Back-illumination interference tomography

Abstract

A back-illuminated interference tomography (BIT) microscope apparatus is disclosed that include a light source; an objective lens defining an objective lens focal plane located a distance fobj from the objective lens, wherein fobj is an objective lens focal length; a tube lens defining a tube lens focal plane located a distance fit from the tube lens, wherein fit is a tube lens focal length; and an image sensor that includes a pixel array to capture and output an image, wherein the pixel array occupies a portion of the tube lens focal plane, wherein diverging light from the light source passes through the objective lens and forms a light image at a distance from the objective lens that is greater than fobj, a reflected light component originating from the light image passes through the objective lens focal plane, through the objective lens and is focused by the tube lens onto the pixel array and further wherein the pixel array captures and outputs an image.

Description

Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 BACK-ILLUMINATION INTERFERENCE TOMOGRAPHYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to U.S. Provisional Patent Application No. 63 / 762,428 filed on February 24, 2025, the contents of which are hereby incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure is directed to correlating sensor, temporal, and navigation data in real time.BACKGROUND

[0003] 1. Introduction

[0004] High-resolution imaging of unlabeled bulk tissues is useful for characterizing biological tissues in vivo. A wide variety of imaging technologies have been introduced over the last few decades to address this need. Optical coherence tomography (OCT), which has gained widespread adoption in medical fields such as ophthalmology, provides depthsectioning and label-free imaging through low-coherence interferometry. Subsequently, full field optical coherence tomography (FF-OCT) was developed to eliminate the need for raster scanning. Scattering-based confocal microscopy, in which out-of-focus scattered light is rejected to achieve optical sectioning, has shown promise in dermatology, and adherent leukocytes have even been observed in human vasculature with this technique.

[0005] Since Zernike’s initial observation and publication on phase contrast, phase contrast microscopy techniques have become ubiquitous in biological research due to their label-free nature, negligible phototoxicity, high speed, and sensitivity to weakly scattering objects. Quantitative phase imaging (QPI) enables the measurement of the refractive index of unaltered tissue structures. Transmission-based illumination with thin, weakly scattering samples has long dominated the field of phase contrast microscopy to avoid the influence of multiple scattering that disrupts phase. Techniques such as gradient light interference microscopy (GLIM) and epi-GLIM have sought to address this using white light interferometry, a Nomarski approach, and phase shifting. Reflected light microscopy is often referred to as incident light, epi-illumination and is a method of choice for fluorescence and for image specimens that remain opaque even when ground to a thickness of 30 micrometers. InAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 epifluorescence microscopy, both the excitation and emission light travel through the same objective.

[0006] When introduced in 2012, Oblique Back-illumination Microscopy (OBM) provided a simple and fast approach to generate phase contrast images in bulk turbid media

[0014] , OBM uses laterally offset illumination sources to produce backscattered light that passes through the focal plane at a net oblique angle, encoding lateral phase gradients at this plane to intensity changes at the detector. Since this seminal work, the field has been further developed to enable quantitative phase imaging using deconvolution. While imaging bulk pathology and stationary in vivo samples has shown promise with qOBM, the requirement of multiple triggered sources and computational expense of reconstruction hinders the speed of this technology, making it difficult to apply to in vivo blood cell imaging.SUMMARY

[0007] According to examples of the present disclosure, a new epi-mode microscopy technique called back-illumination tomography (BIT). BIT generates contrast to weakly scattering objects using back-scattering of a spatially confined source imaged on-axis, beyond the objective focal plane. The technique operates similar to Optical Transmission Tomography (OTT) demonstrated recently and explained by the Gouy phase shift. We offer an alternative description of the contrast mechanism via the expected amplitude point spread function for a partially coherent system. BIT’s epi-mode geometry enables its use in bulk, turbid media, and its simplicity and speed make it appealing for in vivo blood cell imaging and bulk tissue histopathology. A high numerical aperture (NA) objective allows both the creation of spatially coherent backscattered light and a short axial imaging point spread function, which, in combination, allows the visualization of fine tissue structures with optical sectioning. We first demonstrate agreement between simulation based on Streibl’s model and experimental data from imaging scattering TiO2 particles. Next, we image human blood cells in vitro in microfluidic chambers to characterize the relationship between the illumination source image depth and BIT contrast. Finally, we demonstrate two applications of BIT: (1) imaging flowing human blood cells with high speed and high resolution in vivo, and (2) imaging unprocessed bulk human tissue specimens.

[0008] Biological tissues and cells are composed of discrete compartments of biochemical media that often exhibit subtle differences in refractive index. Light propagating through these compartments partially diffracts in a forward direction with a TT / 2 phase shift. We introduce a microscopy technique to image this scattering signal in thick tissues, called Back-illuminationAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 Interference Tomography (BIT). An incoherent source is demagnified and imaged past the focal plane of a high numerical aperture objective lens, producing a small, semicoherent source of backscattered light. This backscattered light undergoes a phase inversion over the narrow depth of field of the microscope, providing interference contrast to weakly scattering objects at the focal plane. BIT offers a fundamentally different source of contrast to conventional illumination and oblique back-illumination microscopy. Compared to these techniques, we show that BIT improves contrast to blood cells in vitro in microfluidic chambers and in vivo in a human capillary. Finally, we apply BIT to unstained, unlabeled bulk human tissue ex vivo and compare side-by-side to adjacent frozen sections stained with Hematoxylin and Eosin. These results demonstrate the potential of BIT to provide high resolution, high speed, 3D imaging of unprocessed biological tissues.

[0009] The development of techniques for high-speed, label -free imaging of bulk scattering media that can resolve the intricate structures of biologic tissue with epi-mode illumination continues to be a challenge. Here we present a novel such microscope configuration called back-illumination interference tomography (BIT). Through the use of a demagnified illumination source imaged beneath the objective focal plane, a partially spatially coherent transmission-like source is created through on-axis back-illumination. Scattering objects undergo a phase inversion about the narrow depth of field of the microscope, yielding enhanced contrast to scattering particles. According to examples of the present disclosure, the present technique yields improved contrast to blood cells in vitro in microfluidic chambers, fundamentally different contrast to conventional illumination techniques and oblique back-illumination microscopy, and even visualize blood cells in vivo in a human capillary. Additionally, the technique is applied to unstained, unlabeled bulk human tissue pathologic specimens, comparing side-by-side to Hematoxylin and Eosin stained adjacent slices. Together these results demonstrate the potential of BIT to provide high resolution, high speed scattering information in biologic tissue.

[0010] According to examples of the present disclosure, a back-illuminated interference tomography (BIT) microscope apparatus is disclosed. The microscope apparatus comprises a light source; an objective lens defining an objective lens focal plane located a distance fot>j from the objective lens, wherein fobj is an objective lens focal length; a tube lens defining a tube lens focal plane located a distance f L from the tube lens, wherein HL is a tube lens focal length; and an image sensor that includes a pixel array to capture and output an image, wherein the pixel array occupies a portion of the tube lens focal plane, wherein diverging light from the light source passes through the objective lens and forms a light image at a distance from theAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 objective lens that is greater than fobj, a reflected light component originating from the light image passes through the objective lens focal plane, through the objective lens and is focused by the tube lens onto the pixel array and further wherein the pixel array captures and outputs an image.

[0011] Various additional features can be included in the BIT microscope apparatus including one or more of the following features. The light source comprises a green light emitting diode (LED). The objective lens is a miniature objective lens, such as a Gradient Index (GRIN) lens, or a reversed aspheric compound lens (RACL). The BIT microscope apparatus further comprises a condensing lens placed less than its focal length away from the light source, reducing the divergence of the light as it enters the objective lens. The BIT microscope apparatus further comprises a flashing illumination source that is on for a short time relative to the movement of the sample, and then off until the next image is acquired. The illumination and detection paths each comprise linear polarizers with a polarizing axis that can be adjusted to modify image contrast. The light source has a small aperture placed in front of it. The light source is a near-infrared wavelength. The light source is a coherent laser source. The light source is a semicoherent light source, such as an LED with a pinhole or a superluminescent diode. The light is delivered through a fiber optic light guide. The objective lens is translated axially with a motor, such as a piezo electric stage or a stepper motor. The multiple images are acquired from different axial planes, and the information is combined to compute quantitative phase maps. The objective is inverted and a sample is placed on a glass window and scanned to acquire large field of view data. The multiple illumination colors are used to spectrally multiplex data from different illumination and imaging planes. The BIT microscope apparatus comprises a set of beamsplitters is used to split different axial imaging planes to different lateral planes on the image sensor. The BIT microscope apparatus further comprises a beamsplitter between the condensing lens and the objective lens. The acquired image is transformed to appear similar to a pathology stained image. The image sensor is a cell-phone camera or is connected to a cell phone for processing and / or image transmission.

