Patent Application
20220283421
The present disclosure relates generally to mobile device microscopy and, more specifically, to systems and methods that can implement Microscopy with Ultraviolet Surface Excitation (MUSE) imaging on a mobile device microscope.
With only small modifications (e.g., an external aspheric compound lens), mobile devices can to be used to perform microscopy as a research and diagnostic tool. Most mobile devices are already equipped for photography and include at least one integrated photographic camera (including at least a camera lens and a high quality digital image sensor). More importantly, these mobile devices also have sufficient computational power for image processing and provide an internet connection for data transfer. Such mobile devices can become microscopes in a cost effective way by mounting a reversed camera lens (obtained for a low cost and/or repurposed from retired technology) as the external compound lens (or external objective lens) in front of the mobile device's camera. Using the reversed camera lens in this manner can optimize both the resolution (dependent on pixel size) and the field of view (dependent on sensor size) without requiring a complex and costly lens assembly. The resolution can be increased even more by using a reversed camera lens designed for image sensors that are smaller than the high quality digital image sensor of the integrated photographic camera in the mobile device.
The focal length of these mobile device microscopes is necessarily limited in order to achieve higher resolution. Limited focal length is not a problem for microscopy imaging of thin samples with transmitted light. However, limited focal lengths are a problem when using techniques like Microscopy with Ultraviolet Surface Excitation (MUSE) imaging, which requires type-C ultraviolet light (UVC) to be delivered to a sample surface. UVC cannot transmit through most glass and polymer materials, so the original MUSE microscope configuration cast the UVC light onto the sample surface with a light emitting diode (LED) placed between the microscope objective and the sample. However, the original MUSE microscope configuration would not work in a mobile device microscope due to very limited spacing between the reversed lens and the sample, so the MUSE microscope requires a significantly more complicated and costly setup.
Provided herein is a solution that allows imaging techniques that can image thick samples, like Microscopy with Ultraviolet Surface Excitation (MUSE), to be used with a mobile device microscope. The systems and method described herein employ a compact and low cost attachment based on frustrated total internal reflection (FTIR) that can be used to perform high resolution MUSE imaging (while still having the functionality of a basic bright-field microscope).
In one aspect, the present disclosure can include an external accessory that allows a mobile device to perform microscopy imaging with Type-C ultraviolet (UVC) light excitation. The external accessory includes a compound lens (also referred to as an objective lens) that can be placed in front of a camera lens of the mobile device. The compound lens forms an imaging relay with the camera lens so that an image of a front focal plane of the compound lens is generated at an image sensor of the camera lens of the mobile device. A UVC light transparent optical window can be placed in front of the compound lens, positioned such that a front surface of the optical window overlaps the front focal plane of the compound lens. One or more light emitting diode (LED) can be placed at one or more side-edges of the optical window and can be configured to emit UVC light through the optical window, where the UVC light undergoes total internal reflection within the optical window. An externally triggered LED driver can power and control the one or more LED.
In a further aspect, the present disclosure can include a system that can be used to perform high resolution MUSE imaging. The system can include a mobile device and an external accessory. The mobile device can have a camera. The external accessory can allow the mobile device to perform microscopy imaging with UVC light excitation. The external accessory includes a compound lens that can be placed in front of a camera lens of the mobile device. The compound lens forms an imaging relay with the camera lens so that an image of a front focal plane of the compound lens is generated at an image sensor of the camera lens of the mobile device. A UVC light transparent optical window can be placed in front of the compound lens, positioned such that a front surface of the optical window overlaps the front focal plane of the compound lens. One or more UVC-generating LED can be placed at one or more side-edges of the optical window and can emit UVC light through the optical window, where the UVC light undergoes total internal reflection within the optical window. An externally triggered LED driver can power and control the one or more LED.
In another aspect, the present disclosure can include a method for performing high resolution MUSE imaging. A biological sample can be placed within an imaging field of an external accessory to a mobile device. The external accessory includes a compound lens configured to be placed in front of a camera lens of the mobile device to form an imaging relay with the camera lens of the mobile device so that an image of a front focal plane of the compound lens is generated at an image sensor of the camera lens of the mobile device. A UVC light transparent optical window can be placed in front of the compound lens and positioned such that a front surface of the optical window overlaps the front focal plane of the compound lens. One or more UVC-generating LED positioned at one or more side-edge of the optical window and can emit UVC light through the optical window, where the UVC light undergoes total internal reflection within the optical window that is frustrated by the biological sample. An externally triggered LED driver can power and control the one or more LED. A photograph can be taken of the imaging field of the compound lens to provide a microscopy image of the biological sample.
The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.
As used herein, the singular forms “a,” “an” and “the” can also include the plural forms, unless the context clearly indicates otherwise.
As used herein, the terms “comprises” and/or “comprising,” can specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups.
