Emission Filter Research Articles

Synthesizing, characterizing, and cold plasma treating of Cr2O3/CuO nanomaterials doped PMMA/PEO for flexible optoelectronic applications

Polymeric nanocomposites are attracting significant attention due to their ability to facilitate innovative uses. This work investigated the possibility of improving performance by examining the impact of dispersing copper oxide (CuO) and chromium (III) oxide (Cr2O3) nanoparticles in a blend of poly (methyl methacrylate) and polyethylene oxide (PEO) on several properties. UV–visible and photoluminescence spectroscopies were used for optical properties, and FE-SEM was used for surface morphology analysis. The results showed that as the CuO–Cr2O3 percentage increased, the extinction coefficient increased, and indirect band gaps decreased. The photoluminescence properties showed two distinct peaks (dual emission). We measured the AC electrical properties with frequencies ranging from 100 Hz to 5 MHz. The composite's AC conductivity and dielectric loss increased significantly at high frequencies (higher than 2 MHz). The dielectric constant is nearly frequency-independent and increases as the concentration of nanoparticles increases. We used a DC plasma sputtering facility to treat the nanocomposites with argon plasma at a pressure of 0.12 mBar for 7 min. We analyzed the films' properties before and after plasma treatment, observing a significant impact on nanocomposite-incorporated nanoparticles. After plasma treatment, the band gap decreased from 5.05 eV to 4.8 eV for the lowest concentration of CuO + Cr2O3 (1.5 wt%) and from 3.55 eV to 2.47 eV for the highest concentration of CuO + Cr2O3 (6 wt%). The changes ranged from 0.25 to 1.08 eV. The films possess features that render them suitable for high-frequency optoelectronic devices as well as optical applications such as emission filters and UV shielding.

Three in one: coordination-induced emission for inherent fluorescent Al-MOF synthesis combined with inner filter effect@aggregation-induced emission mechanisms for designing color tonality and ratiometric sensing platforms.

Three phenomena, namely coordination-induced emission (CIE), aggregation-induced emission (AIE), and inner filter effect (IFE), were incorporated into the design of a ratiometric and color tonality-based biosensor. Blue fluorescent Al-based metal-organic frameworks (FMIL-96) were prepared from non-emissive ligand and aluminum ions via CIE. Interestingly, the addition of tetracycline (TC) led to ratiometric detection and color tonality, as the blue emission at 380nm was quenched (when excited at 350nm) due to IFE, while the green-yellowish emission at 525nm was enhanced due to AIE. Based on that, an ultra-sensitive visual-based color tonality mode with smartphone assistance was developed for detection of TC. The sensor exhibited a linear relationship within a broad range of 2.0 to 85.0μMTC with a detection limit of68.0nM. TC in milk samples was quantified with high accuracy and precision. This integration of smartphone and visual fluorescence in solution isaccurate, reliable, cost-effective, and time-saving, providing an alternative strategy for the semi-quantitative determination of TC on-site.

EnderScope: a low-cost 3D printer-based scanning microscope for microplastic detection.

Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Enhancing simultaneous determination of some angiotensin II receptor antagonists and amlodipine in plasma using HPTLC with fluorescence densitometry: Independent fluorescence detection of the co-administrative drugs in the mixture across various pH conditions

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA’s fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18–300 ng/band, while AIIRAs drugs exhibited a linear range of 6–150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.

Bichromatic tetraphasic full-field optical coherence microscopy.

Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast. We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts. BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast. BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors. BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.