[0012] According to examples of the present disclosure, a method of imaging using a back-illuminated interference tomography (BIT) microscope apparatus is disclosed. The method comprises providing light from a light source; receiving the light by an objective lens defining an objective lens focal plane located a distance fot>j from the objective lens, wherein fobj is an objective lens focal length; receiving the light by a tube lens defining a tube lens focal plane located a distance f L from the tube lens, wherein fiL is a tube lens focal length; and imaging an object using an image sensor that includes a pixel array to capture and output an image ofAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 the object, wherein the pixel array occupies a portion of the tube lens focal plane, wherein diverging light from the light source passes through the objective lens and forms a light image at a distance from the objective lens that is greater than fobj, a reflected light component originating from the light image passes through the objective lens focal plane, through the objective lens and is focused by the tube lens onto the pixel array and further wherein the pixel array captures and outputs an image.

[0013] Various additional features can be included in the method including one or more of the following features. The light source comprises a green light emitting diode (LED). The method further comprises mounting a three degree of freedom translation stage to the light source. The method further comprising mounting a LED heatsink to the translation stage. The method further comprising receiving the light by a condensing lens having a focal length equal to fcL, wherein the light source is located within the focal length of the condensing lens. The method further comprises splitting the light using a beamsplitter arranged between the condensing lens and the objective lens, a portion of the light from the condensing lens passes through the beamsplitter and into the objective lens and further wherein a portion of the reflected light component passing through the objective lens is directed by the beamsplitter to the tube lens. The beamplitter is a 50:50 non-polarizing beamsplitter. The objective lens is an infinity corrected microscope objective. The objective lens is a miniature objective lens, such as a Gradient Index (GRIN) lens, or a reversed aspheric compound lens (RACL). The condensing lens is placed less than its focal length away from the light source, reducing the divergence of the light as it enters the objective lens. The method further comprises a flashing illumination source that is on for a short time relative to the movement of the sample, and then off until the next image is acquired. The illumination and detection paths each comprise linear polarizers with a polarizing axis that can be adjusted to modify image contrast. The light source has a small aperture placed in front of it. The light source is a near-infrared wavelength. The light source is a coherent laser source. The light source is a semicoherent light source, such as an LED with a pinhole or a superluminescent diode.

[0014] According to examples of the present disclosure, when an object is imaged with a large field of view objective, the center of the field of view gives BIT contrast, while the periphery of the image gives Oblique Back-illumination Microscopy (OBM) contrast. In some examples, adjacent frames can be overlapped during scanning of the object, such as pathology samples, so that each piece of pathology tissue is sequentially imaged with BIT (when it's in the middle of the field of view), and then OBM (when it's on the side of the field of view).Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 These modalities provide complementary contrast of the tissue that is useful for generating histology-like images.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1A shows a back-illumination interference tomography (BIT) optical system according to examples of the present disclosure. FIG. IB shows an enlarged region of interest highlighting how BIT achieves partially spatially coherent backscattered illumination according to examples of the present disclosure.

[0016] FIG. 2A shows an image of scattering TiO? beads in polydimethylsiloxane (PDMS) phantom demonstrating axially-dependent bright-dark contrast with BIT due to partially spatially coherent illumination according to examples of the present disclosure. FIG. 2B shows lateral images of a 595 nm TiCh particle at 500 nm axial increments about the objective focal plane demonstrate a transition from relative bright to dark intensity according to examples of the present disclosure. FIG. 2C shows simulated axial intensity profile for 595 nm TiCh bead in PDMS with partially coherent illumination (y = 0.25) according to examples of the present disclosure. FIG. 2D shows a plot of normalized intensity vs. axial position for simulated particle (dashed line, from FIG. 2C) and measured TiCh particle (solid line, from FIG. 2B) according to examples of the present disclosure.

[0017] FIG. 3 A shows a microfluidic setup for in-vitro human blood cell imaging according to examples of the present disclosure. Blood cells were introduced into a microfluidic capillary approximately 100pm deep in a turbid medium. FIG. 3B shows images of the same red blood cell (RBC) as the LED source is moved from the collecting lens vertex (d = 0 mm) to the collecting lens back focal plane (critical illumination, d= -fcL mm) according to examples of the present disclosure. The contrast of RBC membrane decreases as the LED moves closer to critical illumination and source coherence decreases (increasing Onium). This is highlighted with normalized intensity plot profiles taken across the RBC at d= 0 mm (green), and d= -fcL mm (red). FIG. 3C shows images of the same two white blood cells (WBCs) as the LED source is moved from the collecting lens vertex (d = 0 mm) to the collecting lens back focal plane (critical illumination, d = ~ CL mm) according to examples of the present disclosure. At the critical illumination distance (d= -fcL mm), the LED was also translated laterally to generate oblique back-illumination microscopy (OBM) contrast.

[0018] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D show in vivo imaging of human ventral tongue vasculature with BIT according to examples of the present disclosure, where FIG. 4A shows RBCs flowing through human capillary with red arrows denoting the same RBC trackedAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 with passage of time (left to right, top to bottom), FIG. 4B shows zoomed region of interest tracking the same RBC from FIG. 4 A centered within the FOV, FIG. 4C shows WBC (white arrows) with preceding plasma gap and platelet passing through capillary moments later, and FIG. 4D shows zoomed region of interest tracking the same WBC from FIG. 4C centered within the FOV.

[0019] FIG. 5A and FIG. 5B show the pancreas and FIG. 5C and FIG. 5D show the duodenum imaged with conventional frozen section H&E (left) and with label-free BIT imaging of the adjacent bulk tissue face (right) according to examples of the present disclosure. Zoomed regions of interest highlighted with green boxes (H&E) and red boxes (BIT).

[0020] FIG. 6A shows a back-illumination interference tomography optical setup. An LED or other light source is placed within the focal length (JCL) of a condensing lens (CL) according to examples of the present disclosure. Diverging light passes through a beamsplitter (BS), and is imaged deep to the focal plane of an infinity-corrected microscope objective (obj). Back-scattered light is collected by the microscope objective, relayed off the same beamsplitter, and imaged onto a sensor (CMOS) by a tube lens (TL). FIG. 6B shows a region of interest around the object highlighting the LED imaged deep to the focal plane of the objective (dashed line 52) causing back-scattered transmission-like illumination of the object according to examples of the present disclosure. FIG. 6C shows a region of interest around a small scattering object centered at the objective focal plane. The scattered electric field (Es) incorporates an additional phase shift from the Gouy phase function <pG(z), which yields bright-dark contrast when interfered with the transmitted electric field (Er) at the image sensor. Note that dashed lines 50 and 52 denote conjugated planes.

[0021] FIG. 7A shows a schematic showing LED placed within focal length of condensing lens and resulting defocused image of the LED beneath the objective focal length (dashed line 52) according to examples of the present disclosure. FIG. 7B shows the distance from LED to condensing lens (d) in mm vs. distance from objective focal length to LED image in object (<5z) in pm using a thin lens model.

[0022] FIG. 8A shows TiCh particles in a PDMS phantom show bright-dark contrast parity due to interference contrast caused by the Gouy phase function according to examples of the present disclosure. FIG. 8B shows axially defocused images of the same TiCh particle shows characteristic bright-dark contrast caused by interference according to examples of the present disclosure. FIG. 8C shows a quantitative measurement of the normalized intensity of the axially defocused TiCh particle highlighted in FIG. 8B according to examples of the present disclosure.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0023] FIG. 9A shows red blood cell (RBC) imaging in a microfluidic chamber shows enhanced contrast with axial displacement of a 530nm Fiber Optic source from d = 0mm (touching the condensing lens vertex) to d = 12mm (the condensing lens back focal length -critical imaging) according to examples of the present disclosure. Interference contrast appears when the LED is placed within approximately fed?- FIG. 9B show intensity plot profiles from d = 0mm and d = 12mm that show enhanced contrast due back-illumination interference tomography technique according to examples of the present disclosure.

[0024] FIG. 10A shows back-illumination interference tomography compared with different conventional epi-mode illumination techniques as shown in FIG. 10B, FIG. 10C, and FIG. 10D according to examples of the present disclosure. FIG. 10B shows Kohler illumination with LED defocused in object space, FIG. 10C shows on-axis critical illumination with LED placed on-axis at condenser focal plane, and FIG. 10D shows oblique back-illumination capillaroscopy with LED placed axially at condenser focal plane but laterally displaced from optical axis according to examples of the present disclosure.

[0025] FIG. 11A and FIG. 11B shows in vivo, label-free human blood cell imaging with back-illumination interference. Tomography according to examples of the present disclosure, where FIG. 11 A shows a ROI around a single capillary shows high resolution of red blood cells passing through a single capillary at 200 Hz and FIG. 1 IB show smaller ROI manually tacking the same red blood cell (RBC) as it passes through the capillary loop shows characteristic bright-dark interference contrast as the RBC traverses different axial planes.

[0026] FIG. 12A shows a fiber-based back-illumination interference tomography that provides another possible illumination source for the system according to examples of the present disclosure. FIG. 12B shows an arrangement that given the diverging nature of an LED or fiber source, a condensing lens is not necessary to produce a defocused image beneath the objective focal plane according to examples of the present disclosure.

[0027] FIG. 13 A shows an arrangement that uses a piezo motor on either the objective or the object allow fine and precise axially displacement over a linear phase range according to examples of the present disclosure. FIG. 13B shows a combination of two images of the same object collected on opposite side of the objective focal plane yields by subtraction or some other operation provides a tomographic slice that rejects out-of-focus background-noise. 3D reconstructions are possible using sequential collection of different axial slices of an object.

[0028] FIG. 14A an inverted back-illumination interference tomography system can be used to image pathologic slides with or without staining protocols according to examples of theAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 present disclosure. A microscope slide scanner and piezo motor in this configuration would together enable whole-slide imaging of pathologic specimens with 3D reconstructions. FIG.14B shows an example image of bulk tissue shows promise of back-illumination interference tomography for slide-free imaging according to examples of the present disclosure.

[0029] FIG. 15 A shows a back-illumination interference tomography arrangement that can be combined with multiple spectral sources to produce angular illumination, combining back-illumination interference tomography with illumination akin to oblique back-illumination microscopy according to examples of the present disclosure. FIG. 15B shows a highlighted ROI around the LED and object show imaging multiple LEDs with lateral offset into back-scattering tissue producing symmetric and oblique illumination according to examples of the present disclosure.

[0030] FIG. 16 shows simultaneous acquisition of two axial planes using two synchronized axially displaced cameras enables high-speed acquisition of tomographic slices using back-illumination interference tomography according to examples of the present disclosure.

[0031] FIG. 17 shows chromatic aberration using two spectrally distinct illumination sources can be exploited to create multiple focal planes on the same CMOS according to examples of the present disclosure.

[0032] FIG. 18 shows chromatic aberration using two spectrally distinct illumination sources can be exploited to create multiple focal planes according to examples of the present disclosure. A dichroic mirror can be used to separate the two spectral channels onto two separate cameras, enabling high-speed acquisition of tomographic slices using spectral information.

[0033] FIG. 19 shows an arrangement where a prism can be introduced after the converging tube lens to split N images from different axial positions in the sample to N different lateral positions on the image sensor. A beamsplitter or set of beamsplitters can be used for this purpose. The combination of these images can provide background rejection, quantitative phase contrast, and tomographic 3D reconstructions.

[0034] FIG. 20A and FIG. 20B show an arrangement where back-illumination interference tomography can be optimized for different parts of a wide field of view by illumination with an array of laterally displaced light sources according to examples of the present disclosure. Each source will provide a semicoherent back-illuminated source to a small sub-field of the full objective field of view. These images can be stitched together from sequential acquisition in time or multiplexed with color or polarization encoding.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0035] FIG. 21 shows a back-illumination tomography can also be produced without the use of a beamsplitter by illuminating from a source positioned within the focal length of the tube lens on the detection path according to examples of the present disclosure.DETAILED DESCRIPTION

[0036] 2. Methods

[0037] 2.1. Optical System

[0038] FIG. 1A shows a back-illumination interference tomography (BIT) optical system according to examples of the present disclosure. By placing the LED within the focal length (fez) of the collecting lens (CL), diverging illumination is relayed through a beamsplitter (BS) and is subsequently imaged below the obj ective focal plane. One aspect of BIT is the generation of semi-coherent back-illumination, which may alternatively be created by removing the CL entirely. Backscattered light is collected by the same objective and imaged onto a CMOS via a tube lens (TL). FIG. IB shows an enlarged focal plane view that highlights how BIT achieves partially spatially coherent backscattered illumination according to examples of the present disclosure. The LED image is demagnified and imaged at a distance Sz beneath the objective focal plane. Backscattered light from the LED image creates transmission-like illumination. This illumination is partially spatially coherent because of the high objective collecting angle (Oobj) and relatively low source illumination angle (Biiium).

[0039] As shown in FIG. 1A, BIT optical system 100 comprises LED 102, such as a green LED, placed within the back focal length (BFL) of a condensing lens (CL) 104, such as a Thorlabs ACL25416U, a distance d away from the lens vertex 106. LED 102 is mounted on XYZ translation stage 108 by affixing the LED heatsink 110 to XYZ translation stage 108, such as a Thorlabs kinematic mount using J-B Weld SteelStik. Diverging light 112 is relayed through the condensing lens 104 through a 50:50 non-polarizing beamsplitter (BS) 114, such as a Thorlabs CCM1-BS013, undergoing a reverse pass through infinity corrected microscope obj ective 116, such as a Nikon 40X 1.15 NA APO LWD WI IS). For experiments that require axial scanning, infinity corrected microscope objective 116 was mounted on a piezo collar 118, such as a Thorlabs PFM450E or Nanomax XYZ translation stage (Thorlabs MAX313D). An image of the LED is formed at a distance 6z beyond the focal plane of the microscope objective, with a diameter of \DLED\ (1). Note that the dashed lines 120, 122 in FIG. 1A and FIG. IB represent conjugate planes. The conjugate plane defined at the back focal length of CL 104 is decoupled from the position of the LED 102, creating the image of the LED 102 beyond theAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 objective focal plane. The objective focal plane, and thus the object being imaged, is made conjugate with the tube lens back focal plane, where the CMOS sensor is placed.

[0040] Scattered light from the high intensity region defined by the LED image creates net on-axis back-illumination from a spatially confined source. The coherence factor, y, from the LED image is:y — N Amum / N Aobjwhere NAuium. and / NAobj. are the numerical apertures of the effective illumination and objective lens, respectively. The system presented here uses: fd = 16 mm, fobj = 5 mm, and 89 d = -4 mm. Using a thin lens approximation, 6z = 200 / / m and IDLEDI = 75 / / m, resulting in a coherence factor of y = 0.25. Back-illuminated light from the defocused LED image passes through the object near the objective focal plane, yielding transmission-like illumination with a partially coherent source. Light is collected by the high NA objective, reflected off the beamsplitter, and imaged through a tube lens (TL) onto a CMOS sensor (pco.edge sCMOS 5.5 or The Imaging Source DMK 33UX252, for the case of in vivo blood cell imaging ).

[0041] Three different illumination source types were used in this study: a 530nm green LED (Luxeon Star Green Saber Z1 10mm, 92 Im, 500mA), a 660nm red LED (Luxeon Star Deep Red Saber Z1 10mm, 325 mW, 500mA), and a 530nm green fiber source (Thorlabs M530F1 coupled to FT400UMT 0.39 NA, 400 / / m). Note that data collected and presented in FIG.2A, FIG. 2B, FIG. 2C, and FIG. 2D and FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D used the 530nm green LED, FIG. 3A, FIG. 3B, and FIG. 3C used the 530nm green fiber source, and data collected and presented in FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D used the 660nm LED source. The choice of source type was dictated by the need for including hemoglobin absorption signal, steric hindrance that was eased by using a fiber source that allowed d = 0 mm, and empirically observed optimization of contrast for the pathologic tissue specimen in the red portion of the visible spectrum.

