other needs are described herein with respect to illustrative, non-limiting, implementations. Various alternatives, modifications and equivalents are possible.

An embodiment of a phase mask is described that comprises a light blocking layer disposed on a substrate, where the light blocking layer has a number of optically transmissive regions each configured as a first pattern. The first pattern includes two segments that have different phase configurations from each other, and the light blocking layer includes at least three angular orientations of the first pattern.

In some implementations the substrate is constructed of optically transparent glass that may include BK7 glass. The optically transparent glass may also include an anti-reflective coating and the light blocking layer can include a layer of chrome disposed on the substrate. Further, the optically transmissive openings may be radially distributed on the substrate in some cases having six instances of the first pattern distributed around with two instances of the first pattern at each angular orientation.

Also, the two segments may be configured as a circular segment that, in some implementations can have a first side with an arc shape and a second side that with a substantially linear shape. The three angular orientations of the first pattern may include an angle of 0, pi/3, and 2pi/3. In addition, the phase configuration for a first segment may comprise a phase delay of zero and the phase configuration for a second segment may comprise a phase delay of pi, where the second segment may have a longer optical path length than the first segment. In some instances, this is accomplished having a coating of material in the second segment.

Further, an embodiment of confocal microscope is described that includes a light source configured to produce a light beam, and a phase mask. The phase mask has a light blocking layer disposed on a substrate, where the light blocking layer has with a number of optically transmissive regions each configured as a first pattern. The first pattern includes two segments that have different phase configurations from each other and the light blocking layer includes at least three angular orientations of the first pattern. The confocal microscope also includes a device operatively coupled to the phase mask that moves the phase mask to position the optically transmissive openings in a path of the light beam.

In some implementations, the optically transmissive openings include six instances of the first pattern, with two instances of the first pattern at each angular orientation. In some cases, the three angular orientations include an angle of 0, pi/3, and 2pi/3 and may include a phase configuration for a first segment that comprises a phase delay of zero and a phase configuration for a second segment that comprises a phase delay of pi. The phase configurations can include a second segment that has a longer optical path length than the first segment that in some cases some may be accomplished using a coating of material in the second segment.

Additionally, an embodiment of confocal microscope is described that includes a light source configured to produce a light beam, a detector configured to produce 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, where the light blocking layer has with a number of optically transmissive regions each configured as a first pattern. The first pattern includes two segments that have different phase configurations from each other and the light blocking layer includes at least three angular orientations of the first pattern. The confocal microscope also includes a device operatively coupled to the phase mask that moves the phase mask to position the optically transmissive openings in a path of the light from the sample.

In some implementations, the light from the sample is produced from an interaction of the light beam with the sample.

The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they are presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.

DETAILED DESCRIPTION OF EMBODIMENTS

As will be described in greater detail below, embodiments of the described invention 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 enabled for Raman Spectroscopy and/or Fluorescence Spectroscopy.

FIG.1provides a simplified illustrative example of user101capable of interacting with computer110and microscope120. Embodiments of confocal microscope120may include a variety of commercially available microscopes. For example, confocal microscope120may include the DXR confocal enabled Raman microscopes available from Thermo Fisher Scientific.FIG.1also illustrates a network connection between computer110and confocal microscope120, however it will be appreciated thatFIG.1is intended to be exemplary and additional or fewer network connections may be included. Further, the network connection between the elements may include “direct” wired or wireless data transmission (e.g. as represented by the lightning bolt) as well as “indirect” communication via other devices (e.g. switches, routers, controllers, computers, etc.) and therefore the example ofFIG.1should not be considered as limiting.

Computer110may include any type of computing platform such as a workstation, a personal computer, a tablet, a “smart phone”, one or more servers, compute cluster (local or remote), or any other present or future computer or cluster of computers. Computers typically include 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. It will also be appreciated that more than one implementation of computer110may be used to carry out various operations in different embodiments, and thus the representation of computer110inFIG.1should not be considered as limiting.

In some embodiments, computer110may employ a computer program product comprising a computer usable medium having control logic (e.g. computer software program, including program code) stored therein. The control logic, when executed by a processor, causes the processor to perform some or all of the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts. Also in the same or other embodiments, computer110may employ an internet client that may include specialized software applications enabled to access remote information via a network. A network may include one or more of the many types of networks well known to those of ordinary skill in the art. For example, a network may include a local or wide area network that may employ what is commonly referred to as a TCP/IP protocol suite to communicate. A network may include a worldwide system of interconnected computer networks that is commonly referred to as the internet, or could also include various intranet architectures. Those of ordinary skill in the related art will also appreciate that some users in networked environments may prefer to employ what are generally referred to as “firewalls” (also sometimes referred to as Packet Filters, or Border Protection Devices) to control information traffic to and from hardware and/or software systems. For example, firewalls may comprise hardware or software elements or some combination thereof and are typically designed to enforce security policies put in place by users, such as for instance network administrators, etc.