As used herein, the term “and/or” can include any and all combinations of one or more of the associated listed items.
As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts/steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.
As used herein, the term “Microscopy with Ultraviolet Surface Excitation (MUSE) imaging” can refer to a microscopy method utilizing ultraviolet (UV) surface excitation to generate high-quality images of one or more samples.
As used herein, the term “imaging” can refer to methods and technologies for visualizing and examining structures not observable with the naked eye. One example type of imaging is medical imaging, in which visual representations of anatomical structures and biological samples are created for diagnostic, treatment, or research purposes.
As used herein, the term “microscopy” can refer to a type of imaging that examines a sample using a microscope. Types of microscopy can include, for example, bright field microscopy, MUSE, a hybrid between bright field microscopy and MUSE, or the like.
As used herein, the term “microscope”, also referred to as an optical microscope or a light microscope, can refer to an instrument that uses light and a system of lenses to generate magnified images of a sample.
As used herein, the term “ultraviolet (UV) light” can refer to a type of electromagnetic radiation with wavelength shorter than that of visible light, but longer than X-rays. For example, UV light can have a wavelength from 10 nm to 400 nm. One specific type of UV light is Type-C ultraviolet (UVC) light, having a center wavelength from 240 nm to 290 nm.
As used herein, the term “mobile device microscope” can refer to a mobile device (also referred to as a mobile electronic device) with the capability of photography, such as a smartphone, a tablet computer, a digital camera, a laptop computer, or the like, being used as a microscope (either alone or with one or more accessories).
As used herein, the terms “accessory”, “external accessory”, “attachment”, “external attachment”, or the like, can refer to a device that can be attached or added to a mobile device to increase the utility, efficiency, or versatility of the mobile device. For example, an accessory can be attached to the mobile device to increase the ability of the mobile device to operate as a microscope.
As used herein, the term “resolution” can refer to the level of detail contained within an image (represented by the number of pixels, the size of the pixels, or the like). A high resolution image can include more detail than an image with a lesser resolution and, for example, can have pixels sized on the order of microns/micrometers.
As used herein, the term “focal length” can refer to the distance between the optical center of the lens to its focal point.
As used herein, the term “imaging relay” between at least two lenses can refer to the transmission of an image of the front focal plane of one lens to be generated at an image sensor of another lens. The imaging relay can be used to magnify the specimen.
As used herein, the term “photographic camera” can refer to an optical instrument that includes at least one camera lens and at least one digital image sensor. The photographic camera may be integrated within a mobile device.
As used herein, the term “camera lens” (also referred to as a “photographic lens” or “photographic objective”) can refer to an optical lens or assembly of lenses of a photographic camera that can be paired with an image sensor.
As used herein, the term “image sensor” can refer to a device that converts light striking a camera lens into an electronic signal (the electronic signal can be transformed into a digital image by a processor, for example).
As used herein, the term “objective lens” can refer to a lens within an optical system that is located closest to the sample. The objective lens can be a compound lens and/or be part of a compound lens.
As used herein, the term “compound lens” can refer to multiple lenses that are arranged on a common axis. The compound lens can be used to increase magnification of a sample. At least one of the multiple lenses within the compound lens can be an aspheric lens (in these instances, the compound lens is referred to as an “aspheric compound lens”).
As used herein, the term “sample” can refer to a small part used for testing or examination to show what the whole is like. For example, the sample can be a biological sample, in which the whole is an organic material, such as blood, interstitial fluid, tissue, bone, etc.
As used herein, the term “total internal reflection” can refer to a phenomenon in which light traveling through one transparent medium reaches an interface with a second, less dense transparent medium and fully reflects back into the denser medium at the angle of incidence (the angle at which the light hits the interface between the two media). Frustrated total internal reflection can refer to a phenomenon in which some of the light hitting the interface between the two transparent media is not reflected. This can occur when a third medium with a higher refractive index abuts the interface between the first two media.
As used herein, the term “exogenous” can refer to a substance originating outside of a body.
As used herein, the term “excite”, “excitation” or the like, can refer to raising the energy of a particle, atom, nucleus, or molecule above its ground or baseline state.
As used herein, the term “histology” can refer to the study of microscopic biological structures.
As used herein, the term “stain”, “staining” or the like, can refer to the use of one or more selected dyes on specimens to increase the visibility of certain structures. Examples of stains can include a fluorescent probe, a fluorophore, or the like.
As used herein, the terms “fluorescent probe” and “fluorophore” can refer to a fluorescent material, molecule, substance, or the like that can change its fluorescence emission (re-emit light) upon light excitation.
