Smartphone microscopes offer accessibility for advanced imaging, particularly in settings with limited access to traditional microscopes. Existing designs often compromise between cost, performance, and functionality. Many require complex sample preparation (e.g., thin sections), limiting their utility. This paper introduces Pocket MUSE, an ultra-compact smartphone fluorescence microscope designed for versatility, high imaging performance, simplicity, and low cost. Two primary smartphone microscope design strategies exist: using multiple optical elements (expensive and complex) or a single lens (limited performance and functionality). While single-lens designs are cost-effective, improvements are needed to enhance their performance and functionality. Previous advancements included using a reversed smartphone camera lens to reduce aberrations and increase field of view, and colored polymer lenses to replace expensive filters. However, mechanical aspects like focusing remain costly and complex. Sample preparation also presents a challenge; conventional smartphone microscopes usually require flat, thin, stained samples, which is not always feasible in mobile settings. To address these issues, the researchers integrate Microscopy with Ultraviolet Surface Excitation (MUSE), originally used in benchtop microscopes, with a smartphone microscope. MUSE utilizes sub-285 nm UV light, which is strongly absorbed by biological structures, providing strong optical sectioning near the surface and eliminating the need for thin samples. A wide range of common fluorescent dyes are excitable by this UV light, enabling simple multichannel fluorescence microscopy. The UV light is blocked by common optical materials, negating the need for excitation light filters, simplifying the design. Furthermore, MUSE sample preparation is extremely fast and simple. Integrating MUSE into a compact smartphone microscope presents an engineering challenge due to limited working distance. Pocket MUSE uses frustrated total internal reflection (TIR) to deliver light, using a UVC transparent optical window as a sample holder and waveguide, eliminating the need for a focusing mechanism. Finally, the authors improve the resolution to submicron levels via optical design optimization and introduce simple, versatile sample processing strategies.

The introduction adequately reviews existing smartphone microscope designs, highlighting the trade-offs between cost, performance, and functionality. It emphasizes the limitations of complex sample preparation and the need for simpler, more versatile methods. The review then focuses on previous work using MUSE (Microscopy with Ultraviolet Surface Excitation) for benchtop microscopy, establishing its potential for slide-free histology. However, a more comprehensive review of existing single-lens smartphone microscope designs and their specific limitations regarding resolution, field of view, and fluorescence capabilities would strengthen this section. The authors should also include a more detailed discussion of the previous limitations encountered when attempting to integrate MUSE into a compact smartphone microscope design.

Pocket MUSE consists of four main components: an objective lens (a reversed aspheric compound lens, RACL), a sample holder (a fused quartz glass optical window), UV LEDs, and a 3D-printed base plate. The RACL provides a wide field of view (~1.5 x 1.5 mm²). The sample holder is pre-aligned to the focal plane of the lens, eliminating the need for focusing mechanics; focusing is done via the smartphone camera's focus adjustment. Frustrated TIR (total internal reflection) is used to deliver UV light from two miniature UV LEDs positioned against the optical window, providing uniform illumination across the field of view. Resolution is improved by using a smaller RACL with a shorter focal length, increasing magnification and effective resolution via denser spatial sampling on the smartphone's camera sensor. The study performed benchmark tests comparing Pocket MUSE's image quality to conventional MUSE imaging (Nikon Plan APO 10x/0.45 objective), bright-field imaging (Keyence BZ-X810 microscope with various objectives), and bright-field imaging using a standalone 1/7" RACL on a smartphone. Histology imaging was demonstrated on various tissue samples using a single-dip staining protocol, and pseudo-H&E color-remapping was performed. Whole-mount fluorescent immunohistochemistry (IHC) was demonstrated on a brain slice using Alexa Fluor 488-conjugated GFP antibodies. Plant and environmental sample imaging was performed, showcasing the visualization of intrinsic fluorescence. Bright-field and hybrid (bright-field plus fluorescence) imaging modes were demonstrated, showing capabilities for various samples. Mucosal smear imaging was tested, showcasing simple preparation and imaging capabilities. Selective bacteria imaging was demonstrated, differentiating Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) bacteria using dual-fluorescent staining (DAPI and WGA-AF594). Fabrication involved sourcing parts from online vendors, modification of components (e.g., sanding LEDs), and 3D printing of the base plate. Alignment was an iterative process involving sanding the base plate until the sample was in focus using the smartphone camera's focus adjustment. Pocket MUSE images were acquired using the default smartphone camera app or third-party apps for advanced control. Data processing involved converting raw image data (e.g., DNG) to 24-bit RGB formats using software such as Adobe Camera Raw or Raw-Therapee. The study used various staining protocols, including those using rhodamine B, DAPI, acridine orange, and Alexa Fluor 488. The authors also included detailed procedures for sample preparation for different types of samples (e.g., whole-mount samples, cytology samples, bacteria samples).

Pocket MUSE achieved submicron resolution, comparable to a 10x objective on a conventional benchtop microscope, while maintaining low cost and ease of fabrication. The use of a smaller RACL with a shorter focal length significantly improved resolution compared to previous RACL designs. Frustrated TIR illumination provided uniform illumination across a large field of view despite the limited working distance. The device produced high-quality histology images similar to those from benchtop MUSE systems, enabling pseudo-H&E color-remapping. Successful whole-mount fluorescent IHC imaging demonstrated its potential for research and teaching. Imaging of plant and environmental samples highlighted the versatility of the system, leveraging intrinsic fluorescence and simple staining protocols. Bright-field, fluorescence, and hybrid imaging modes were all successfully demonstrated. The simple sample preparation for mucosal smears reduced the time required for imaging and allowed visualization of cells on cotton fiber matrices. Pocket MUSE successfully differentiated bacteria populations in fluid samples using differential fluorescent labeling (DAPI and WGA-AF594), opening possibilities for selective bacteria imaging and quantification. The cost of the optical add-on for the smartphone was estimated to be in the range of $20-50 depending on regional pricing. The device proved easy to use, requiring minimal training for non-professionals.

Pocket MUSE addresses the limitations of existing smartphone microscopes by providing a low-cost, high-performance solution for various microscopy applications. The improved resolution and wide field of view, combined with the simplified sample preparation offered by MUSE, make it suitable for diverse applications ranging from point-of-care diagnostics to environmental monitoring. The system's ease of fabrication and operation makes it accessible to a wide range of users. The successful demonstration of whole-mount IHC and the ability to differentiate bacteria populations highlight the system's potential for research and diagnostics. The limitations of single FOV imaging and the need for additional image processing for non-uniform illumination are acknowledged, but these limitations are often outweighed by the advantages of affordability and accessibility for many applications. The simplicity and low cost of Pocket MUSE makes it particularly promising for resource-limited settings. The combination of MUSE with smartphone technology opens avenues for more widespread access to advanced microscopy. The potential integration with other diagnostic technologies such as microfluidic devices further expands the potential impact of this device.

Pocket MUSE is a significant advancement in low-cost fluorescence microscopy. Its high performance, ease of use, and versatility make it a valuable tool for research, education, and point-of-care diagnostics, especially in resource-constrained environments. Future research could focus on improving magnification capabilities, developing automated image stitching techniques, and exploring new applications in areas such as rapid disease diagnosis and environmental monitoring.

The current design of Pocket MUSE is limited to a single field of view, requiring manual repositioning for larger samples. While frustrated TIR illumination achieves relatively uniform illumination, some non-uniformity might exist, necessitating image processing for some applications. The availability of suitable reversed aspheric compound lenses could affect the reproducibility of the device. The study does mention safety considerations regarding UV light exposure and recommends using protective equipment.