Methods for microscopy with ultraviolet surface excitation (MUSE) imaging
Inventors
Assignees
Case Western Reserve University
Publication Number
US-12281985-B2
Publication Date
2025-04-22
Expiration Date
2040-07-16
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Abstract
Described herein are methods for imaging a fluorescent bioassay (including a substrate, such as dispersed cellular sample, exposed to one or more exogenous fluorophore and/or fluorescent probe that accumulate in a structure of interest). The bioassay can be excited with Type-C ultraviolet (UVC) light produced by one or more light emitting diode (LED). The UVC can have a center wavelength that causes emission by the fluorescent bioassay. A digital optical device can collect a signal emitted from the fluorescent bioassay in response to the excitation. The methods relate in particular to Microscopy with Ultraviolet Surface Excitation (MUSE) imaging.
Core Innovation
The invention provides methods for Microscopy with Ultraviolet Surface Excitation (MUSE) imaging, particularly enabling use on mobile device microscopes by introducing a compact and low-cost external accessory. This accessory includes a UVC-transparent optical window/sample holder, one or more Type-C ultraviolet (UVC) light emitting diodes (LEDs), and is designed to allow UVC excitation of exogenous fluorophores or fluorescent probes in biological samples. The methods include exposing biological samples to fluorophores, exciting them with UVC light delivered uniformly to the sample surface via frustrated total internal reflection, and collecting the emission signal with a digital optical detector such as a mobile phone camera.
The problem being addressed is the difficulty of implementing MUSE imaging with mobile device microscopes due to the necessity of placing a UVC light source between the sample and the objective lens—a challenge because of the extremely limited space in mobile device microscopes. Most conventional UVC LEDs cannot fit in this narrow gap, making traditional MUSE setups incompatible with mobile platforms. Furthermore, delivering UVC light effectively and uniformly to the sample surface in this constrained geometry was previously not possible with standard device designs.
The solution leverages an external accessory that employs a thick (greater than 0.5 mm) UVC-transparent optical window as both a sample holder and a waveguide for total internal reflection. UVC light is coupled in from the side of the window and, upon contact with a sample, frustrated total internal reflection allows localized excitation near the surface. A compact housing aligns this system with the mobile device camera, enabling both high-resolution fluorescence (MUSE) imaging and basic bright-field imaging. The device and methods allow for simple, portable, and cost-effective MUSE imaging without the need for bulky instrumentation or multiple excitation sources and filters.
Claims Coverage
There are two independent inventive features covered by the claims.
Method for imaging a dispersed cellular sample using an external accessory with UVC illumination and digital detection
This method comprises: - Exposing a dispersed cellular sample to one or more exogenous fluorophores and/or fluorescent probes that accumulate in a structure of interest. - Positioning the sample in contact with a top face of an optical window as part of an external accessory, the accessory including the optical window and UVC LED(s). - Positioning the external accessory in focus with a microscope having one or more digital image sensors. - Exciting the fluorophores and/or probes within the sample using UVC light from the LEDs, with the UVC delivered through side faces of the optical window and uniformly delivered to the sample surface via frustrated total internal reflection (FTIR). - Collecting emitted light from the sample, emitted out the bottom face of the optical window, with the digital image sensor to create an image of the structure of interest.
Method for imaging a fluorescent bioassay using an external accessory with UVC illumination and signal collection by a digital optical detecting device
This method comprises: - Obtaining a fluorescent bioassay. - Positioning the bioassay in contact with a top face of an optical window of an external accessory, the external accessory comprising the optical window and UVC LED(s). - Positioning the external accessory in focus with a digital optical detecting device. - Exciting the fluorescent bioassay with UVC light from the LED(s), the UVC light transmitting through one or more side faces of the optical window and being delivered uniformly to the surface via frustrated total internal reflection. - Collecting the emission signal from the fluorescent bioassay through a bottom face of the optical window towards the digital optical detecting device.
The inventive features lie in methods for imaging biological samples and fluorescent bioassays using an external accessory for mobile or digital microscopes. The accessory provides UVC excitation via side-coupled LEDs and a UVC-transparent optical window employing frustrated total internal reflection, allowing sample excitation and emission collection in a compact, portable format.
Stated Advantages
Enables MUSE imaging with mobile device microscopes by overcoming the problem of limited space for UVC illumination between the sample and objective lens.
Provides a compact, low-cost, and portable solution for high-resolution fluorescence imaging with only minor modifications to existing mobile device microscope designs.
Allows for imaging of thick samples by sampling the surface directly, expanding the range of applications for mobile device microscopes.
Enables both fluorescence (MUSE) and bright-field imaging capabilities with the same device.
The external accessory and components can be produced easily at low material cost with widely available tools.
Provides a uniform and efficient method for delivering UVC light to the sample surface via a UVC-transparent optical window using frustrated total internal reflection.
Documented Applications
Slide-free histology imaging of tissue samples for research and diagnostic purposes.
Imaging of plant, fungus, algae, and environmental samples, including detection of cell structures and diagnosis of plant diseases.
Bright-field and hybrid imaging (overlaying fluorescence and bright-field images) for enhanced visualization of biological samples.
Imaging and diagnosis using mucosal smears, such as Pap smears and cheek swab cytology.
Selective imaging of bacteria and differentiation of bacterial populations in liquid samples, including Gram-specific identification and viability differentiation.
Diagnosis of conditions in blood samples, including detection of blood borne pathogens or parasites such as malaria, with either direct imaging or prep with specific stains.
Detection of microorganisms in body fluids, food, or environmental samples, including tuberculosis identification in sputum samples.
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