Label-free imaging and classification of live P. falciparum enables high performance parasitemia quantification without fixation or staining.

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that deep learning enables both life stage classification and accurate parasitemia quantification of ordinary brightfield microscopy images of live, unstained red blood cells. We tested our method using both a standard light microscope equipped with visible and near-ultraviolet (UV) illumination, and a custom-built microscope employing deep-UV illumination. While using deep-UV light achieved an overall four-category classification of Plasmodium falciparum blood stages of greater than 99% and a recall of 89.8% for ring-stage parasites, imaging with near-UV light on a standard microscope resulted in 96.8% overall accuracy and over 90% recall for ring-stage parasites. Both imaging systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results establish that label-free parasitemia analysis of live cells is possible in a biomedical laboratory setting without the need for complex optical instrumentation. We anticipate future extensions of this work could enable label-free clinical diagnostic measurements, one day eliminating the need for conventional blood smear analysis.

Label-free hematology analysis using deep-ultraviolet microscopy.

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins.

Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.

Microscopy with ultraviolet surface excitation (MUSE): A novel approach to real-time inexpensive slide-free dermatopathology.

Traditional histology relies on processing and physically sectioning either frozen or formalin-fixed paraffin-embedded (FFPE) tissue into thin slices (typically 4-6 µm) prior to staining and viewing on a standard wide-field microscope. Microscopy using ultraviolet (UV) surface excitation (MUSE) represents a novel alternative microscopy method that works with UV excitation using oblique cis-illumination, which can generate high-quality images from the cut surface of fresh or fixed tissue after brief staining, with no requirement for fixation, embedding and histological sectioning of tissue specimens. We examined its potential utility in dermatopathology. Concordance between MUSE images and hematoxylin and eosin (H&E) slides was assessed by the scoring of MUSE images on their suitability for identifying 10 selected epidermal and dermal structures obtained from minimally fixed tissue, including stratum corneum, stratum granulosum, stratum spinosum, stratum basale, nerve, vasculature, collagen and elastin, sweat glands, adipose tissue and inflammatory cells, as well as 4 cases of basal cell carcinoma and 1 case of pseudoxanthoma elasticum deparaffinized out of histology blocks. Our results indicate that MUSE can identify nearly all normal skin structures seen on routine H&E as well as some histopathologic features, and appears promising as a fast, reliable and cost-effective diagnostic approach in dermatopathology.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation.

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

Computational On-Chip Imaging of Nanoparticles and Biomolecules using Ultraviolet Light.

Significant progress in characterization of nanoparticles and biomolecules was enabled by the development of advanced imaging equipment with extreme spatial-resolution and sensitivity. To perform some of these analyses outside of well-resourced laboratories, it is necessary to create robust and cost-effective alternatives to existing high-end laboratory-bound imaging and sensing equipment. Towards this aim, we have designed a holographic on-chip microscope operating at an ultraviolet illumination wavelength (UV) of 266 nm. The increased forward scattering from nanoscale objects at this short wavelength has enabled us to detect individual sub-30 nm nanoparticles over a large field-of-view of >16 mm2 using an on-chip imaging platform, where the sample is placed at ≤0.5 mm away from the active area of an opto-electronic sensor-array, without any lenses in between. The strong absorption of this UV wavelength by biomolecules including nucleic acids and proteins has further enabled high-contrast imaging of nanoscopic aggregates of biomolecules, e.g., of enzyme Cu/Zn-superoxide dismutase, abnormal aggregation of which is linked to amyotrophic lateral sclerosis (ALS) - a fatal neurodegenerative disease. This UV-based wide-field computational imaging platform could be valuable for numerous applications in biomedical sciences and environmental monitoring, including disease diagnostics, viral load measurements as well as air- and water-quality assessment.

Microscopy with ultraviolet surface excitation for rapid slide-free histology.

Histological examination of tissues is central to the diagnosis and management of neoplasms and many other diseases and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides-a process that can require hours or days, contributes to cost and delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that, within minutes, provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin and eosin histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280 nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA sequencing). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings and to provide opportunities for rapid histology in research.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers.

Nanoscale imaging using deep ultraviolet digital holographic microscopy.

A deep ultraviolet off-axis digital holographic microscope (DHM) is presented. The microscope has been arranged with as least as possible optical elements in the imaging path to avoid aberration due to the non-perfect optical elements. A high resolution approach has been implemented in the setup using oblique illumination to overcome the limitation introduced by the optical system. To examine the resolution of the system a nano-structured template has been designed and the result confirms the submicron and nanoscale resolution of the arranged DHM setup.

Feasibility of a tetracycline-binding method for detecting synovial fluid basic calcium phosphate crystals.

OBJECTIVE: Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. METHODS: A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. RESULTS: After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. CONCLUSION: With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source.

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.

Effects of fiber-optic probe design and probe-to-target distance on diffuse reflectance measurements of turbid media: an experimental and computational study at 337 nm.

Fiber-optic probes are widely used in optical spectroscopy of biological tissues and other turbid media. Only limited information exists, however, on the ways in which the illumination-collection geometry and the overall probe design influence the interrogation of media. We have investigated both experimentally and computationally the effect of probe-to-target distance (PTD) on the diffuse reflectance collected from an isotropically (Lambertian) scattering target and an agar-based tissue phantom. Studies were conducted with three probes characterized by either common (single-fiber) or separate (two bifurcated multifiber probes) illumination and collection channels. This study demonstrates that PTD, probe design, and tissue scattering anisotropy influence the extent of the transport of light into the medium, the light-collection efficiency, and the sampling volume of collected light. The findings can be applied toward optimization of fiber-optic probe designs for quantitative optical spectroscopy of turbid media including biological tissues.

Photography of the living human cornea in ultraviolet light.

The living human cornea was photographed in ultraviolet light of bandpass 315-326 nm produced by a xenon arc light source and multilayer interference filters. The image was captured on black and white film using a quartz fluorite lens. The photographs revealed structural details not seen in visible wavelengths.

Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation.

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 microM for two-photon, and 220 microM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.

Phase separation in polyfluorene-polymethylmethacrylate blends studied using UV near-field microscopy.

In this paper we present a near-field microscopy study of thin films of a phase-separated blend of the fluorescent conjugated-polymer poly(9,9-dioctylfluorene) [PFO] with the non-fluorescent polymer polymethylmethacrylate [PMMA]. A scanning near-field optical microscope (NSOM) was used to generate (blue) fluorescence from the PFO following UV excitation at 362 nm. A range of different concentrations of PFO in PMMA were studied ranging from 1 to 50% PFO in PMMA by mass. By studying both the shear force and fluorescence images we were able accurately to determine the distribution of PFO in the PMMA. We found that phase separation occurs over a number of different length-scales between 5 micro m and 250 nm. We show that at PFO concentrations of 1%, the PFO lies on top of the PMMA. At a PFO relative concentration of 50%, the PMMA phase extends through the whole thickness of the film to the underlying substrate. We use such samples to discuss the resolution of NSOM when imaging thick organic films. Furthermore, we confirm that the length-scales of phase separation can be modified via control over spin-casting protocols.

Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching.

We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.

Use of UV ParaLens adapter for detection of acid-fast organisms.

Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears.