Pocket MUSE: an affordable, versatile and high-performance fluorescence microscope using a smartphone

The study addresses the challenge of creating a low-cost, compact, and high-performance smartphone microscope that minimizes trade-offs among cost, imaging performance, and functionality, while simplifying sample preparation. Existing smartphone microscopes either use multi-element systems with high cost/complexity or single-lens add-ons with limited performance and difficulty incorporating fluorescence. Moreover, conventional microscopy requires complex sample preparation (e.g., thin sections), which is impractical outside labs. The authors propose integrating Microscopy with Ultraviolet Surface Excitation (MUSE) into a compact smartphone microscope—Pocket MUSE—to achieve strong surface optical sectioning, simple multichannel fluorescence without costly filters, and rapid, user-friendly sample preparation. The goal is to deliver submicron-resolution, multichannel fluorescence imaging over a wide field of view using affordable, readily sourced components, suitable for point-of-care, education, environmental studies, and at-home health monitoring.

The paper situates Pocket MUSE within prior smartphone microscopy efforts: multi-element systems or attachments to benchtop microscopes provide full capabilities but at high cost and complexity; single-lens add-ons are compact/low-cost but suffer from aberrations, limited resolution, and difficulty implementing epifluorescence. Innovations to improve compact designs include reversed smartphone camera lenses to reduce aberrations and increase FOV, and colored polymer lenses to replace bulky filters. However, mechanical components (focusing/positioning) and sample preparation remain bottlenecks. For sample processing, specialized systems (e.g., microfluidic chambers) can simplify workflows but are sample-specific. MUSE, previously demonstrated for slide-free histopathology on benchtop systems, offers strong surface optical sectioning with sub-285 nm UV, excitation of common dyes (DAPI, fluorescein, rhodamine) emitting across the visible spectrum, and natural blocking of UV by common optics obviating excitation filters. The authors leverage these properties to simplify smartphone-based fluorescence microscopy and sample preparation.

Design and components: Pocket MUSE comprises four main parts: a reversed aspheric compound lens (RACL) placed directly in front of the smartphone camera, a 0.5 mm-thick fused quartz glass sample holder pre-aligned to the focal plane, two miniature 275–285 nm UV LEDs for excitation, and a 3D-printed base plate (FDM, <2 g material). The LEDs are powered via the smartphone’s USB/Lightning (with OTG) through a step-up regulator (e.g., Pololu U3V12F9). The sample adheres to the window by surface tension, enabling focus without mechanical adjustments; fine focus uses the phone’s autofocus/manual focus. Objective lens selection: A smaller-focal-length RACL (<1.5 mm) is used to increase effective magnification (e.g., shifting from 1:1 to ~1:2 conjugation) while preserving optical resolution, improving sampling on typical smartphone sensors (which are otherwise pixel-limited). Zemax simulations with Largan patent designs confirmed good PSF across a ~1 mm central FOV. Empirical tests using lenses scavenged from aftermarket camera modules identified a 1/7" RACL (e.g., Largan 40069A1) paired with an iPhone 6s+ (Sony IMX315, F/2.2, 2.65 mm focal length), achieving <1 µm effective resolution (>25% contrast on USAF-1951), resolving to Group 9 Element 2 (0.87 µm), over ~1.5 × 1.5 mm² FOV. Estimated f-number ~1.8 (NA ~0.27). Frustrated TIR illumination: To overcome limited working distance and LED package size constraints, the authors implemented frustrated total internal reflection (TIR). UV light is side-coupled into the fused quartz sample holder; above the glass-air critical angle it guides via TIR. Contact with the specimen (glass-sample interface) alters the critical angle, allowing refractive leakage to illuminate the surface. A dual-LED configuration placed on opposing edges compensates for absorption losses near each LED, yielding uniform excitation (modeled and measured) within ±10% across ~3 mm. The arrangement avoids precise angular alignment required by TIRF. Sample preparation and imaging modes: - Slide-free histology: Single-dip staining (e.g., 0.05% w/v Rhodamine B + 0.01% w/v DAPI in 50% methanol, 5–20 s), rinse, and surface contact imaging. Pseudo-H&E color remapping applied for familiar contrast. - Whole-mount IHC: Fixed Thy1-GFP mouse brain slice (500 µm) stained overnight in buffer with Alexa Fluor 488-conjugated anti-GFP (1% v/v) and propidium iodide, followed by washes; RGB channel unmixing in ImageJ/FIJI. - Plants/environmental: Intrinsic and dye-induced fluorescence (DAPI, rhodamine B; iodine for absorptive contrast) on cut surfaces of plant tissues; imaging of algae and micro-animals. - Bright-field and hybrid modes: Bright-field via ambient transillumination (white wall/paper). Hybrid mode enables simultaneous fluorescence and bright-field by turning on UV during transillumination, aiding identification of features (e.g., WBC nuclei among RBCs with acridine orange). - Cytology/mucosal smears: Cotton swab dipped in 10% v/v CytoStain + propidium iodide, quick rinse, smear on window or image directly on cotton fibers. - Bacteria: Labeling in fluid with DAPI (nucleic acid) and WGA-AF594 (peptidoglycan; Gram-positive), enabling visual color-based discrimination of Gram status; analysis by bivariate histograms of RGB ratios. Fabrication and alignment: Lenses harvested from aftermarket camera modules; UV LED packages thinned (to ~1 mm) and fused quartz windows cut/polished to ~10×10 mm². Custom PCBs connect LEDs to a DC up-regulator with push-button. Base plate and retainer designed in SolidWorks, printed in PLA. For alignment, the base plate is intentionally thick so the RACL focal plane starts ~150 µm below the sample surface; iterative sanding of the phone-side base plate with 1000–3000 grit brings the sample surface into focus tolerance; smartphone focus range accommodates tens of microns variability. Imaging and data processing: Images acquired mainly on iPhone 6s+; exposure times 10 ms–1 s (typical hundreds of ms at ISO 400). Raw capture via third-party apps (e.g., Halide) and conversion to 24-bit RGB (e.g., Camera Raw, RawTherapee). Background light reduction by dimming/foil. Bacterial image analysis used median filtering, background subtraction, thresholding, and plotting of R/G and R/B ratios in Matlab.

- Achieved submicron effective resolution on a smartphone: resolved USAF-1951 Group 9 Element 2 (0.87 µm) with a 1/7" RACL on iPhone 6s+, over ~1.5 × 1.5 mm² FOV; lens estimated f/# ~1.8 (NA ~0.27). - Optical modeling and experiments show uniform dual-LED frustrated TIR excitation with <±10% intensity variation across ~3 mm, suitable for ~1.5 mm FOV imaging. - Pocket MUSE produces histology-quality images comparable to benchtop MUSE (10×/0.45 NA), and pseudo-H&E color remapping reproduces familiar contrast for thick, slide-free specimens. - Demonstrated whole-mount IHC imaging (Thy1-GFP brain slice) using standard fluorophore-conjugated antibodies (Alexa Fluor 488) with channel unmixing, highlighting neurons and nuclei. - Plant and environmental imaging: intrinsic and dye-enhanced fluorescence reveals structures (e.g., xylem labeling by rhodamine; DAPI labeling of polysaccharide-rich structures; iodine stains starch), comparable against benchtop fluorescence, MUSE, and bright-field. - Bright-field and hybrid imaging enable concurrent visualization of structural and fluorescent features; example: acridine-orange-stained WBC nuclei highlighted amid RBCs in smears. - Mucosal cytology imaging via rapid, seconds-long workflows; high-contrast visualization of cytoplasm (CytoStain), nuclei and bacteria (propidium iodide), on glass or directly on cotton fibers. - Selective bacteria imaging: color-based differentiation of Gram-negative (E. coli; DAPI-dominated blue-gray) and Gram-positive (B. subtilis; WGA-AF594 red plus DAPI-bright endospores orange) within mixtures; quantified via bivariate RGB ratio histograms. - Cost and accessibility: material cost of optical add-on approximately $20–$50 in small volumes; device is compact, durable, 3D-printable, and simple to assemble and operate. - Safety: UV exposure during regular operation estimated low (<0.01 mW/cm² at 10 cm), with most UV contained and absorbed; standard PPE recommended.

The integration of MUSE into a single-lens smartphone microscope addresses key limitations of prior designs by enabling surface-confined, multichannel fluorescence imaging without bulky filter sets or complex sample preparation. Using a short focal length RACL increases magnification to overcome pixel-limited resolution, achieving near-diffraction-limited sampling on common smartphone sensors. Frustrated TIR side-coupling solves the working distance constraint, delivering uniform sub-285 nm excitation in a highly compact form factor. Together, these innovations enable slide-free histology, IHC, plant and environmental imaging, cytology smears, and bacteria discrimination with minimal user training and rapid workflows. The results demonstrate imaging performance comparable to a benchtop 10× objective for many tasks, validating the approach for field use, education, and point-of-care diagnostics. The hybrid bright-field/fluorescence mode further enhances interpretability by co-locating fluorescent markers with natural morphology. Overall, Pocket MUSE significantly advances the practicality and versatility of smartphone microscopy.

Pocket MUSE introduces a compact, low-cost, and user-friendly smartphone fluorescence microscope that combines a short-focal-length RACL with dual-LED frustrated TIR MUSE illumination to achieve submicron effective resolution over a ~1.5 mm FOV. It simplifies sample preparation and operation, enabling rapid slide-free histology with pseudo-H&E mapping, whole-mount IHC, plant and environmental imaging, cytology smears, and bacteria discrimination. The platform’s affordability and ease-of-use make it suitable for resource-limited settings, point-of-care diagnostics, education, and mobile sensing applications. Future work may include extending magnification options, image stitching for larger areas, broader smartphone compatibility and standardized lens sourcing, and integration as a readout for paper-based microfluidics, immunosensors, microarrays, and lateral flow assays.

- Magnification and image stitching are limited in the current compact design; higher magnification modalities are not integrated. - Lens sourcing: suitable RACLs are typically OEM components; small-quantity procurement can be challenging and may require salvaging from aftermarket parts. - Illumination uniformity depends on dual LEDs; single-LED configurations are cheaper but less uniform (requiring post-processing corrections). - UV safety considerations necessitate careful assembly to minimize leakage and use of PPE; although measured exposure is low, cumulative exposure risks exist. - Performance depends on smartphone sensor characteristics; while increased magnification mitigates pixel-limiting, results may vary across devices. - Working distance is short; samples must conform to the window surface, focusing on surface features (MUSE optical sectioning limits depth).