Abstract 76: Correlated multi-scale 3D optical microscopy of tumor tissues

Abstract Three-dimensional (3D) optical microscopy in combination with advanced tissue clearing methods permits the interrogation of intact large-sized tumor tissues. There are many optical microscopy methods (e.g. light-sheet and confocal microscopy) offering 3D tumor images at varying levels of spatial resolution (e.g. organ, tissue, cell, and molecule level). However, it is a challenge to correlatively integrate various resolution 3D images obtained by different optical microscopy of tumor tissue. In this project, we developed a correlated multi-scale 3D optical microscopy method that enables the acquisition of macroscopic to microscopic spatial information from a single tumor tissue in 3D. Firstly, we used light sheet microscopy to image a whole large-sized, cleared tumor tissue and analyzed the results to identify regions of interests (ROIs). After defining ROIs in the 3D whole tumor image, we added a UV-activatable visible dye, spiropyran, in the agarose gel surrounding the cleared tumor and exposed the sample to a UV light sheet horizontally at the height (z-axis) of the tumor where the ROIs present. The activated dye showed ‘purple’ colored lines in the agarose gel and we physically marked the gel along the visible colored lines. We further sectioned the tumor at the ROI positions using a vibratome after reversing tissue clearing process. After immunofluorescence staining and aqueous-based tissue clearing, confocal microscopy allowed the localization of various cell types in the ROIs of tumor macrosections. To validate the method for multi-scale evaluation of cancer immunotherapy, we tested it with control and DMXAA-treated mouse mammary tumor tissues. We successfully acquired the 3D images of whole tumors by capturing autofluorescence signals with a 633 nm excitation laser and its matched emission filter. Based on the autofluorescence tumor images, we defined two ROIs for each control and treated tumor and performed optical marking and physical sectioning of the tumors as described above. Next, we stained the macrosections (400 µm thick) with DAPI, CK8-DL488, CD45-DL550, and ER-TR7-DL680 to visualize cancer, immune, and stromal cells in the ROIs at cellular resolution. We quantitatively analyzed the correlated multi-scale 3D image data for assessment of the effects of DMXAA treatment on the tumor microenvironment including eradiation of cancer cells, immune infiltration, and remodeling of tumor stroma. In summary, this new microscopy method will provide comprehensive spatial information of tumor tissues and benefit a broad cancer research field such as the discovery of new tumor biomarkers and the development of effective cancer therapies. Citation Format: Jingtian Zheng, Steve Seung-Young Lee. Correlated multi-scale 3D optical microscopy of tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 76.

High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters.

Hyperspectral imaging (HSI) technologies have enabled a range of experimental techniques and studies in the fluorescence microscopy field. Unfortunately, a drawback of many HSI microscope platforms is increased acquisition time required to collect images across many spectral bands, as well as signal loss due to the need to filter or disperse emitted fluorescence into many discrete bands. We have previously demonstrated that an alternative approach of scanning the fluorescence excitation spectrum can greatly improve system efficiency by decreasing light losses associated with emission filtering. Our initial system was configured using an array of thin-film tunable filters (TFTFs, VersaChrome, Semrock) mounted in a tiltable filter wheel (VF-5, Sutter) that required ~150-200 ms to switch between wavelengths. Here, we present a new configuration for high-speed switching of TFTFs to allow rapid time-lapse HSI microscopy. A TFTF array was mounted in a custom holder that was attached to a piezoelectric rotation mount (ThorLabs), allowing high-speed rotation. Switching between adjacent filters was achieved using the internal optics of a DG-4 lightsource (Sutter Instrument), including a pair of off-axis parabolic mirrors and galvanometers. Output light was coupled to a liquid lightguide and into an inverted widefield fluorescence microscope (TI-2, Nikon Instruments). Initial tests indicate that the HSI system provides a 15-20 nm bandwidth tunable excitation band and ~10-20 ms wavelength switch time, allowing for high-speed HSI imaging of dynamic cellular events. This work was supported by NIH P01HL066299, R01HL169522, NIH TL1TR003106, and NSF MRI1725937.

A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein.

Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo. To develop intensiometric sensors with the capacity for ratiometric quantification, dual-FP Matryoshka sensors were generated by using a single cassette with a large Stokes shift (LSS) reference FP nested within the reporter FP (cpEGFP). Here, we present a genetically encoded calcium sensor that employs green apple (GA) Matryoshka technology by incorporating a newly designed red LSSmApple fluorophore. LSSmApple matures faster and provides an optimized excitation spectrum overlap with cpEGFP, allowing for monochromatic coexcitation with blue light. The LSS of LSSmApple results in improved emission spectrum separation from cpEGFP, thereby minimizing fluorophore bleed-through and facilitating imaging using standard dichroic and red FP (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new FP, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

Mechanistic investigation of photodynamic therapy using Visudyne in human KB carcinoma cells

Photodynamic therapy is a therapy that utilizes a specialized drug, a photosensitizer, which is not toxic by itself, but upon activation with light, is activated and leads to the production of singlet oxygen species. This production of singlet oxygen species starts a cascade that leads to cell death. Photodynamic therapy is a potential treatment for cancer patients, and it might be helpful to promote human health by providing alternative treatment to cancer. In-vitro study with Visudyne (VD) in KB carcinoma cells unveiled the mechanism of cell injury to cell death upon photodynamic therapy (PDT). In this study, we investigated real time monitoring of the production of singlet oxygen (SO), reactive oxygen species (ROS), mitochondria integrity and chromatin integrity in VD treated live cells by irradiating with a mercury lamp light source and 665–675 nm and 480–560 nm emission filter. Upon PDT we also observed cytoskeleton remodeling and cytochrome –C release using immunofluorescence techniques. We investigate the cytotoxicity of PDT and performed quantitative analysis for SO, ROS and mitochondrial potential change using colocalization analysis between VD and mitochondria.