[0042] 2.2. Overview of contrast mechanism

[0043] FIG. 2A shows an image of scattering TiO? beads in polydimethylsiloxane (PDMS) phantom demonstrating axially-dependent bright-dark contrast with BIT due to partially spatially coherent illumination according to examples of the present disclosure. FIG. 2B shows lateral images of a 595 nm TiO? particle at 500 nm axial increments about the objective focal plane demonstrate a transition from relative bright to dark intensity according to examples of the present disclosure. FIG. 2C shows simulated axial intensity profile for 595 nm TiCh bead in PDMS with partially coherent illumination (y = 0.25) according to examples of the presentAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 disclosure. FIG. 2D shows a plot of normalized intensity vs. axial position for simulated particle (dashed line, from FIG. 2C) and measured TiCh particle (solid line, from FIG. 2B) according to examples of the present disclosure.

[0044] The 3D Optical Transfer Function (OTF) for a partially spatially coherent telecentric optical imaging system with circular source size ps and circular pupil size ppwas derived previously in Streibl. In this model, the OTFs for imaging the phase, Tp( , 17), and absorption, TA(JJ., if), of an object are.

[0045] Image formation can then be described as the convolution of the object’s phase P(r) and absorption (r) spatial distribution with the system point spread function and an added background intensity term (B). The system point spread functions are the Fourier transforms of the phase and absorption OTFs, TP(r), and T / i(r), respectively:

[0046] For partially spatially coherent illumination on the axis, the OTF exhibits odd symmetry about the J] = 0 plane. This results in minimal contrast to phase objects centered around the objective focal plane. However, for objects axially displaced a small distance away from this plane (approximately / i the depth of field), the symmetry is broken. This results in high contrast, as observed by Zernike on phase contrast. In addition, the OTF incorporates a TT phase inversion for scattered objects centered axially around the objective focal plane on the observed field. Consequently, there is an intensity inversion for scattering objects that occurs, centered around the objective focal plane. This phenomenon vanishes for incoherent illumination (y — 1). Though this effect is commonly observed in transmission microscopy, BIT is novel in that it enables epi-mode imaging of these scattering objects using a simple illumination modification.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0047] Far away from the center of the object field of view, which is typically 100 - 200 micrometers, the light collected by the objective lens passes through the focal volume at a net oblique angle, causing oblique back-illumination microscopy contrast. BIT imaging there for provides axial interferometric phase contrast in the center of the field of view and simultaneously lateral phase contrast at the periphery of the field of view. These sections may be analyzed using different reconstruction techniques based on their different contrast mechanisms.

[0048] 2.3. TiC>2 Scattering Measurement and Simulation

[0049] To evaluate the contrast generated in BIT, a simple scattering-only phantom was fabricated by mixing TiCh particles in a Polydimethylsiloxane (PDMS) phantom. TiCh particles were dissolved at a concentration of 1 mg / mL to mimic tissue scattering of approximately 's-Umm'1. With the 530nm LED illumination, from LED 102, placed at approximately d = 4 mm, an axial scan of TiCh particles was acquired in 500nm axial increments. The results of this experiment are shown in FIG. 2A and FIG. 2B. Note that images are processed with a flat-field correction algorithm adopted from Ford et al., whereby images are divided by an 80-pixel Gaussian blurred version of themselves to generate a 32-bit corrected image, and contrast adjusted from [0.9,1.1] to improve visualization and generate near-uniform illumination across the FOV.

[0050] We use the above equations to simulate the axial intensity distribution for a spherical TiCh particle in PDMS with y = 0:25 (FIG. 2C). The profile of this simulation is compared to experimentally measured imaging data in FIG. 2D. Note that each of the images, experimental and simulated data, underwent intensity normalization using the flatfield corrected algorithm described above.

[0051] 2.4. In-vitro static blood cell imaging experiment

[0052] Tissue-mimicking microfluidic chambers were fabricated following a previously developed protocol. Briefly, using spin-coating, SU8-3010 photoresist (PR) is deposited uniformly on a 76.2mm silicon wafer (University Wafers #447) and exposed to UV light through a photomask in the shape of branching microvasculature (FIG. 3 A). The PR is cured, producing 25 pm tall channels on the device mold. Separately, PDMS is doped with TiCh and India ink to approximate tissue optical properties ( .s' = 1.7mm'1and )J.a= 0.017mm'1). The turbid PDMS is cast over the device mold, cured, cut out, and channels are cut through the device base with a blunt-tipped 20 gauge needle. Separately, a thin membrane of PDMS approximately 100 pm thick, also doped with TiCh and India ink, is spin coated on an additional silicon wafer coated with a soft-baked layer of PR (S-1813). The device base is plasma bondedAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 to the thin membrane and the second silicon wafer is released from the finished device using an acetone bath to dissolve the soft-baked PR layer. This produces a PDMS microfluidic device with optical properties mimicking human tissue and 25 pm tall capillary channels embedded beneath 100 pm of turbid media.

[0053] Whole blood, purchased from ZenBio (SER-WB 10ML-SDS), was loaded into a ImL syringe and connected to the microfluidic device using PEEK tubing and Luer adapters (IDEX 1569L, P-659, F-247, F-333NX). After initial blood flow was produced with a syringe pump, the flow was stopped and whole blood inside the microfluidic device was imaged while mostly stationary (only undergoing Brownian motion). For a fixed field of view containing a RBC of interest, the distance from the 530 nm green fiber tip to the condensing lens (d) was varied with an image taken at 2 mm increments of axial translation (FIG. 3B). The LED position was varied across the full axial range of interest, from lens vertex (d = 0 mm) to the back-focal plane of CL (critical imaging, d = -12 mm = -fed). Plot profiles at the two extremes are shown. The experiment was repeated again, with a pair of white blood cells (FIG. 3C). Finally, for the white blood cells, with the fiber tip at critical illumination d= -fcL, the fiber was laterally translated to generate comparative oblique-back illumination microscopy images.

[0054] 2.5. In vivo human blood cell imaging

[0055] The BIT microscope was inverted and mounted to image the ventral surface of a human tongue to image blood cells flowing through superficial capillaries in vivo. Using a custom pneumatic suction objective cap, capillaries were fixed during imaging. Compared to previous studies, to reduce the bulkiness of the system, a smaller and lower-cost image sensor was used (The Imaging Source DMK 33UX252). Videos were acquired at 200 fps, with an exposure time of 1 ms, a gain of 19.7, and a gamma correction of 4. 530 nm green LED illumination was used to enable simultaneous acquisition of absorption and phase signal from blood cells.

[0056] 2.6. Ex vivo human pathologic tissue imaging

[0057] The BIT microscope was adapted for ex vivo tissue pathology imaging. A piezoelectric stage (Thorlabs PFM450E) was installed on the microscope objective to enable axial scanning. A matte black spray paint was applied to the internal brass collar of this stage to reduce stray reflected light. For comparative transmission images of sliced tissue, we added a Kohler illumination system with a white light LED (Luezon Star 6500K Saber Z 1 10 mm 154 Im @ 500 mA). A 660 nm LED was used for BIT imaging in epi-mode, as shown in FIG. 1 A and FIG. IB. A flip mirror at 45° was installed after the beamsplitter and tube lens along with an RGB sensor (The Imaging Source DFK 38UX304) to enable color acquisition ofAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 Hematoxylin and Eosin (H&E) stained tissue samples. This approach enabled conventional microscope slides to be imaged with white illumination in transmission mode, and easily swapped for bulk tissue imaging with epi-illumination in BIT.

[0058] Discarded tissue specimens were obtained from Whipple procedures was frozen in O.C.T. compound (Fisher Healthcare Tissue-Plus O.C.T. Compound 23-730-571), sectioned in a cryostat with 15 pm slices, mounted on (+) charged 75 mm slides (Tedpella - Diamond 260382-1), stained following conventional H&E protocol (Abeam ab245880), and preserved using mounting media (Tedpella Histomount 19479) and a coverslip (Tedpella 260140). The remaining tissue on the chuck was thawed in room temperature PBS, placed within a 35 mm glass bottom culture dish (Cellvis D35-28-1.5-N) and gently compressed using a coverglass window. This surface is unstained and unsectioned bulk tissue, but is the directly adjacent tissue face to corresponding sliced and stained H&E images.