Mobile devices can to be used to perform microscopy using an external aspheric compound lens (e.g., a reversed camera lens designed for a smaller image sensor) mounted in front of the mobile device's camera. Higher resolution can be achieved with this microscopy set up while sacrificing the focal length, which is not a problem for microscopy imaging of thin samples with transmitted light, but becomes a problem when imaging thick samples. Microscopy with Ultraviolet Surface Excitation (MUSE) imaging is a type of imaging that can be used to image thick samples, but requires type-C ultraviolet light (UVC) to be delivered to a sample surface to excite the sample. UVC cannot be transmitted through most glass and polymer materials, so the UVC must be delivered between the microscope objective and the sample. Delivery between the microscope objective and the sample is impossible using a mobile device microscope due to very limited spacing between the compound lens and the sample. The systems and methods described herein provide a solution that allows imaging techniques like MUSE imaging to be used with a mobile device microscope.
The systems and methods described herein employ a compact and low cost external accessory for a mobile device that can excite a sample based on frustrated total internal reflection (FTIR) and this excitement can be used to perform high resolution MUSE imaging. (It should also be noted that the external accessory leaves the mobile device with the capabilities of a basic bright-field microscope.) The external accessory requires only minor structural modifications based on the original design concepts of MUSE imaging and mobile device microscopes. The external attachment uses a UVC-transparent optical window/sample holder as the total internal reflection waveguide, allowing for uniform delivery of UVC light to the sample (held onto the microscope by surface tension) through the space between the objective lens and the sample. Traditionally, a thin optical window (<0.25 mm—quartz or fused silica) has been used as the sample holder, but the external attachment uses a thick optical window (>0.5 mm, similar to or thicker than the width of a conventional UVC LED emitter) as both the sample holder and the total internal reflection (TIR) waveguide. When the sample is in contact with the optical window, TIR is disrupted and the UVC light leaks out to the sample. Using a UVC LED with a few milliwatt optical power is sufficient to generate detectable fluorescence signals and create MUSE contrast with a mobile device microscope.
With only minor modifications, many mobile devices can be used as high performance microscopes for research and diagnostic applications. One of the easiest and most cost effective ways to create a mobile device microscope involves mounting a reversed camera lens in front of the mobile device camera. Such mobile device microscopes are very useful to take bright field microscopy images of thin samples, but suffer when imaging thick samples (using methods like Microscopy with Ultraviolet Surface Excitation (MUSE) imaging). MUSE imaging requires type-C ultraviolet light (UVC), having a center wavelength from 240 nm to 290 nm, which cannot transmit through most glass and polymer materials, to be delivered to a sample surface to excite the sample. So, the UVC must be delivered between the reversed camera lens and the sample, which would not work in a mobile device microscope due to very limited spacing between the reversed camera lens and the sample. The external accessory 10 as shown in
The external accessory 10 uses the original design concepts of MUSE microscopy and mobile device microscopes with only minor modifications. As such, the external accessory 10 is a compact and low cost attachment to a mobile device. The external accessory 10 includes an objective lens 12 (or compound lens), an optical window 14, one or more light emitting diodes (LEDs) 16 and/or 17, and an LED driver 18. At least a portion of one or more of the objective lens 12, the optical window 14, the one or more LEDs 16 and/or 17, and the LED driver can be encased in one or more housings (illustrated as housing 19). The housing 19, in some instances, can be configured to attach to a mobile device to position the external accessary 10 relative to the mobile device. In other instances, the housing 19 can simplify focusing by ensuring that a focal spot is pre-aligned, making the configuration very convenient to work with in the field when a stable work bench is not available. Moreover, it will also be understood that the LED driver 18 need not be physically connected to the LED 17. Instead, the LED driver 18 can be coupled to any part of the external accessory 10 to power and/or control the LEDs 16, 17. The components of the external accessory 10 can be constructed at a low cost—for example, the whole assembly can be produced easily for under $30 in material cost with widely-available tools.
The external accessory 10 can be attached to a mobile device 22 to form a system 20 as shown in
The objective lens 12 (or compound lens) can be configured to be placed in front of a camera lens 24 of the mobile device 22 (so that the objective lens 12 center aligns with the camera lens 24, which may include an automatic process). For example, the housing 19 can be specifically configured for the mobile device 22 to ensure that the objective lens 12 aligns with the camera lens 24. The objective lens 12 (or compound lens) can be an inexpensive lens and/or an aspherical lens, such as a reverse camera lens from an older-model smartphone, an infinite corrected microscope objective, or the like. While the inexpensive lens and/or the aspheric lens generally has good microscopy performance, the inexpensive lens can generally have a very limited focal length; in order to achieve higher resolution, the inexpensive lens is designed for an image sensor with an even shorter focal length (<1 mm, for example). The purpose of the objective lens 12 (or compound lens) is to form an imaging relay with the camera lens 24 so that an image of a front focal plane of the objective lens 12 (or compound lens) is generated at an image sensor 26 of the camera lens 24 of the mobile device 22.