(Invited) hand-Held Florescence Microscope for Single Micro/Nano Particle Imaging

Fluorescence microscopes are commonly used to detect micro and nano biological entities (blood cells, microbes, functionalized particles etc.). Several experimental assays have been developed for biomarker measurements using this microscopy instrument. Common examples include cell viability analysis, surface receptor based specific blood cell enumeration, phagocytosis determination etc. However, the laboratory-based bench top fluorescence microscope suffers from high capital cost, maintenance, experienced staff for its operation and lack of portability. To address several of these challenges, here we have developed a hand-held, portable, 3D printed fluorescence microscope to enable single cell enumeration and nanoparticle imaging operated by smartphone.3D printed platform is composed of top and bottom portions. The bottom portion includes excitation optics including an LED and excitation filter. While the top portion houses an emission filter, and a lens, while a smartphone can be placed on top portion for imaging. The excitation and emission filters can be swapped out for different fluorophores used. We have used the platform for imaging leukocytes collected from the whole blood. Blood samples were procured from Robert Wood Johnson Medical Hospital. We ran the leukocytes on our photonics platform and laboratory florescence microscope concurrently for a comparative analysis and found a high correlation of R2 = 0.99 in between cell counting results. Further, swapping different lenses can achieve high resolution for the imaging system. The resolution was verified by US Airforce Resolution Chart. This allowed us to image fluorescent nanoparticles up to 800nm at various concentrations and excitation modalities.This platform can be used for single-cell analysis, specific leukocytes enumeration and nanoparticles imaging. The demonstrated results can enable significant applications to develop many biomedical and diagnostic assays.

NIRDye 812: A molecular platform tailored for multimodal bioimaging applications of targeted fluorescence- and photoacoustic-guided surgery.

The primary treatment for malignant tumors remains to be surgical removal of the diseased tissue. The presence or absence of residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased tissue from healthy tissue unambiguously in real time is crucial. Near infrared-I (NIRI) fluorescence-emitting targeting biomolecular constructs such as anticancer antibody-fluorophore conjugates, namely cetuximab-IRDye® 800CW (CTB-IRDye® 800CW), are FDA-approved for clinical trial usage in the fluorescence-guided resection of diseased tissue due to affording improved direct visualization of tumor tissue when compared to the use of either the unaided eye under standard white light illumination (WLI) surgical techniques or non-targeting fluorophores. Unfortunately, though helpful, CTB-IRDye® 800CW affords limited (i) identification of diseased tissue and (ii) tumor margin delineation, because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) as a result of its spectral/photophysical profiles poorly aligning with the fixed optical windows of pre-/clinical setups. As such, CTB-IRDye® 800CW is more prone to affording incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye® 800CW to achieve PA signals that could result in higher TBRs. However, in clinical trial practice, using IRDye® 800CW for PA imaging also yields subpar TBRs due to it affording low PA signals. To overcome such limitations, we developed NIRDye 812, a structurally-modified topological equivalent of IRDye® 800CW, to confer it the capability to yield both higher TBRs and superior PA signal than that of the equivalent CTB-conjugate and fluorophore IRDye® 800CW itself, respectively. To do so, we substituted the oxygen atom at its meso-position with a sulfur atom. CTB-NIRDye 812 demonstrated a red-shifted absorption wavelength at 796nm and a peak NIR-I fluorescence emission wavelength at 820nm, which better dovetails with the fixed windows of preinstalled fixed emission filters within commercial pre-/clinical NIR-I fluorescence imaging instruments. Overall, CTB-NIRDye 812 provided a∼2-fold increase in TBRs compared to those of CTB-IRDye® 800CW in vivo. Also, NIRDye 812 displayed an ∼60% higher PA signal than that of IRDye® 800CW. Collectively, we achieved our goal of improving upon the spectral/photophysical and PA properties of IRDye® 800CW via introducing a subtle modification to its electronic core such that its CTB immunoconjugate could potentially allow for fast track or breakthrough designation by the FDA due to its near-identical structure displaying considerably improved efficacy.