[0059] 3. Results and Discussion

[0060] 3.1. TiC>2 Scattering Measurement and Simulation

[0061] To characterize the observed contrast generated by the BIT system, a simple scattering phantom comprising TiCh particles in PDMS was imaged. FIG. 2A, FIG. 2B, FIG.2C, and FIG. 2D show the results of this experiment. First, in FIG. 2A, we observe that the scattering-only TiCh particles exhibit a bright-to-dark intensity variation with respect to the background intensity. To further highlight this, FIG. 2B shows one such TiCh particle with a ~595nm measured diameter imaged at five distinct axial locations centered about the objective focal plane. When placed beyond the objective focal plane (~z defocus), the particle appears bright, as it is translated to the focal plane the contrast decreases (due to the 3D OTF odd symmetry about the = 0 plane), and when translated to within the focal plane ( +z defocus), the same particle becomes dark. This behavior is expected for partially spatially coherent illumination of a scattering object.

[0062] We next simulated the scenario of a 595nm TiO? particle in PDMS with a coherence factor of y = 0:25, predicted by our thin-lens model. FIG. 2C shows the resulting simulation in an axial xz plot. Taking axial plot profiles and mean intensity measurements through the axial focus of the measured particle (solid line) and the simulated particle (dashed line), we observe good agreement for the predicted axial range over which a particle of this size and index of refraction undergoes intensity inversion, as shown in FIG. 2D.

[0063] Discrepancies between the measured and simulated data are likely due to vibrations of the optical table dithering the axial and lateral position, multiple scattering events not accounted for in the simulation (which uses the first Bom approximation), and the TiCh particleAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 imaged is not a perfect sphere (as is assumed in the simulation). Still, the agreement between the experiment and the simulation suggest that the signal arises from transmission-mode illumination with a partially spatially coherent source. The defocused, demagnified LED image beneath the particle produces transmission-like illumination from a spatially confined source due to net back-illumination.

[0064] 3.2. Imaging static blood cells

[0065] Blood cells were imaged in vitro in a tissue-realistic microfluidic chamber emulated capillaries in skin. In this setup, the blood flow could be stopped, allowing for the same blood cells to be imaged as the configuration of the BIT system was changed. Of particular interest is the contrast sensitivity to the variable d, the distance of the illumination source from the condensing lens (CL) vertex.

[0066] FIG. 3A shows a schematic of the microfluidic chamber experiments used for this experiment according to examples of the present disclosure. Blood flow was stopped, and a field of view with a flat RBC that shows its characteristic bilobed shape was selected for imaging, as shown in FIG. 3B. The fiber illumination source was brought in contact with the lens vertex (d= 0mm), and axially translated at 2 mm steps until reaching critical illumination at the back focal plane of the CL (d= -fed). As this occurs, the contrast of the blood cell varies significantly, dropping off when |d| > 4mm. Plot profiles across the red blood cell (RBC) are taken at the two extremes (green: d = 0mm, red: d = -fed), showing how visibility of the RBC membrane is enhanced with BIT illumination. Interestingly, due to the green illumination chosen for this experiment, it appears that absorption-only information becomes present at critical illumination (d= -fed).

[0067] Next, we sought to study a predominately phase-only object. A pair of white blood cells (WBCs) were located stationary in the microfluidic chamber and imaged undergoing the same axial source translation as conducted in FIG. 3B. The results of this are shown in FIG.3C. Again, visibility of the cells, and even subcellular components, are significantly enhanced with |d| < 4 mm. The contrast degrades rapidly as |d| increases, and the phase-only cells are nearly invisible with critical illumination. Finally, with the source axially positioned for critical illumination (d= -fed), the source was subsequently laterally translated to produce an oblique back-illumination microscopy (OBM) configuration. The cells now demonstrate the characteristic lateral bright-to-dark intensity gradient caused by lateral phase gradients in OBM. However, it is clear from these data that on-axis defocused back-illumination in BIT produces a fundamentally different contrast as compared to the laterally offset, critically imaged OBM configuration.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0068] 3.3. In vivo human blood cell imaging

[0069] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D show in vivo imaging of human ventral tongue vasculature with BIT according to examples of the present disclosure, where FIG. 4A shows RBCs flowing through human capillary with arrows denoting the same RBC tracked with passage of time (left to right, top to bottom), FIG. 4B shows zoomed region of interest tracking the same RBC from FIG. 4A centered within the FOV, FIG. 4C shows WBC (arrows 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445) with preceding plasma gap and platelet passing through capillary moments later, and FIG. 4D shows zoomed region of interest tracking the same WBC from FIG. 4C centered within the FOV.

[0070] FIG. 5A and FIG. 5B show the pancreas and FIG. 5C and FIG. 5D show the duodenum imaged with conventional frozen section H&E (left) and with label-free BIT imaging of the adjacent bulk tissue face (right) according to examples of the present disclosure. Zoomed regions of interest highlighted with boxes 501, 502, 503, 504, 505 (H&E) and boxes 510, 511, 512, 513, 514 (BIT).

[0071] Further investigating the use case of blood cell imaging with BIT, we imaged the ventral tongue capillaries of a human participant. Data from this experiment are shown in FIG.4A, FIG. 4B, FIG. 4C, and FIG. 4D, with corresponding visualizations (visualization 001). FIG. 4A shows RBCs flowing through a capillary loop with the same RBC denoted with the arrows 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420. In FIG. 4B, a smaller ROI is selected that tracks with this same RBC centered in the frame. The capillary loop itself was observed to have a geometry projecting in the axial direction. As the RBC passes through this capillary loop, its axial position with respect to the focal plane of the objective varies. Even though it is a primarily absorptive object at the 530nm wavelength used in this experiment, the measured intensity of the RBC varies from bright to dark, just as observed with the TiCh particle experiments. Thus, BIT is capable of generating partially spatially coherent transmission-like illumination in vivo. The visibility of the resulting cells is remarkable.

[0072] The other major cellular components of blood are platelets and WBCs. Thus, we sought to find and image these objects with BIT in vivo as well. FIG. 4C and FIG. 4D show this, whereby a white blood cell passes through the capillary loop proceeded by a plasma gap and a platelet (visualization 002). Thus, BIT shows great promise in the field of non-invasive, label-free blood cell imaging.

[0073] 3.4. Ex vivo human pathologic tissue imagingAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0074] Another use case for BIT is in pathology, where label-free contrast to bulk tissue can be achieved that would be compatible not only with ex vivo pathologic specimens, but also with in vivo bulk tissue prior to resection. To preliminarily test this, we imaged adjacent regions of tissue with conventional frozen section and H&E staining and label-free bulk tissue BIT imaging. The results of this experiment are shown in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D, with pancreas shown in FIG. 4 A and FIG. 4B and duodenum shown in FIG. 4C and FIG. 4D. Tissue types are paired between BIT and H&E column-wise, with zoomed in ROIs shown as highlighted in boxes in the right-most columns. It is clear from these results that BIT is capable of imaging nuclei and surrounding tissue structures, despite having no absorption-based contrast mechanism and without transmission illumination typically required for phase contrast techniques. Further studies across different tissue types, with both benign and diseased tissue, in vivo and ex vivo will further elucidate the potential of BIT in this field. With sufficient training data generated via this adjacent tissue imaging technique, a generative adversarial network could be trained to convert BIT images to H&E stained images, as has been shown in other similar recent works

[0016] ,

[0075] FIG. 6A presents the basic components of a back-illumination interference tomography (BIT) optical setup 1. An LED or other light source 10 is placed within the focal length (fcr) of a condensing lens 12. The point source or diverging light 70 from light source 10 passes through the condensing lens 12, which reduces the divergence of the diverging light 70, i.e., the condensed light 72 is closer to parallel. Because the light source 10 is within the fcr of the condensing lens 12 the focal length of the condensed light 72 is fci. That is, the condensing lens 12 causes the condensed light to appear to have originated from a point source at the focal plane 50 of the condensing lens 12. A portion of the condensed light 72 passes through beamsplitter 14 and into an objective lens 16. The objective lens 16 has a focal length of fobj such that the objective lens focal plane 52 is the distance fobj from the plane of the objective lens 16. The objective lens 16 may be an infinity corrected microscope objective.