The optical window 14 can be configured to be located/placed in front of (or above) the objective lens 12 (or compound lens)—in other words, between the objective lens 12 (or compound lens) and the sample 28 (which can, in some instances, be a biological sample and/or may be stained. The optical window 14 can be positioned such that a front surface of the optical window overlaps the front focal plane of the objective lens 12 (or compound lens). In some instances, at least a portion of the optical window 14 can include a sample holder. However, the optical window 14 may include additional hardware to facilitate positioning or holding the sample 28. In some instances, the sample 28 can be held onto/within the sample holder by surface tension (e.g., provided by liquid 29). The optical window 14 can be transparent to UVC light. For example, the optical window 14 can be at least partially made of quartz, fused silica, and/or UV transparent sapphire.
One or more light emitting diode (LED) (e.g., LED 16 and/or LED 17) can be positioned at one or more side-edges of the optical window 14. The one or more LED (e.g., LED 16 and/or LED 17) can be configured to emit UVC light through the optical window 14. As the UVC light transmits through the optical window 14, the UVC light can excite at least a portion of the sample 28 (e.g., the stain associated with the sample 28). As an example, the optical window 14 can exhibit a transmittance greater than 50% at the center wavelength of the one or more LEDs (e.g., LED 16 and/or LED 17).
The one or more LEDs (e.g., LED 16 and/or LED 17) are powered and controlled by an externally triggered LED driver 18 (in some instances, the LED driver 18 can power and control the one or more LEDs, but in other instances, the LED driver 18 can be linked to/paired with a separate LED controller that can, for example, regulate current to the LEDs). The LED driver 18 can be triggered by a mechanical button (which is pressed electronically, by a person, or the like), a digital signal from the mobile device 22, a flash light signal from the mobile device 22, or the like. The LED driver 18 can be powered by an external battery or other external power source, in some instances. In other instances, the LED driver 18 can be powered by an integrated battery of the mobile device 22 (in instances when the LED driver 18 is connected to the mobile device 22). It should be understood that in instances where the LED driver 18 and LED controller are separate, each may be powered by an integrated battery of the mobile device 22 and/or powered by an external battery or other external power source.
In order to acquire good microscopy images, many modalities require thin samples, which are difficult to prepare in many scenarios. The external accessory 10 allows for imaging using thick samples by sampling the surface directly, enabling a broad range of applications that could not be done with mobile device microscopes previously. Since MUSE imaging requires illumination with UVC light and UVC light cannot transmit through most glass and polymer materials, it is impossible to use conventional microscope lenses or mobile device camera lenses to deliver the light. In traditional MUSE microscope configurations, UVC light is cast onto the sample surface by an LED placed between the microscope objective and the sample. However, with limited spacing between the objective lens 12 (or compound lens) and the sample 28, the traditional approach would require a significantly more complicated and costly setup.
The external accessory 10 is designed based on the optical concept of frustrated total internal reflection (FTIR). FTIR is easy to implement and has been used in many consumer applications, such as touch sensing and transparent drawing boards. The optical window 14 of the external accessory 10 facilitates the FTIR, acting as the optical window, the sample holder, and a total internal reflection waveguide, as shown in
The external accessory 10 only requires minor structural modifications compared to the original design concepts of MUSE imaging and traditional mobile device microscopes. Instead of using a thin optical window (<0.25 mm—quartz or fused silica) as the sample holder (like with traditional MUSE imaging), the external accessory 10 uses a thick optical window 14 (>0.5 mm, similar to or thicker than the width or length of a conventional UVC LED emitter) as both the sample holder and the TIR waveguide. The UVC LED emitter (e.g., used as elements 16 and/or 17) can have an optical power of a few milliwatts.
The external accessory 10 employs the one or more LEDs 16 and/or 17 on one or more lateral side-edges of the optical window 14 to emit and transmit UVC light 32 through the optical window. The UVC light 32 emitted from one or more of the LEDs 16, 17 is guided through the optical window 14 of the attachment 10 using TIR. When the UVC light contacts at least a portion of the sample 28 (in contact with the optical window 14 through the liquid 29), the TIR is disrupted and the UVC light 32 can leak out into the sample 28. The leaked UVC light 32 can excite the sample to create an excited emission 34. The emitted light 36 is transmitted through the objective lens 12 (or compound lens), through the camera lens 24, to the image sensor 26. The UVC LED emitter (e.g., used as elements 16 and/or 17) can have an optical power of a few milliwatts and be sufficient to generate detectable fluorescence signals and create MUSE imaging contrast with a mobile device camera.