Dispersion Managed Mode-Locking in All-Fiber Polarization-Maintaining Nd-Doped Laser at 920 nm

We present a comprehensive study of dispersion managed ultrashort-pulse generation in Nd-doped polarization maintaining all-fiber laser at a 920 nm wavelength. We implement a linear laser scheme with a chirped fiber Bragg grating (CFBG) serving as a semitransparent mirror and semiconductor saturable absorber mirror (SESAM) as a mode-locker and as a second non-transparent mirror. Complete 1064 nm emission filtering is ensured by 920/1064 wavelength division multiplexer. The dispersion compensation by CFBG was sufficient to reach anomalous net dispersion in the shortest laser scheme allowing us to investigate mode-locking regimes in the −0.05 ps <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ÷ 0.24 ps <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> net dispersion range by varying the length of the passive fiber. With this approach we demonstrate high order harmonic mode-locking at dispersion closer to zero and large energy span near-parabolic shape single pulse operation in anomalous dispersion regime. The investigation is supported by numerical simulations used to optimize the laser resonator design and to investigate intracavity pulse evolution.

Thin and Scalable Hybrid Emission Filter via Plasma Etching for Low-Invasive Fluorescence Detection

Hybrid emission filters, comprising an interference filter and an absorption filter, exhibit high excitation light rejection performance and can act as lensless fluorescent devices. However, it has been challenging to produce them in large batches over a large area. In this study, we propose and demonstrate a method for transferring a Si substrate, on which the hybrid filter is deposited, onto an image sensor by attaching it to the sensor and removing the substrate via plasma etching. Through this method, we can transfer uniform filters onto fine micrometer-sized needle devices and millimeter-sized multisensor chips. Optical evaluation reveals that the hybrid filter emits light in the 500 to 560 nm range, close to the emission region of green fluorescent protein (GFP). Furthermore, by observing the fluorescence emission from the microbeads, a spatial resolution of 12.11 μm is calculated. In vitro experiments confirm that the fabricated device is able to discriminate GFP emission patterns from brain slices.

An Innovative Miniaturized Cell Imaging System Based on Integrated Coaxial Dual Optical Path Structure and Microfluidic Fixed Frequency Sample Loading Strategy

On-site rapid detection of cells is especially of concern for many fields, image-based detection results can directly reflect the multi-dimensional information such as cell contour and aggregation state. This article proposes an innovative miniaturized cell imaging system based on integrated coaxial dual optical path structure and microfluidic fixed frequency sample loading strategy, which performs synchronous imaging of bright field mode and fluorescence field mode on cells. The bright field optical path and fluorescence field optical path are integrated into the coaxial dual optical path structure based on the dichroic mirror with splitting and steering functions. The movement of the shutter’s curtain is controlled by a micro-motor, so that the plane mirrors fixed on the curtain with different light angle can be switched to the optical axis, and the fast switching of the bright field optical path and the fluorescence field optical path can be implemented. A drawer-type filter module can be pulled out of the coaxial dual optical path structure, the emission filter and the dichroic mirror in this module can be replaced according to the fluorescence emission peak of the sample to be tested, thereby expanding the detectable cell types of this system. In addition, based on the force analysis and calculation of the fluid, a fixed frequency was obtained to drive the sample loading. The accumulated kinetic energy of the fluid during this period can be offset by the closing of the pinch valve, and the flow rate of the sample in the imaging field can be rapidly reduced to close to zero to avoid the tailing effect in the image. It is experimentally confirmed that the detection accuracy of this system, the registration matching accuracy of cells and fluorescent dots, the cell survival rate, and the detection sensitivity of cell viability. This system balances the tradeoff between portability, flux and accuracy, offers more functionality in terms of technology and application for on-site cell detection.

Concurrent bioimaging of microalgal photophysiology and oxidative stress.