[0076] The portion of the condensed light 72 that passes through the objective lens 16 can be referred to as the objective light 74. The objective light 74 having been modified by the objective lens 16, it creates a light image 18 deep to the objective lens focal plane 52. That is, the objective light 74 passes through the objective lens focal plane 52 and forms the light image 18 on the side of the focal plane 52 opposite the objective lens 16 side.

[0077] FIG. 6B is a detail portion of FIG. 6A showing a portion of objective lens 16 adjacent a scattering object 24, the objective light 74, light image 18 and back-scattered light 76. A portion of the back-scattered light 76 interacts with the scattering object 24. Whether havingAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 interacted with the scattering object 24 or not, most or all of the back-scattered light 76 is collected by the objective lens 16 and reflected off the beamsplitter 14 onto a complementary metal-oxide semiconductor (CMOS) sensor 22 by a tube lens 20. The tube lens 20 has a focal length of frL. The sensor 22 may include a pixel array (not shown) or other means permitting the sensor to acquire an image. The focal plane 54 of the tube lens 20 may be coplanar with the pixel array such that the image captured by the sensor 22 is captured at the tube lens focal plane 54.

[0078] FIG. 6B highlights the back-scattered illumination 76 from the LED light image 18 deep to the focal plane 52 of the objective 16 that illuminates and interacts with the object 24.

[0079] FIG. 6C isolates the scattering object 24 of FIG. 6B centered on the objective focal plane 52 and being illuminated by the back-scattered light 76. The back-scattered light 76 encountering the object 24 results in transmitted light 78 and scattered light 80, depending upon the physical parameters of the object 24. The scattered light 80 undergoes an additional TT phase shift, resulting in an intensity inversion for an axial phase gradient when combined with the transmitted light 78 at the image sensor 22. Due to the Gouy phase shift about the objective focal plane 52, the combined scattered 80 and transmitted 78 field results in constructive or destructive interference and corresponding bright or dark intensity measurements at sensor 22 when the object is a Rayleigh length (ZR) above or below the focal plane 52.

[0080] FIG. 7A shows details of a BIT apparatus on accordance with an embodiment isolating the LED light source 10 showing how it is placed within the focal length (f'ci.) of condensing lens 12 and the resulting defocused LED light source image 18 beneath the focal plane 52 of the objective lens 16. FIG. 7B is a graph of the relationship between the distance d from the LED light source 10 to the condensing lens 12 in mm vs. the distance 6z from the objective focal plane 52 to the light source image 18 in micrometers (pm) using a thin lens model. The thin lens model allows for the calculation of the approximate image plane of an objective, given the focal length of the lens and the position of the object. 1 / f = l / d_object + l / d_image . For reference, it is called a “Gaussian thin lens equation” on wikipedia: https: / / en.wikipedia.org / wiki / Thin_lens and in the textbook ref: Hecht, Eugene (1987). Optics (2nd ed.). Addison Wesley. § 5.2.3. ISBN 0-201-11609-X.

[0081] FIG. 8A is an image from the CMOS sensor 22 of titanium dioxide (TiO?) particles in a polydimethylsiloxane (PDMS) tissue-mimicking phantom taken with an embodiment of the BIT apparatus. The image shows bright-dark contrast parity due to interference contrast caused by the Gouy phase function. FIG. 8B presents five CMOS sensor 22 images of a singleAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 PDMS phantom having variable axially defocused images of the same TiCh particle showing characteristic bright-dark contrast resulting from the Gouy interference. FIG. 8C illustrates a quantitative measurement of the normalized intensity of the axially defocused TiCh particle highlighted in FIG. 8B. The axial distance in FIG. 8C is the Sz in FIG. 7A, i.e., a measure in pm of the distance between the light source image 18 and the focal plane 52 of the objective lens 16.

[0082] FIG. 9A presents CMOS sensor 22 images of about a dozen red blood cells (RBCs) in a microfluidic chamber; the images are identical except that the distance d from the LED light source 10 to the condensing lens 12 varies between 0 mm and 12 mm in 2 mm steps. Thus, d is a measure of the axial displacement of a 530 nanometer (nm) fiber optic light source 32 from the condensing lens 12 from d = 0 mm (touching the condensing lens 12) to d = 12 mm. For this condensing lens 12, / CL = 12 mm. Thus, d is varied across the entire CL distance. As seen in FIG. 9A, useful interference contrast appears when the light source 10 or 32 is placed within approximately / ci / 2, i.e., within half the focal length or about 6 mm, of the condensing lens 12.

[0083] FIG. 9B compares the intensity plot profiles for d = 0 mm (dashed line) and d = 12 mm (solid line). The dashed line (light source touching condensing lens) represents enhanced contrast resulting from the BIT technique in accordance with an embodiment presented herein while the dashed line represents poor contrast because d, at 12 mm, is too large. Referencing FIG. 9B, setting d to the fcL of 12 mm results in the light source image being disposed on the objective focal plane 52 and, thus, not providing semi-coherent back-lighting.

[0084] FIG. 10A presents an image of a capillaroscopy utilizing the present BIT apparatus and technique. FIG. 10B is an image of Kohler illumination of the same capillaroscopy process with LED light source defocused in object space, FIG. 10C is a capillaroscopy image taken with critical illumination of the LED light source placed on-axis at the condenser focal plane. FIG. 10D shows oblique back-illumination capillaroscopy with the LED light source placed axially at the condenser focal plane but laterally displaced from the optical axis. Referencing FIG. 6A, this lateral displacement is generated by moving the light source along the CL 12 plane 50 away from the optical axis of the objective lens. This has the effect of producing an image of the light source at the objective focal plane 52, but laterally offset from the objective optical axis. Compared to the BIT contrast in FIG 10A, the oblique back-illumination technique in FIG 10D shows inferior contrast to small phase object, such as the intercellular granules present in the imaged white blood cell.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0085] FIG. 11 A presents a series of images of in vivo, label-free human blood cell imaging with the present BIT technique. The images of FIG. 11A were taken of a region-of-interest around a single capillary to show high resolution of red blood cells passing through a single capillary from a video acquired at 200 frames per second The images of FIG. 1 IB are of a smaller region-of-interest manually tracking the same RBC as it passes through the capillary loop and illustrating characteristic bright-dark interference contrast as the RBC traverses different axial planes. The bright-to-dark intensity change confirms the interferometric contrast that is fundamentally different from the oblique back-illumination microscopy technique and also highlights the opportunity for quantitative axial phase contrast estimation by combining multiple images from different axial planes.

[0086] FIG. 12A presents the BIT technique and apparatus utilizing a fiber optic light source 32 as an alternative illumination source for the system. The fiber optic light source image 34 presented below the focal plane 52 of objective lens 16 will be an image of the tip of the fiber optic light source 32. FIG. 12B is another alternative apparatus, useful with either an LED light source 10 or a fiber optic light source 32. Given the diverging nature of an LED or fiber source, a condensing lens is not necessary to produce a defocused light source image 18 beneath the objective focal plane. Thus, FIG. 12B shows the apparatus without a condensing lens. In such a setup, it may be advantageous to have the LED light source 10 or fiber-optic light source 32 closer to the beam splitter 14 relative to the setup that includes a condensing lens 12.

[0087] FIG. 13A utilizes a stepping motor 36, e.g., a piezoelectric motor, controlled by a controller 38. The stepping motor 36 may be connected to the objective lens 16 or the structure supporting the objective lens 16 such that fine and precise axial displacement of the objective lens 16 up or down relative to FIG. 13 A, over a linear phase range may be imparted to the lens 16 via the controller 38. Alternatively, the stepping motor 36 may be attached to whatever structure supports the scattering object 24 such that the controller 38 causes axial displacement of the scattering object 24 relative to the stationary objective lens 16. Since the focal plane 52 of the objective lens 16 is a set distance from the objective lens 16, i.e., fobj, axial movement of the objective lens 16 relative to the scattering object will result in axial movement of focal plane 52 relative to the scattering object 24. Multiple images of the same object 24 may be acquired with relative movement of the objective lens 16 versus the scattering object 24 and, thus, movement of focal plane 52 relative to the scattering object 24.