The external attachment 10 can excite a sample based on frustrated total internal reflection (FTIR) and this excitement can be used to perform high resolution MUSE imaging. (It should also be noted that the external accessory leaves the mobile device with the capabilities of a basic bright-field microscope.) The external attachment uses a UVC-transparent optical window/sample holder as the total internal reflection waveguide, allowing for uniform delivery of UVC light to the sample (held onto the microscope by surface tension) through the space between the objective lens and the sample. Traditionally, a thin optical window (<0.25 mm—quartz or fused silica) has been used as the sample holder, but the external attachment uses a thick optical window (>0.5 mm, similar to or thicker than the width of a conventional UVC LED emitter) as both the sample holder and the total internal reflection (TIR) waveguide. When the sample is in contact with the optical window, TIR is disrupted and the UVC light leaks out to the sample. Using a UVC LED with a few milliwatts of optical power is sufficient to generate detectable fluorescence signals and create MUSE contrast with a mobile device microscope.
The external attachment device 10 can be small to allow for portable imaging with the mobile device 22. For example,
Another aspect of the present disclosure can include methods 60-80, as shown in
The methods 60-80 are illustrated as a process flow diagram with flow chart illustrations. For purposes of simplicity, the methods are shown and described as being executed serially; however, it is to be understood and appreciated that the present disclosure is not limited by the illustrated order, as some steps could occur in different orders and/or concurrently with other steps shown and described herein. Moreover, not all illustrated aspects may be required to implement the methods.
Referring now to
Referring now to
The following example shows the use of an example of Microscopy with Ultraviolet Surface Excitation (MUSE) imaging implemented on a mobile device. The following example is for the purpose of illustration only and is not intended to limit the appended claims.
Aspheric compound lenses, 285-nm LEDs, fused silica optical windows and other general supplies were purchased from various online vendors and modified as follows: 1. aspheric compound lenses were gently removed from the aftermarket replacement cameras using plastic tweezers; 2. quartz windows of the LEDs were removed using a razor blade and the height of the LED packaging was further reduced to −1 mm (from −1.25 mm) by manual sanding with a file (180 Grit); 3. fused silica optical windows were cut into ˜10×10 mm2 squares using a diamond scribe, with two opposite edges polished sequentially using 40/30/12/9/3/1/0.3 μm grade lapping films. The base plate and the sample holder retainer were designed with Solidworks, and 3D printed with polylactide using an FDM printer (Snapmaker). The modified LEDs were soldered on customized printed circuit board (PCB) adaptors (designed with Autodesk EAGLE, fabricated by OSHPark.com). The LEDs were wired to a DC up-regulator with a push button switch in between. The components were assembled as shown in
An easy and robust alignment procedure was developed to tolerate the limited accuracy of inexpensive components (e.g., 3D printing and optical window thickness) and allow nonprofessionals to align the system. It is critical to align the sample holder to the focal plane of the reversed aspheric compound lens (RACL). To tolerate variations from the manufacturing process, the base plate is designed to be slightly thicker, so the focal plane of the RACL offsets ˜150 μm below the sample surface. Alignment of Pocket MUSE is an iterative process where the baseplate surface facing the smartphone is sanded with 1000-3000 grit sandpaper until the sample surface is in focus. Taking advantage of the focus adjustment function of smartphone cameras, the focal plane of the microscope can swing by tens of microns, reducing the accuracy needed from the sanding step. The thickness of the base plate (measured with a caliper) and alignment of the system (evaluated qualitatively by image sharpness) is verified regularly (e.g., every ˜30 μm) until good alignment is achieved.
The Pocket MUSE component is mounted onto the smartphone with double sided tape. The DC up-regulator is either connected to the smartphone USB outlet (for Android phones), Lightning outlet (for iPhones, with an On-The-Go (OTG) converter) or an external battery. After samples are loaded on the sample holder by surface tension, microscopy images can be taken directly with the default smartphone camera apps. For advanced controls of imaging parameters (e.g., ISO (gain), exposure time, focus, output format, etc.), it is helpful to use third-party or customized camera apps (e.g., Halide). For MUSE imaging, UV illumination should be enabled with the push button switch before the focus and exposure adjustments. Exposure time varies between 10 ms and 1 s depending on the sample type and dye concentration. Smaller ISO (gain) is desired for better signal to noise ratio. To prevent background light, external lights can be dimmed or aluminum foil can be used to cover the microscope. Bright-field transillumination is achieved by facing the smartphone towards a white scattering surface (e.g., white wall, printing paper, etc.). Instability by hand is usually well tolerated because relative sample motion with respect to the smartphone is extremely small, especially for exposure times shorter than 250 ms.