The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis and represents a major cause of oxidative stress in phototrophs, having detrimental effects on the photosynthetic apparatus, limiting cell growth, and productivity. Several methods have been developed for the quantification of cellular ROS, however, most are invasive, requiring the destruction of the sample. Here, we present a new methodology that allows the concurrent quantification of ROS and photosynthetic activity, using the fluorochrome dichlorofluorescein (DCF) and in vivo chlorophyll a fluorescence, respectively. Both types of fluorescence were measured using an imaging Pulse Amplitude Modulation (PAM) fluorometer, modified by adding a UVA-excitation light source (385nm) and a green bandpass emission filter (530nm) to enable the sequential capture of red chlorophyll fluorescence and green DCF fluorescence in the same sample. The method was established on Phaeodactylum tricornutum Bohlin, an important marine model diatom species, by determining protocol conditions that permitted the detection of ROS without impacting photosynthetic activity. The utility of the method was validated by quantifying the effects of two herbicides (DCMU and methyl viologen) on the photosynthetic activity and ROS production in P. tricornutum and of light acclimation state in Navicula cf. recens Lange-Bertalot, a common benthic diatom. The developed method is rapid and non-destructive, allowing for the high-throughput screening of multiple samples over time.

A very low-cost pulse-amplitude modulated chlorophyll fluorometer

The quantum yield of photosystem II (ϕPSII) is an important metric to determine a plant’s light use efficiency and to detect plant stresses. Commercial instruments that measure ϕPSII exist, but their cost may prevent widespread adoption. We present the design and realization of a chlorophyll fluorometer capable of measuring ϕPSII that uses a minimal set of optical components and relies on low-cost electronics with total component costs below USD 300. A consumer grade 405 nm laser diode obviates the need for optical elements in the excitation path, and an avalanche photodiode ensures high sensitivity. In addition, an emission filter and a collimating lens are needed. We demonstrate sensor linearity and actinic light immunity with fluorescence reference standards, and we compare our device to a commercial instrument to assess accuracy: With three plant species (Spathiphyllum, Heuchera, and Hosta) and under actinic light variations from zero to 450 μmolm−2s−1, reported ϕPSII from both instruments correlated with R2=0.87. Implications of our design choices, their costs, and alternatives are discussed.

Utilization of Natural Zeolite as Emission Filter in Catalytic Converter of Diesel Engine

The performance of natural zeolites in a catalytic converter to reduce the emission contents of diesel engine was studied in this research. Diesel engines are engines that use a high compression ratio to carry out a combustion process that will produce high contents of carbon monoxide (CO), nitrogen oxides (NOx), and sulfur oxide (SO2) in diesel engine exhaust gases. One solution to reduce the contents of exhaust gas compounds in diesel engines is to use catalytic converter technology. In this study, the catalytic converter used natural zeolite as an emission filter. The catalytic converter was designed in the form of a pipe made from iron plates and hollow balls that were used as natural zeolite holders. A diesel engine emission test was conducted using a gas analyzer with engine speed variations. The results show the highest reduction efficiency in the emission contents of diesel engine (carbon monoxide (CO), nitrogen oxides (NOx), and sulfur dioxide (SO2)) of 46.14%, 22.77%, and 90.56%, respectively.

Deep-learning-augmented computational miniature mesoscope.

Fluorescence microscopy is essential to study biological structures and dynamics. However, existing systems suffer from a trade-off between field of view (FOV), resolution, and system complexity, and thus cannot fulfill the emerging need for miniaturized platforms providing micron-scale resolution across centimeter-scale FOVs. To overcome this challenge, we developed a computational miniature mesoscope (CM2) that exploits a computational imaging strategy to enable single-shot, 3D high-resolution imaging across a wide FOV in a miniaturized platform. Here, we present CM2 V2, which significantly advances both the hardware and computation. We complement the 3 × 3 microlens array with a hybrid emission filter that improves the imaging contrast by 5×, and design a 3D-printed free-form collimator for the LED illuminator that improves the excitation efficiency by 3×. To enable high-resolution reconstruction across a large volume, we develop an accurate and efficient 3D linear shift-variant (LSV) model to characterize spatially varying aberrations. We then train a multimodule deep learning model called CM2Net, using only the 3D-LSV simulator. We quantify the detection performance and localization accuracy of CM2Net to reconstruct fluorescent emitters under different conditions in simulation. We then show that CM2Net generalizes well to experiments and achieves accurate 3D reconstruction across a ~7-mm FOV and 800-μm depth, and provides ~6-μm lateral and ~25-μm axial resolution. This provides an ~8× better axial resolution and ~1400× faster speed compared to the previous model-based algorithm. We anticipate this simple, low-cost computational miniature imaging system will be useful for many large-scale 3D fluorescence imaging applications.

