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.
Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical ...margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 μm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.
Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can ...rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and NLM evaluation can begin within 3 min of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in a blinded reading of NLM images compared with the gold standard of formalin-fixed, paraffin-embedded H&E histology. A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10 × 10 mm with 1-4 mm thickness, stained for 2 min for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 s. NLM images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pretesting, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides. Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on NLM compared with paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7-99.1%; 95% confidence interval) and 100.0% (97.0-100.0%), respectively. Interobserver agreement for NLM reading had a Fleiss κ = 0.95. The high cancer detection accuracy and rapid specimen preparation suggest that NLM may be useful for intraoperative evaluation in radical prostatectomy.
Models are required to accurately predict mass and energy balances in a bioregenerative life support system. A modified energy cascade model was used to predict outputs of a multi-crop (tomatoes, ...potatoes, lettuce and strawberries) Lunar greenhouse prototype. The model performance was evaluated against measured data obtained from several system closure experiments. The model predictions corresponded well to those obtained from experimental measurements for the overall system closure test period (fivemonths), especially for biomass produced (0.7% underestimated), water consumption (0.3% overestimated) and condensate production (0.5% overestimated). However, the model was less accurate when the results were compared with data obtained from a shorter experimental time period, with 31%, 48% and 51% error for biomass uptake, water consumption, and condensate production, respectively, which were obtained under more complex crop production patterns (e.g. tall tomato plants covering part of the lettuce production zones). These results, together with a model sensitivity analysis highlighted the necessity of periodic characterization of the environmental parameters (e.g. light levels, air leakage) in the Lunar greenhouse.
Microscopy with ultraviolet surface excitation (MUSE) typically has an optical sectioning thickness significantly larger than standard physical sectioning thickness, resulting in increased background ...fluorescence and higher feature density compared to formalin-fixed, paraffin-embedded physical sections. We demonstrate that high-index immersion with angled illumination significantly reduces optical sectioning thickness through increased angle of refraction of excitation light at the tissue interface. We present a novel objective dipping cap and waveguide-based MUSE illuminator design with high-index immersion and quantify the improvement in optical sectioning thickness, demonstrating an e
section thickness reduction to 6.67 µm in tissue. Simultaneously, the waveguide illuminator can be combined with high or low magnification objectives, and we demonstrate a 6 mm
field of view, wider than a conventional 10x pathology objective. Finally, we show that resolution and contrast can be further improved using deconvolution and focal stacking, enabling imaging that is robust to irregular surface profiles on surgical specimens.
A design for a low-cost, heterodyne, frequency domain-diffuse optical spectroscopy system is presented and validated. The system uses a single wavelength of 785 nm and a single detector to illustrate ...the capability, but is built in a modular fashion to make it easily expandable to additional wavelengths and detectors. The design incorporates methods to allow software-based control over the system operating frequency, laser diode output amplitude, and detector gain. Validation methods include characterization of electrical designs as well as determination of the system stability and accuracy using tissue-mimicking optical phantoms. The system requires only basic equipment for its construction and can be built for under
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In the present study, CR39 track etch detector was calibrated with a new system and the total charge changing cross-section of 300 A MeV Fe26+ ion beam in aluminum target was measured. The CR39 ...nuclear track detectors were used to identify the incident charged particles and their fragments using an optical microscope DM6000 M and automated image analyzer system installed with Leica QWin Plus software. The CR39 detectors before and after the target were calibrated and found to have the same charge response; the charge resolution in both of the detectors were 0.19e and 0.20e, respectively. The calibration points were fitted with a polynomial of degree one and all the points are within the limits of the experimental errors. The response functions were also obtained and fitted with a polynomial of degree three which are quite good throughout Z/β = 4.6 to 41.4. The value of the total charge changing cross-section is σtot = (1663 ± 236) mb. The total charge changing cross-section was compared with the experimental results of others and also fitted by the Bradt-Peters geometrical cross-section.
► A new system of optical microscope DM6000 M with Leica QWin Plus software was used in the present measurement. ► New features of Leica QWin Plus were explored to achieve a higher accuracy. ► Better charge resolution and improved threshold were achieved in the present measurements. ► The calibration curves of CR39 detectors were obtained and fitted with first degree polynomial. ► The total charge changing cross-section of 300 A MeV Fe26+ ion beam in aluminum target was measured.
Tissue clearing methods render biological tissues transparent while maintaining tissue structure, enabling visualization of entire tissues. Recent developments in tissue clearing have predominantly ...emphasized preserving intrinsic fluorescent proteins or aqueous-based tissue clearing and so typically involve complex procedures and long processing times. The utilization of tissue clearing protocols in standard of care histology settings has been less well explored, and protocols for rapid clearing of human tissue specimens are limited. This study presents a novel rapid clearing protocol and demonstrates a low-cost tissue processor for high volume rapid tissue clearing that can be intergraded into standard histology workflow. We demonstrate rapid clearing in dermatological specimens, including both nonmelanoma and melanoma excisions.
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