[0088] A number of useful outputs utilizing images collected by the CMOS sensor 22 may be achieved by the relative axial displacement of the conjugate imaging plane corresponding to CMOS sensor 22 relative scattering object 24. For example, FIG. 13B presents two imagesAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 of the same scattering object 24 collected on opposite sides of the objective focal plane 52 yielding, through subtraction, a tomographic slice that rejects out-of-focus background noise. Further, sequential collection of images by CMOS sensor 22 for different axially shifted slices of an object 24 may be reconstructed into a 3D representation of the object. This being a tomographic technique, a computer may be utilized to perform and optimize any combination of standard tomographic processes on a set of images or slices. Further, the 3D representation of the object may be transformed to look like a conventional pathology stained tissue or analyzed by a computer algorithm for clinical assessment.

[0089] FIG. 14A illustrates use of the BIT system to image a scattering object 24, e.g., a pathology specimen, disposed on a microscope slide 44 with or without staining protocols. The microscope slide 44 may be disposed on a scanner bed 40, which scanner bed 40 can be moved manually or by motors (not shown) along one or both the x-axis and y-axis of the scanner bed 40. Such movement permits different portions of the scattering object 24 to be aligned with the primary axis 64 of the BIT system. Movement of the scanner bed 40 relative to the z-axis, which is parallel to the BIT primary axis 64, may also be possible. In the event that the scanner bed 40 has an adjustable z-axis, it may be possible to eliminate the piezo electric stepping motor 36 attached to the objective lens 16 because the movement of the sample in the z-direction achieves the same result as the stepper motor 36 of FIG. 13 A. Movement of the scanner bed 40 may be controlled utilizing a scanner bed controller 42. The scanner bed 40, bed controller 42 and, if needed, piezo motor 36 in this BIT configuration would together permit whole-slide imaging of pathologic specimens with 3D reconstructions of slice images. That is, movement of the scanner bed 40 by scanner bed controller 42 coordinated with image capture by the CMOS sensor 22 would permit multiple z-axis slices and multiple x, y-axes images. Images collected by the CMOS sensor 22 could, using various tomographic techniques, be combined to achieve many different useful results.

[0090] FIG. 14B is an example single BIT image of bulk biological tissue which shows promise of back illumination interference tomography for slide-free imaging. Cellular membranes and subcellular compartments, such as small granules and the nucleus are clearly visible. Sharp optical sectioning is achieved without the use of any spatial or coherence gating, as is required with confocal microscopy and optical coherence microscopy, respectively. Unlike oblique back-illumination microscopy, which produces a directional bright-to-dark contrast for each lateral phase gradient object, the contrast of the object in BIT imaging is relatively insensitive to its position around the field of view.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02

[0091] FIG. 15A illustrates a BIT system utilized in combination with multiple differentiated light sources to image biological sample 30. LED light source 10R may comprise one set of electromagnetic frequencies, e.g., ‘red’ light, while LED source 10G may consist of a different set of electromagnetic frequencies, e.g., ‘green’ light. As illustrated in FIG. 15 A, these two differentiated light sources may be axially aligned or offset from axis 54. These differentiated frequencies may simultaneously travel through the objective lens 16 and result in a red-light source image 18R and a green-light source image 18G, as best illustrated in FIG. 10B, sending two sources of back-scattered light 76R and 76G interacting with the scattering object 24. Back-scattered light 76G is normal to the scattering object 24 while back-scattered light 76R provides angularly offset illumination of object 24. Beamsplitter 15 may be configured such that back-scattered light 76R is directed through tube lens 20R and as image light 82R to CMOS sensor 22R while back-scattered light 76G is directed through tube lens 20G and as image light 82G to CMOS sensor 22G. Such an arrangement could be used to combine back-illumination interference tomography with oblique back-illumination microscopy illumination. Each of FIG. 15A and 15B show how the placement of the multiple LED light sources 10G, 10R relative to the primary axis 54 results in a mirror-image LED light source image 18G, 18R about the primary axis 54. Both the axially aligned light and lateral offset light, as noted, results in back-scattered light 76G, 76R interacting with the scattering object 24 and producing symmetric and oblique illumination.

[0092] FIG. 16 illustrates a BIT system utilized in combination with multiple CMOS sensors permitting synchronized, high-speed acquisition of tomographic slices. The arrows representing light in other figures is not included in FIG. 16 to clearly show the apparatus. Light source 10, beamsplitter 14 and objective lens 16 are arranged in a similar setup to FIG.6A. However, a second beamsplitter 14’ divides the light it receives between a first CMOS sensor 22-1 and a second CMOS sensor 22-2. Each CMOS sensor 22 is displaced axially from the focal plane of the tube lenses 20-1 and 20-2. This results in the conjugate image planes of each sensor in the object plane to be axially displaced above or below the objective focal plane 50 by a distance Azo. In FIG. 16, CMOS sensor 22-2 is placed at plane 58-2, which is within the focal length of tube lens 20-2, resulting in a conjugate imaging plane beyond the focal plane 50 of the objective lens, i.e., at a point +Azo past the objective lens plane 50. CMOS sensor 22-1 is placed at plane 58-1, which is beyond the focal length of tube lens 20-1, resulting in a conjugate imaging plane within the focal plane 50 of the objective lens, i.e., at a point -Azo in front of the objective lens plane 50. Such an arrangement could be used to simultaneouslyAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 acquire two synchronized axially displaced planes using BIT and combined using the techniques described in FIG. 13, to produce high speed tomographic slices of phase contrast.

[0093] FIG. 17A illustrates a BIT system utilized in combination with multiple light sources. For example, a LED light source 101 emitting EM frequencies in the near-infrared (NIR) range and a LED light source 10R emitting EM frequencies in the red range have their frequencies combined by, for example, a NIR dielectric beam mirror 26. The combined 10R, 101 light may travel through the objective lens 16 and be received by the CMOS sensor 22. In this embodiment, CMOS sensor 22 is capable of receiving images at different focal planes. For example, CMOS sensor 22 may be axially moved such that its sensor plane 58 aligns with the red sensor plane 58R to focus and image the red image light 82R and thence moved axially to align with the NIR sensor plane 581 to focus and image the NIR image light 821. Thus, such a BIT setup permits exploitation of chromatic aberration using two spectrally distinct illumination sources to create multiple focal planes on the same CMOS sensor.

[0094] FIG. 18 illustrates a BIT system utilized in combination with multiple light sources and multiple CMOS sensors. A LED light source 101 emitting, for example, EM frequencies in the near-infrared range and a LED light source 10R emitting, for example, EM frequencies in the red range have their EM waves combined by, for example, a NIR dielectric beam mirror 26. These two light sources can be said to create spectrally distinct light sources. The optional condensing lens 12, beamsplitter 14 and objective lens 16 may be arranged in a similar setup to FIG. 6A, resulting in a combined 18R, 181 light source image deep to the focal plane 52 of the objective lens 16. The back-scattered combined light passes through objective lens 16 reflects off beamsplitter 14 and then encounters a component 28, e.g., a dichroic mirror, that divides the light it receives between a first CMOS sensor 22-1 and a second CMOS sensor 22-2. Each CMOS sensor 22 has a different focal plane 58 at which a sensor image is captured. CMOS sensor 22-2 has a focal plane 58-2 that receives an image and CMOS sensor 22-1 has a focal plane 58-1 resulting in another image. The images of CMOS sensors 22-1 and 22-2 will be of the same object 24 but at different focal planes, this being achieved utilizing two spectral channels. Such an arrangement could be used to simultaneously acquire two synchronized axially displaced planes using BIT and, thus, high speed acquisition of tomographic slices.

[0095] FIG. 19 shows an arrangement similar to that shown in FIG. 13 A with the addition of a a prism 60 after the converging tube lens 20 to split N images from different axial positions in the sample to N different lateral positions on the image sensor 22 using lens 62 and lens 64. The combination of these images can provide background rejection, quantitative phaseAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 contrast, and tomographic 3D reconstructions. A beamsplitter or set of beamsplitters can be used for this purpose.