As described herein, embodiments of the described invention include a phase mask for SIM with a confocal microscope. In the described embodiments, 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 an SLM, as well as being easier to implement (e.g. the phase mask does not need the additional optical components and software required by the SLM in order to operate effectively).

FIG.2provides a simplified illustrative example of confocal microscope120that includes phase mask200. Confocal microscope120includes elements typically found in commercially available confocal microscopes such as source215that produces light beam217. Source215may include any type of light source used for confocal microscopy that includes, but is not limited to, a laser, Light emitting Diode (LED), broad band, or other type of source known to those of ordinary skill in the art. Embodiments of confocal microscope120may also include beam splitter225that selectively reflects light in a specified wavelength range to objective lens227and sample205, and is transmissive within a specified wavelength range to allow light to pass through to lens229through aperture223(e.g. a “pinhole” type aperture) to detector235. Detector235may include any type of detector typically found in commercially available confocal microscopes such as a CCD, photomultiplier, or other type of detector known to those of ordinary skill in the art. Those of ordinary skill in the related art will also appreciate that theFIG.2is provided for the purposes of example and that other elements and/or configurations of confocal microscope120are considered within the scope of the described invention. For example, prior to reaching detector235the light may first pass through a spectrograph to disperse the light into a spectrum.

In the example ofFIG.2, phase mask200is positioned in the path of light beam217to pattern the excitation light delivered to sample205. However, it will also be appreciated that phase mask200can be positioned in the path of light219to pattern the light from sample205onto detector235(e.g. light that is emitted, scattered, etc. as a result of an interaction of light beam217with sample205).

FIG.3provides an illustrative example of a top view of phase mask200. Embodiments of phase mask200include substrate307constructed from an optically transmissive material with known optical characteristics. For example, substrate307may include a 60 mm×60 mm area constructed from a type of optical glass used in lenses and other optical components, such what is referred to as “crown glass” that has good optical and mechanical characteristics, and is resistant to chemical and environmental damage. One particular type of crown glass useful for phase mask200includes glass with a borosilicate additive, such as BK7 glass available from Schott AG.

As illustrated inFIG.3, substrate307includes light blocking region305that may include any type of configuration capable of blocking the transmission of light through substrate307. One example includes a configuration that comprises a deposition of a layer of chrome material on the surface of substrate307(e.g. may be the top surface or bottom surface). Also,FIG.3illustrates light blocking region305as a substantially circular ring, however it will be appreciated that the ring configuration is exemplary and other configurations may be utilized (e.g. a substantial portion the surface area of one side substrate307may include light blocking region305, or light blocking region305may be configured as a linear strip).

Further,FIG.3illustrates a plurality of optically transmissive regions in light blocking region305, illustrated as pattern310. As described above, the ring configuration of light blocking region305and the arrangement of each of patterns310, as illustrated inFIG.3, is exemplary and should not be considered as limiting. In some embodiments, pattern310may include a linear strip of light blocking region310or, as described above, may include a substantial portion the surface area of the surface of substrate307where the arrangement of patterns310may be substantially linear. For example, in the ring or linear configuration, pattern310may include a pattern covering region of about 4 mm×4 mm. For a linear embodiment this may include a linear configuration of light blocking region305comprising about 4 mm×24 mm

FIG.3also illustrates fastener320that may include a nut/bolt configuration or any other fastener configuration known to those of ordinary skill in the art.FIG.4illustrates a side view of phase mask200that includes substrate307having a substantially planar configuration and substantially consistent thickness that is held in place by fastener320operatively coupled to base405. In some embodiments, base405utilizes clamping mechanism407to operatively connect phase mask200to a translation device such as a motor that rotates phase mask200about the axis around fastener320(e.g. a stepper motor). In embodiments where phase mask200includes a linear arrangement of patterns310, the translation device is constructed and arranged to provide linear motion to phase mask200. It will also be appreciated that the translation device may include other types of elements known in the related art, such as a piezo, etc.

In addition,FIG.3illustrates 6 instances of pattern310at various angles relative to the optical path of light beam217(e.g. when the instance of pattern310is positioned in the optical path). Each instance of pattern310is at a location of light blocking region305indicated by position indicator303. Light blocking region305also includes pattern313at a first position indicated by position indicator303that is substantially circular to permit substantially all of light beam217to pass through substrate307, and a region without a light transmission pattern at position2indicated by position indicator303that blocks substantially all of light beam217from passing through substrate307.