Unlike scientific cameras, smartphone camera apps usually automatically process raw image data and save the data as 24-bit RGB color images. Therefore, data processing (e.g., white balance, digital filters, etc.) can take place even before (e.g., in preview mode) an image is acquired. Although it is difficult to determine the actual data processing algorithm performed by different smartphones, such information is not required for most Pocket MUSE applications. Still, it is possible to use third-party camera apps (e.g., Halide on iOS and ProCam on Android) to save raw (unprocessed) image data, which is especially beneficial when extended dynamic range, lossless data, and advanced processing are needed. To visualize camera raw data, it is necessary to first convert the data (e.g., DNG file) into 24-bit RGB formats (e.g., TIFF). Data conversion can be performed with software such as Adobe Camera Raw (in Photoshop) and RawTherapee (in GIMP). These programs are commonly used for non-scientific photo editing, so they could be easily adapted by non-professional users.
Excised mouse tissue was obtained from unrelated studies with IACUC approval. The tissue was either used fresh directly following dissection, or fixed in 4% paraformaldehyde overnight and stored in 1X phosphate buffered saline (PBS) at 4° C. Other animal and plant samples were collected from the subject's kitchen (e.g., vegetables, meat, etc.), university campus (e.g., algae, pine needles, etc.) and backyard (e.g., garden plants, roundworms, etc.). All samples were manually cut or torn with tweezers into smaller pieces (<3×6×3 mm3). For each sample, at least one relatively flat imaging surface is created. Staining solutions were prepared by dissolving dyes in 30-70% v/v alcohol. One commonly used staining solution in this study is 0.05% w/v rhodamine B and 0.01% w/v DAPI in 50% v/v methanol, which was used for most histology samples and some plant samples. The sample is immersed in the staining solution for 5-20 s, rinsed with tap water and briefly dried with an absorbent material (e.g., tissue paper). Pseudo H&E color mapping was performed using the method described previously.
For the IHC staining demonstration, a piece of fixed Thy1-GFP (Jackson Laboratory, CAT #011070) mouse brain slice (500-μm thick) was obtained from unrelated studies with IACUC approval. A universal buffer (e.g., for blocking, staining and washing) containing 3% v/w bovine serum albumin, 1% v/w Triton X-100, 0.05% v/w sodium azide and 1×PBS was prepared ahead of time. For blocking, the brain slice was first incubated in an excess amount of the universal buffer for ˜2 h at 37° C. For whole-mount staining, the blocking buffer was then replaced with 500 μL fresh universal buffer containing 1% v/v GFP Polyclonal Antibody (Alexa Fluor 488 conjugate, Thermo Fisher Scientific, CAT #A-21311) and 0.05% w/v propidium iodide. The sample was shaken at 37° C. for 16 hours. After staining, the sample was washed again in an excess amount of the universal buffer for ˜2 h at 37° C., followed by a 30 min wash in PBS. Channel unmixing was performed using ImageJ.
Blood samples were collected from a subject with a consumer lancing device (for blood glucose monitoring). The experiment was determined as a non-human subject research project by Case Western Reserve University's Internal Review Board (IRB) and was conducted under the consent of the subject who provided the sample. 100 μL of blood was mixed in 100 μL PBS containing 4 mM ethylenediaminetetraacetic acid and 0.01% w/v sodium azide. For nuclei staining, 10 μL of the blood sample was mixed with 1 μL of 0.1% w/v acridine orange in 50% v/v methanol. For dense blood smear imaging, 1 μL of the stained sample was dropped on the sample holder and air dried prior to imaging. For thin blood smear imaging, the stained sample was further diluted 10 times with PBS prior to imaging. Similarly, cheek swab samples were collected from one subject using consumer cotton swabs. The experiment was also determined as a non-human subject research project by the university's IRB and was conducted under the consent of the subject who provided the sample. After swabbing the inner surface of the cheek, the cotton swab was dipped in a staining solution containing 10% v/v CytoStain (Richard-Allan Scientific) and 0.01% w/v propidium iodide for 5 s. The cotton swab was then briefly rinsed with tap water and dried with an absorbent material. The stained cell lesions were either imaged after being smeared on the sample holder surface, or directly on the cotton swab.
To test non-specific bacterial labeling, a random mixture of bacteria was collected from the supernatant of a mouse tissue specimen that was improperly stored in non-sterile PBS at 4° C. for 6 months. The sample was diluted 10 times with PBS, and 100 μL of the sample was mixed with 10 μL of 0.1% w/v acridine orange in 50% v/v methanol. 2.5 μL of the mixture was dispensed on the Pocket MUSE sample holder and the aliquot was imaged directly with Pocket MUSE. To test Gram-specific bacterial labeling, Escherichia coli (E. coli) was generously provided from an unrelated study. Bacillus subtilis (Ehrenberg) Cohn (B. subtilis) was ordered from American Type Culture Collection (ATCC, CAT #23857). Both bacteria were cultured in lysogeny broth overnight at room temperature. For the experiment, 4 samples were prepared as follows: 1. 500 μL PBS as a control; 2. 100 μL E. coli culture in 400 μL PBS; 3. 100 μL B. subtilis culture in 400 μL PBS; 4. 50 μL E. coli culture and 50 μL B. subtilis culture in 400 μL PBS. Each sample was mixed with a 100 μL staining solution, containing 0.05% w/v DAPI and 0.1% w/v WGA-AF594 in 50% methanol. 2.5 μL of each mixture was imaged with Pocket MUSE with the same camera configuration. Distribution of pixel values was plotted using Matlab.
To ensure low cost and ease of production, Pocket MUSE features a simple design while maintaining the ability to obtain high-quality images. It consists of only 4 major components: an objective lens, a sample holder, UV LED light sources and a base plate (
By eliminating all adjustment mechanisms, even first-time users can easily operate Pocket MUSE. To image, samples (tissue or fluid) are attached to the sample holder by surface tension. As the sample holder is pre-aligned to the focal plane of the objective, the sample is always in focus during normal operation. In addition, similar to conventional smartphone photography, Pocket MUSE is designed to take quality images while holding the phone with one hand. This provides extra convenience for applications in the field, where a stable working bench is not always available. After imaging, the sample holder can be easily cleaned using cotton swabs and common solvents (e.g., 70% isopropanol). For heavy duty cleaning or sterilization, the sample holder can also be detached from the device.
The microscope compartment of Pocket MUSE is improved over previous RACL smartphone microscope designs. In an early-phase implementation, the RACL design delivered good resolution and a large FOV while maintaining relatively low cost (lens cost <$10). The principle behind this design is simple and robust. Because smartphone camera lenses are capable of telecentric imaging, stacking a pair of such lenses face-to-face creates 1:1 finite image conjugation (object size:image size) between their back focal planes, corresponding to the object plane (sample surface) and the image plane (sensor surface) of the microscope. However, this original design had a critical limitation. While a common smartphone camera lens often has f-numbers around 1.5-2.4 (corresponding to a numerical aperture of ˜0.2-0.3), and provides ˜1-2-μm optical resolution, the actual resolution is pixel-limited because a typical smartphone camera sensor often has a pixel size of ˜1.5-2 μm. The pixels are grouped in 2×2 as part of RGB Bayer filter configuration, further reducing the effective pixel size to ˜3-4 μm.
Improving the resolution of previous RACL designs would further expand the capabilities and potential applications for smartphone microscopes. The original RACL manuscript suggested that a smartphone with a large sensor and small pixel size (e.g., Nokia 808) can help improve the effective resolution 10. However, it is not a universal solution for most smartphones. Here, the effective resolution of the RACL design can be further improved using a smaller RACL with a shorter focal length (e.g., <1.5 mm). Through Zemax optical simulation, it was confirmed that a smaller RACL can effectively reduce the image conjugation ratio (e.g., from 1:1 to 1:2) while maintaining good optical performance over a >1 mm FOV. While preserving optical resolution, the smaller RACL increases the magnification of the system, and in turn, boosts the effective resolution through denser spatial sampling when projected onto the cellphone camera sensor. As the simulation did not take into account the specific optics and sensor size for each smartphone, a wide range of lens samples were obtained from aftermarket consumer products and tested them with different smartphones. Among the lenses tested, 1 μm effective resolution and 1.5×1.5 mm2 FOV can be achieved using a smaller RACL designed for 1/7″ image sensors. By comparison, this optical design greatly outperforms a conventional benchtop microscope with a high quality 10× objective. Therefore, these lenses were chosen for the Pocket MUSE design.
A smaller RACL often has a large entrance aperture (>3 mm diameter) and a short working distance (<1 mm). Within this narrow working distance, it is necessary to fit a sample holder (optical window). Because conventional sub 285 nm UV LEDs often have package sizes (3.5×3.5×1 mm3) that are even larger than the RACL, implementing the original MUSE illumination configuration becomes nearly impossible due to limited spatial clearance. To overcome this problem, frustrated TIR was identified as an effective approach to deliver light to the sample surface. In the configuration, LED-based sub 285 nm UV illumination is coupled into the sample holder (a 0.5 mm thick quartz optical window) from the side faces of the optical window. Above the glass-air critical angle, the coupled light bounces between the two glass surfaces through TIR. When a sample is present, the glass-air interface turns into a glass-sample (glass-water) interface. It changes the TIR critical angle and allows some light to refract out of the glass, facilitating sample illumination. In addition, the TIR illumination was further optimized by implementing two LEDs. Because a significant amount of light is absorbed by sample regions closer to the LED, a single LED could not effectively illuminate the entire FOV. Through optics-based simulation, a >50% energy drop across 2 millimeters of sample was noted, causing significantly non-uniform illumination. To compensate for this drop, another LED was added on the opposite edge of the optical window. Through both modeling and experiments, it was shown that relatively uniform illumination (<±10% variation across 3 mm) can be achieved with the dual-LED setup.