[0096] FIG. 20 A and FIG. 20B show an arrangement similar to FIG. 15A and FIG. 105B where back-illumination interference tomography can be optimized for different parts of a wide field of view by illumination with an array of laterally displaced light sources according to examples of the present disclosure. Each source will provide a semicoherent back-illuminated source to a small sub-field of the full objective field of view. These images can be stitched together from sequential acquisition in time or multiplexed with color or polarization encoding.

[0097] FIG. 21 shows a back-illumination tomography can also be produced without the use of a beamsplitter by illuminating from a light source 10 positioned within the focal length of the tube lens 20 on the detection path according to examples of the present disclosure. The light from the light source 10 passes through an objective lens 16, which has a focal length of fobj such that the objective lens focal plane 52 is the distance fobj from the plane of the objective lens 16, to create a light image 18 deep to the objective lens focal plane 52. The objective lens 16 may be an infinity corrected microscope objective. A controller 38 may be connected to the objective lens 16 or the structure supporting the objective lens 16 such that fine and precise axial displacement of the objective lens 16 up or down relative to FIG. 21, over a linear phase range may be imparted to the lens 16 via the controller 38. Alternatively, a stepping motor may be attached to whatever structure supports the scattering object such that the controller 38 causes axial displacement of the scattering object elative to the stationary objective lens 16.

[0098] 4. Conclusion

[0099] Back-illumination interference tomography is a microscope technique that uses a demagnified, defocused, on-axis source imaged deep to the microscope objective plane. Backscattered light from the spatially confined source provides net transmission-like illumination of the object with partially spatially coherent illumination, and enhanced interferometric contrast following theory predicted by Streibl

[0019] , The ability to generate partially spatially coherent back-illumination using an LED and turbid media such as tissue is surprising, but its simplicity and results are remarkable. It is clear that the scattering properties of the medium are critical to BIT’s success, and this is an area that requires significant further investigation. Additionally, the spatial coherence that enhances contrast in BIT inevitably will degrade with object depth and source diffusion. Regardless, BIT appears to work not only in vitro, but also enables remarkable visibility of blood cells in vivo and pathology specimens ex vivo. The simplicity of BIT is appealing, as its lack of scanning parts, triggered or swept sources allow its speed to be limited only by the sensor frame rate. At the potential cost of imagingAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 speed however, investigation of quantitative phase imaging with BIT is enticing. Finally, with sufficient training data, generative adversarial network based image-to-image transformation will enable rapid conversion from BIT images H&E images. The implications for in vivo tissue analysis intraoperatively prior to tissue resection could be profound.

[0100] The embodiments set forth below represent the necessary information to enable those skilled in the art to practice the embodiments and illustrate the best mode of practicing the embodiments. Upon reading the following description in light of the accompanying drawing figures, those skilled in the art will understand the concepts of the disclosure and will recognize applications of these concepts not particularly addressed herein. It should be understood that these concepts and applications fall within the scope of the disclosure and the accompanying claims.

[0101] It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of the present disclosure. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0102] It will be understood that when an element such as a layer, region, or substrate is referred to as being “on” or extending “onto” another element, it can be directly on or extend directly onto the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly on” or extending “directly onto” another element, there are no intervening elements present. Likewise, it will be understood that when an element such as a layer, region, or substrate is referred to as being “over” or extending “over” another element, it can be directly over or extend directly over the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly over” or extending “directly over” another element, there are no intervening elements present. It will also be understood that when an element is referred to as being “connected” or “coupled” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. In contrast, when an element is referred to as being “directly connected” or “directly coupled” to another element, there are no intervening elements present.

[0103] Relative terms such as “below” or “above” or “upper” or “lower” or “horizontal” or “vertical” may be used herein to describe a relationship of one element, layer, or region to another element, layer, or region as illustrated in the Figures. It will be understood that theseAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 terms and those discussed above are intended to encompass different orientations of the device in addition to the orientation depicted in the Figures.

[0104] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes,” and / or “including” when used herein specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0105] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Claims

Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 What is Claimed is:

1. A back-illuminated interference tomography (BIT) microscope apparatus comprising:a light source;an objective lens defining an objective lens focal plane located a distance fobj from the objective lens, wherein fobj is an objective lens focal length;a tube lens defining a tube lens focal plane located a distance f L from the tube lens, wherein fiL is a tube lens focal length; andan image sensor that includes a pixel array to capture and output an image, wherein the pixel array occupies a portion of the tube lens focal plane,wherein diverging light from the light source passes through the objective lens and forms a light image at a distance from the objective lens that is greater than fobj, a reflected light component originating from the light image passes through the objective lens focal plane, through the objective lens and is focused by the tube lens onto the pixel array and further wherein the pixel array captures and outputs an image.

2. The BIT microscope apparatus of claim 1, further comprising a condensing lens placed less than its focal length away from the light source, reducing the divergence of the light as it enters the objective lens.

3. The BIT microscope apparatus of claim 2, further comprising a flashing illumination source that is on for a short time relative to the movement of the sample, and then off until the next image is acquired.

4. The BIT microscope apparatus of claim 1, wherein the illumination and detection paths each comprise linear polarizers with a polarizing axis that can be adjusted to modify image contrast.

5. The BIT microscope apparatus of claim 1, wherein the light source emits light at a near-infrared wavelength.

6. The BIT microscope apparatus of claim 1, wherein the light source is a coherent laser source or a semi coherent light source.Attorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 7. The BIT microscope apparatus of claim 1, wherein the light is delivered through a fiber optic light guide.

8. The BIT microscope apparatus of claim 1, wherein the objective lens is translated axially with a motor.

9. The BIT microscope apparatus of claim 1, wherein the multiple images are acquired from different axial planes, and the information is combined to compute quantitative phase maps.

10. The BIT microscope apparatus of claim 1, wherein the objective is inverted and a sample is placed on a glass window and scanned to acquire large field of view data.

11. The BIT microscope apparatus of claim 1, wherein the multiple illumination colors are used to spectrally multiplex data from different illumination and imaging planes.

12. The BIT microscope apparatus of claim 1, wherein a set of beamsplitters is used to split different axial imaging planes to different lateral planes on the image sensor.

13. The BIT microscope apparatus of claim 1, further comprising a beamsplitter between the condensing lens and the objective lens.

14. The BIT microscope apparatus of claim 1, wherein the acquired image is transformed to appear similar to a pathology stained image.

15. A method of imaging using a back-illuminated interference tomography (BIT) microscope apparatus comprising:providing light from a light source;receiving the light by an objective lens defining an objective lens focal plane located a distance fobj from the objective lens, wherein fobj is an objective lens focal length;receiving the light by a tube lens defining a tube lens focal plane located a distance fiL from the tube lens, wherein fir is a tube lens focal length; andAttorney Docket No. 0184.0302-PCT Client Reference No. P18147-02 imaging an object using an image sensor that includes a pixel array to capture and output an image of the object, wherein the pixel array occupies a portion of the tube lens focal plane, wherein diverging light from the light source passes through the objective lens and forms a light image at a distance from the objective lens that is greater than fobj, a reflected light component originating from the light image passes through the objective lens focal plane, through the objective lens and is focused by the tube lens onto the pixel array and further wherein the pixel array captures and outputs an image.

16. The method of claim 15, further comprising receiving the light by a condensing lens having a focal length equal to fcL, wherein the light source is located within the focal length of the condensing lens.

17. The method of claim 16, further comprising splitting the light using a beamsplitter arranged between the condensing lens and the objective lens, a portion of the light from the condensing lens passes through the beamsplitter and into the objective lens and further wherein a portion of the reflected light component passing through the objective lens is directed by the beamsplitter to the tube lens.

18. The method of claim 16, wherein the condensing lens is placed less than its focal length away from the light source, reducing the divergence of the light as it enters the objective lens.

19. The method of claim 15, further comprising a flashing illumination source that is on for a short time relative to the movement of the sample, and then off until the next image is acquired.

20. The method of claim 15, wherein the illumination and detection paths each comprise linear polarizers with a polarizing axis that can be adjusted to modify image contrast.

Images