In some embodiments, phase mask200includes 3 angular orientations, where there are 2 instances of pattern310per angle one instance of pattern310that includes deposited layer segment315and substrate segment317, as well as a second instance of pattern310that has two occurrences of substrate segment317. Also, pattern310may include two segments arranged as “slit” shaped elements (e.g. a slit includes a long narrow opening), which may sometimes be referred to as “circular segments”. Further, in some embodiments the diameter of the circular segments of pattern310are matched to the back-aperture diameter of objective lens227. For example, for an Olympus 100×0.9 NA, that diameter would be >=3.24 mm, and for a long working distance Olympus 100×0.8 NA, that diameter would be >=2.88 mm. Also in the presently described example, one side of each circular segment may have an arc shape such as, for instance, a substantially circular shape (e.g. about ¼ of a circle), and a second side that is substantially linear.

Importantly, in some instances of pattern310there is an optical path length difference between the two segments of pattern310. In other words, the segments have a different optical path length from each other that creates a phase difference in light passing through (e.g. the segment with a longer optical path length produces a phase delay in the portion of light beam217that passes through it relative to the segment with the shorter optical path length). In some embodiments, the optical path difference may be created using a deposition of additional material onto substrate307(e.g. substrate307comprises two substantially planar surfaces with consistent thickness) in one of the segments of pattern310to produce deposited layer segment315that combined with substrate307comprises the longer optical path when compared to substrate segment317of pattern310that only includes substrate307. The deposited material may be the same material as substrate307or other suitable material. Alternatively, or in combination with the deposition, removal of material from substrate307in a segment may be used to shorten to optical path length of one of the segment of pattern310.

For example, the refractive index difference between BK7 glass used for substrate307and air creates an optical path difference between the segments of pattern310. Since the refractive index of BK7 glass is 1.52 at an excitation wavelength of 532 nm, light travels more slowly through BK7 than it does in air. Therefore, an optical path difference can be generated through controlled deposition of BK7 onto one of the segments. Those of ordinary skill in the art appreciate that where n is refractive index, and d is length, and if d1=d2 (e.g. the light travels the same distance in air as it does while it is traveling through the BK7), then the Optical Path Difference (OPD) is equal to n1*d−n2*d (may also be expressed as d=OPD/(n1−n2)). In the present example, n1 is 1.0003 for air and n2 is 1.52 for BK7 at an excitation wavelength of 532 nm (e.g. the values are wavelength dependent). For SIM applications a pi phase difference between the segments is highly desirable, which equates to an OPD of 266 nm for an excitation wavelength of 532 nm. Solving for d, a deposition of BK7 material at a thickness of 512 nm onto one of the segments produces the desired pi phase difference. Therefore, when portions of light beam217passes through substrate segment317and deposited layer segment315that includes a coating of 512 nm of BK7 glass, then the phase difference between the light traveling through segments315and317will be pi.

In the embodiment illustrated inFIG.3, the 6 instances of pattern310include 3 different angles of 0, pi/3, and 2pi/3, where for each angle, pattern310includes substrate segment317with phase delay of 0 and deposited layer segment315with a phase delay of pi. In the described embodiments, the 6 instances of pattern310are useful for performing structured illumination by allowing light beam217to successively pass through each instance of pattern310as it is positioned in the optical path of light beam217(e.g. rotated into the optical path in the case of a circular arrangement as illustrated inFIG.3, or linearly translated for a linear arrangement). For example, embodiments of confocal microscope120equipped with phase mask200and computer110to process the images, can implement SIM to image a sample and obtain a spatial resolution of 150 nm, that is a 2× improvement over the diffraction limit of 300 nm of a typical confocal microscope.

FIG.5provides an illustrative example of a comparison of SIM-Raman image510and associated SIM-Raman data515collected using phase mask200and confocal image520and associated confocal data525collected using a standard confocal microscopy configuration, where both SIM-Raman image510and confocal image520have the same field of view of a substrate comprising an arrangement of vertical and horizontal lines separated by a 250 nm pitch. The example ofFIG.5clearly demonstrates that upon visual inspection SIM-Raman image510has superior resolution to confocal image520, which is further reinforced by SIM-Raman data515that illustrates superior intensity discrimination for data collected along data line505over confocal data525.

FIG.6provides a further illustrative example of a comparison of SIM-Raman image610and associated SIM-Raman data615collected using phase mask200and confocal image620and associated confocal data625collected using a standard confocal microscopy configuration, where both SIM-Raman image610and confocal image620have the same field of view of a substrate comprising an arrangement of overlapping carbon nanotubes. Again, the example ofFIG.6clearly demonstrates that upon visual inspection SIM-Raman image610has superior resolution to confocal image620, which is further reinforced by SIM-Raman data615that illustrates superior intensity discrimination for data collected along data line605over confocal data625(e.g. SIM-Raman image610clearly resolves two separate carbon nanotubes, whereas confocal image620blurs the image of the two together except for the distal ends of the nanotubes).

Having described various embodiments and implementations, it should be apparent to those skilled in the relevant art that the foregoing is illustrative only and not limiting, having been presented by way of example only. Many other schemes for distributing functions among the various functional elements of the illustrated embodiments are possible. The functions of any element may be carried out in various ways in alternative embodiment.