Underwater Multispectral Laser Serial Imager for Spectral Differentiation of Macroalgal and Coral Substrates

The advancement of innovative underwater remote sensing detection and imaging methods, such as continuous wave laser line scan or pulsed laser (i.e., LiDAR—Light Detection and Ranging) imaging approaches can provide novel solutions for studying biological substrates and manmade objects/surfaces often encountered in underwater coastal environments. Such instruments can be used shipboard or coupled with proven and available deployment platforms as AUVs (Autonomous Underwater Vehicles). With the right planning, large areas can be surveyed, and more extreme and difficult-to-reach environments can be studied. A prime example, and representing a certain navigational challenge, is the under ice in the Arctic/Antarctic or winter/polar environments or deep underwater survey. Among many marine biological substrates, numerous species of macroalgae can be found worldwide in shallow down to 70+ m (clear water) coastal habitats and are essential ecosystem service providers through the habitat they provide for other species, the potential food resource value, and carbon sink they represent. Similarly, corals also provide important ecosystem services through their structure and diversity, are found to harbor increased local diversity, and are equally valid targets as “keystone” species. Hence, we expand current underwater remote sensing methods to combine macroalgal and coral surveys via the development of a multispectral laser serial imager designed for classification via spectral response. By using multiple continuous wave laser wavelength sources to scan and illuminate recreated benthic environments composed of macroalgae and coral, we show how elastic (i.e., reflectance) and inelastic (i.e., fluorescence) spectral responses can potentially be used to differentiate algal color groups and certain coral genus. Experimentally, three laser diodes (450 nm, 490 nm, 520 nm) are sequentially used in conjunction with up to 5 emission filters (450 nm, 490 nm, 520 nm, 580 nm, 685 nm) to acquire images generated by laser line scan pattern via high-speed galvanometric mirrors. Placed directly adjacent to a large saltwater imaging tank fitted with optical viewports, the optical system records target substrate spectral response using a photomultiplier preceded by a filter and is synchronously digitized to the scan rate by a high sample rate Analog-to-Digital Converter (ADC). Acquired images are normalized to correct for imager optical effects allowing for fluorescence intensity-based pixel segmentation via intensity thresholding. Overall, the multispectral laser serial imaging technique shows that the resulting high resolution data can be used for detection and classification of benthic substrates by their spectral response. These methods highlight a path towards eventual pixel-wise spectral response analysis for spectral differentiation.

Seeing and Understanding the Light: Student‐Built Fluorescence Microscopes for Dynamic Calcium Imaging in the Undergraduate Physiology Lab Class

Students often treat laboratory equipment as a black box with little knowledge of how scientific instrumentation works to gather data. We are creating interdisciplinary classrooms where students gain confidence to build and understand tools to solve biological questions as future scientists. We describe student construction, use and assessment of our Do‐It‐Yourself (DIY) fluorescence imaging microscope. Students were first introduced to principles of fluorescence microscopy and Drosophila neurogenetics through lecture material, readings and online resources. Mixed teams of 2‐3 Biology and Engineering students assembled their own imaging microscopes with the following parts: 20X objective lens, excitation and emission filters and dichroic mirror, CMOS camera, 3‐D printed holders and housings for these parts, and a student‐built LED light source (total parts/microscope cost ~$1000). Construction was guided by pre‐recorded build videos and assistance from course instructors and teaching assistants. Student teams tested their microscope by collecting images from prepared slides with GFP fluorescence in fly synaptic boutons. Teams also acquired images from living fly larval preparations in two simple experiments. These compared fluorescence intensity before and during application of a high potassium saline that: 1) released GFP‐labelled neuropeptide from fly synaptic boutons, and 2) activated glutamate neurons expressing GCaMP. They analyzed images for fluorescence intensity and adjusted and colorized images with ImageJ. Assessment of the student learning experience was conducted for two Spring 2021 undergraduate laboratory courses at Cornell University (BioNB/BME/ECE 4910, Principles of Neurophysiology and BioNB 4300, Experimental Molecular Biology) and a Fall 2021 undergraduate Neurobiology laboratory course (Neurobiology 340) at Hobart and William Smith colleges. Students rated statements on all the following topics highly positive: knowledge of fluorescence imaging principles, affective experience of building and understanding the instrumentation, working in teams and clarity of guidance during the microscope build and use sessions. We continue our DIY efforts to expand the teaching, learning and research toolbox for physiology students and faculty.