Slide-free histology is one of the most well-established MUSE applications. Therefore, as the first demonstration, Pocket MUSE is shown to be fully capable of producing high quality histology images similar to those acquired from benchtop MUSE systems. With a single-dip staining process followed by brief tap-water washing, high image contrast was achieved on a large variety of tissue samples (e.g., kidney, muscle, etc.) within minutes (
Pocket MUSE is also a promising tool for imaging various plants (e.g., vegetables, algae, etc.) and environmental samples (e.g., micro-animals, synthetic pollutants, etc.). Many samples (e.g., cilantro, micro-plastic particles, etc.) are intrinsically fluorescent when excited around 265-285 nm. These samples are capable of generating structural contrast without any staining. Compared to conventional bright-field imaging, Pocket MUSE reveals more of the cellular morphology in bulk plant structures. As with animal tissues, plant tissue could also be stained to produce additional micro-structural contrast with a single dip staining process. For instance, DAPI effectively labels polysaccharides moieties (found in, e.g., cell walls, root saps and starch) in addition to cell nuclei, while rhodamine demonstrates accumulation in the xylem. It was also observed that some absorptive staining (e.g., iodine-stained starch) could be effectively incorporated with fluorescent stains to create additional color contrast between different plant structures.
Pocket MUSE can also easily acquire bright-field images when UV illumination is not enabled. This provides a simple and effective method for visualizing naturally colored thin samples (e.g., blood smears). A conventional fluorescence microscope requires switching the filter cube to an open setting for bright-field microscopy which is difficult in a compact smartphone microscope. Because Pocket MUSE does not rely on filters, no mechanical switching is required to change between fluorescence and bright-field imaging. Trans-illumination bright-field microscopy can be realized simply by directing the sample holder towards a bright diffusive surface (e.g., white wall, printing paper, etc.) in the far field. Regular room light and/or natural light (>100 lumen/m2) provide sufficient illumination.
Overlaying the fluorescence and bright-field images is a common and useful technique to highlight the structures of interest in biological samples. With Pocket MUSE, fluorescence and bright-field contrasts can be combined through a single capture, simply by enabling the UV illumination during bright-field imaging (hybrid mode). As an example, with a thin blood smear, white blood cells (WBC, fluorescence) can be highlighted in a crowd of red blood cells (RBC, bright-field) by simply mixing in a small amount of fluorescent nuclei dyes (e.g., 0.01% w/v acridine orange) in the specimen. It is also possible to apply similar approaches to dense blood samples for cytological quantification and infection evaluation.
Mucosal smears are used in many medical diagnostic applications such as Pap smears. Mucosal smear preparation for Pocket MUSE is extremely simple and can be performed within 30 seconds. The specimen is collected with a cotton swab that is then dipped in a dye (e.g., propidium iodide with CytoStain™), briefly washed in tap water and smeared onto the sample holder. Compared to bright-field cytology staining, MUSE fluorescence results in a significantly higher contrast between the cell bodies, nuclei and the background. Cell morphology can be clearly visualized over the majority of the FOV due to low aberrations at the edges. Although only a single FOV could be imaged at a time, a larger population of cells could be rapidly reviewed by repeated repositioning of the same swab. In addition, as conventional mucosal smear cytology imaging requires cells to be attached to a flat glass surface, Pocket MUSE allows cells to be imaged directly on the cotton fiber matrices. Finally, because MUSE captures the surface, some volumetric aspects of cell morphology can be visualized.
Fluorescent staining has been widely used to examine bacteria in liquid samples. As a preliminary demonstration, a bacterial suspension was labeled with fluorescent dyes (e.g., acridine orange) in a simple mixing step. Individual bacteria are smaller than the resolution limit of Pocket MUSE, but their presence can be effectively visualized when if sparsely dispersed in a fluid sample. Suspended bacteria show a distinct twinkling in preview mode due to their movement in and out of the focal plane. In addition, with bacteria-specific fluorescent probes, Pocket MUSE could also differentiate different populations of microorganisms. As a preliminary demonstration, nucleic acid stain (DAPI, which labels all bacteria) combined with peptidoglycan staining (wheat germ agglutinin Alexa Fluor 594 conjugate (WGA-AF594), which labels gram-positive bacteria) can differentiate Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) bacteria populations based on the color of microbe particles, which could be further quantitatively assessed using signals found in the different color channels of the RGB image.
From the above description, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
This application claims priority to U.S. Provisional Application Ser. No. 62/874,688, filed Jul. 16, 2019, entitled “Improved Microscopy Devices, Systems, and Methods” and to U.S. Provisional Application Ser. No. 62/936,757, filed Nov. 18, 2019, entitled “Systems and Methods for Improved Light Sheet Microscopy”. The entirety of these provisional applications is hereby incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/042214 | 7/16/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62874688 | Jul 2019 | US | |
| 62936757 | Nov 2019 | US |
