Excitation Filter Research Articles

Design of a Mini Fluorescence Microscope

Microscopy is one of the most commonly used imaging methods in scientific research. The fluorescence microscopes which are used to determine the cell and tissue content at the molecular level, provides high-resolution images of the fluorochrome marked molecules. They are used in many areas with this feature. However, the use of fluorescence microscopy in biomedical field is limited due to various factors such as cost, size and difficulty of use. Also for some applications of fluorescence microscopy there are no need for very high quality and detailed images. Therefore, a small, practical and cost-effective mini fluorescent microscope is needed CITATION Mar17 \l 1055 (Sanz, Picazo-Bueno, Granero, & Mico, 2017) . In this study, a practical, portable and cost-effective fluorescent microscope was designed for specific proposes. Unlike the common fluorescent microscopes in this mini version one emission and one excitation wavelength is used. From top to bottom the design consists of a LED which is emitting the chosen wavelength of light, an excitation filter that can be used if necessary, a sample holder, a practical focus system developed in this project to be used for focus adjustment, a single-focus microscope objective lens, an emission filter and a camera. Plates holding these elements were formed using laser cutter, these plates are fixed in the form of layers with the appropriate materials. Filters and LED light source were determined and supplied according to the specific fluorescent dye to be used. These elements were tested by spectrophotometer and assembled together in the design. The LED power driver used in the design was supplied and the connections with the LED are completed. The focal point was calculated as 4 mm and the focal length as 44 mm so optical magnification 0.27 um/pixel was obtained. This means a 610 times magnification on a 15.6-inch screen. Using the mini fluorescent microscope a fluorochrome sensitive to 360nm wavelength was imaged. And the image from emitted light at 460nm wavelength was obtained. The entire design cost 2500 TL (434 USD). The dimensions of the fluorescence microscope are 120mm x 120mm x 170mm, 2448 c m 3 . Its weight is 1 kg. This mini fluorescence microscope can be used as an alternative to traditional fluorescent microscopes in developing countries and geographies where resources are scarce, because it is cost-effective and portable. As it is practical and user friendly, it can reduce the need for expert person. Also for point of care applications, this microscope can be an alternative for conventional fluorescence microscopy.

A Low-Cost and Portable Smart Instrumentation for Detecting Colorectal Cancer Cells

Fluorescence imaging is a well-known method for monitoring fluorescence emitted from the subject of interest and provides important insights about cell dynamics and molecules in mammalian cells. Currently, many solutions exist for measuring fluorescence, but the application methods are complex and the costs are high. This paper describes the design and development of a low-cost, smart and portable fluorimeter for the detection of colorectal cancer cell expressing IRFP702. A flashlight is used as a light source, which emits light in the visible range and acts as an excitation source, while a photodiode is used as a detector. It also uses a longpass filter to only allow the wavelength of interest to pass from the cultured cell. It eliminates the need of both the dichroic mirror and excitation filter, which makes the developed device low cost, compact and portable as well as lightweight. The custom-built sample chamber is black in color to minimize interference and is printed with a 3D printer to accommodate the detector circuitry. An established colorectal cancer cell line (human colorectal carcinoma (HCT116)) was cultured in the laboratory environment. A near-infrared fluorescent protein IRFP702 was expressed in the colorectal cancer cells that were used to test the proof-of-concept. The fluorescent cancer cells were first tested with a commercial imaging system (Odyssey® CLx) and then with the developed prototype to validate the result in a preclinical setting. The developed fluorimeter is versatile as it can also be used to detect multiple types of cancer cells by simply replacing the filters based on the fluorophore.

A Miniature 3D Printed LED-Induced Fluorescence Detector for Capillary Electrophoresis and Dual-Detector Taylor Dispersion Analysis.

Taylor dispersion analysis (TDA) provides absolute determination of diffusion coefficients for analytes ranging from small molecules to particulate matter. TDA has seen a resurgence in recent years, as modern commercial capillary electrophoresis (CE) instrumentation is well equipped to meet the precision flow requirements of TDA. Discontinuous flow velocities, which occur during sample injection, can lead to substantial inaccuracies in single-point detection TDA. Dual-point detection allows TDA to be carried out under continuous flow in the volume between the detection points, but dual-point fluorescence detection has not previously been feasible within the confines of commercial CE instrumentation. Here, we describe a compact light-emitting diode (LED)-induced fluorescence detector designed for online, dual-point capillary detection within a commercial CE system. The three-dimensional (3D) printed detector houses an inexpensive LED excitation source, a bandpass excitation filter, an integral 3D printed pinhole collimator, and a ball lens, which collects fluorescence emission. Multivariate optimization of operating conditions yielded a detection limit of 613 ± 13 pM for CE of fluorescein disodium salt solution in borate buffer. The miniature size of the device allowed integration of two detectors within a commercial CE system without modification to the instrument, thereby enabling dual-detector assays including TDA and CE-TDA. Monitoring of the bioconjugation reaction between fluorescein isothiocyanate (FITC) and a model protein illustrates the utility of direct, calibration-free size determination, which enabled the resolution of fluorescence originating from free FITC from that of protein-bound FITC. TDA detection coupled to CE enabled the determination of peak identities without the need for standard solutions.

Universal fluorescence module for intraoperative fluorescein angiography-a technical report.

Even in specialized centers, suboptimal aneurysm clipping can be as high as 12%. Intraoperative fluorescence angiography with indocyanine green and, more recently, fluorescein sodium have been shown to be a good method for intraoperative flow assessment. However, the cost with the apparatus it entails limits its widespread use. We have developed a low-cost universal fluorescence module (FM) designed to visualize fluorescein and perform intraoperative angiography. The purpose of this paper is to describe this device as well as to present our early experience with its use in the treatment of cerebral aneurysms. A FM was designed and built using a cyan-blue narrow bandpass (460 to 490nm) excitation filter and a yellow-orange longpass (blocking wavelengths under 520nm) barrier filter mounted on a 3D-printed holding tray in a specific disposition to perfectly match the light source and the objective lens of the surgical microscope. It allowed switching from white light to fluorescence mode in a simple and sterile fashion. Its perfect attachment to the microscope was possible by reusing the lens fittings extracted from used original drape sets that would otherwise be discarded. Four patients underwent aneurysm clipping using the FM at two institutions from April to September 2018. A bright green fluorescence against a dark background was observed after intravenous bolus of fluorescein. Blood vessels became obviously distinct from non-contrast-filled structures such as clipped aneurysms and the brain. Vascular anatomy could be appreciated without any distortion, including perforating arteries. Intraoperative fluorescence angiography was successfully performed with the use of this universal FM after intravenous injection of fluorescein sodium. This simple and low-cost device may be useful in resource-limited centers, where other sorts of intraoperative angiography are not available.

Simple ultraviolet microscope using off-the-shelf components for point-of-care diagnostics.

At the primary care setting, where there are often no or minimal laboratories, examinations often consist of self-testing and rapid diagnostics. Because of this, medical devices must be simple, robust, and easy to operate. To address these concerns, an alternate fluorescence microscope design uses ultraviolet (UV) excitation, since fluorescent dyes that are excitable in the visible region are also excitable by UV. This may allow for the removal of typical excitation, emission, and dichroic filters as optical components absorb UV wavelengths and UV is not detected by silicon based detectors. Additionally, UV has a very low penetration into samples, which may allow for controlling the depth of excitation, and thus the imaging volume. Based on these ideas, we developed a simple fluorescence microscope built completely from off-the-shelf components that uses UV to image fluorescently stained samples. The simple opto-mechanical design of the system may allow it to be more compact and easy to use, as well as decrease the overall cost of the diagnostic device. For biological validation, we imaged whole blood stained with acridine orange and performed a two-part white blood cell differential count.

Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging.

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

Therapeutic application of light emitting diode: Photo-oncomic approach

As a new light source, light emitting diode (LED) with high brightness and lower cost has been rapidly developed in medical application and light therapy. LED phototherapy can activate target cells with appropriate power and adequate energy density. This review provides general information on therapeutic applications of blue, green, yellow, red, and infrared LED in medical treatments for various physical abnormalities and on bio-imaging. The bio-imaging system is improved by decreasing the number of microscopes apparatuses including neutral-density filter, excitation filters and mechanical shutters. The numbers of excitation photons are increased and the fluorescent excitation efficiency is improved at cellular level. In the target tissue, the therapeutic effect of LEDs is dependent on incident photons irrespective of the system used to generate these photons. Photomodulated light from LED device is delivered in pulsed mode with specific pulse sequences and time. Too low or too high dose of energy may be ineffective at all. Clinical applications of LED light depending on different wavelengths are summarized. The author's photo-oncomic experiments using a specific blue light emitting diode were introduced, showing that blue LED possessed anti-proliferative and anti-metastatic abilities in cancer cells and mice. As a promising light source, photo-oncomic approach of blue LED could be applied to treat cancers and inflammatory diseases.

Development of a low-cost and portable smart fluorometer for detecting breast cancer cells.

Instruments that allow the detection of fluorescence signal are invaluable tools for biomedical and clinical researchers. The technique is widely used in cell biology to microscopically detect target proteins of interest in mammalian cells. Importantly, fluorescence microscopy finds major applications in cancer biology where cancer cells are chemically labelled for detection. However, conventional fluorescence detection instruments such as fluorescence imaging microscopes are expensive, not portable and entail potentially high maintenance costs. Here we describe the design, development and applicability of a low-cost and portable fluorometer for the detection of fluorescence signal emitted from a model breast cancer cell line, engineered to stably express the green fluorescent protein (GFP). This device utilizes a flashlight which works in the visible range as an excitation source and a photodiode as the detector. It also utilizes an emission filter to mainly allow the fluorescence signal to reach the detector while eliminating the use of an excitation filter and dichroic mirror, hence, making the device compact, low-cost, portable and lightweight. The custom-built sample chamber is fabricated with a 3D printer to house the detector circuitry. We demonstrate that the developed fluorometer is able to distinguish between the cancer cell expressing GFP and the control cell. The fluorometer we developed exhibits immense potential for future applicability in the selective detection of fluorescently-labelled breast cancer cells.

Automated sulfides quantification by multispectral optical microscopy

The mining industry needs effective techniques to meet the future challenges of resources extraction. As the deposits become more and more complex, a very good knowledge of an orebody is necessary. Mineralogical characterization is an essential contribution to improve the knowledge on the ore and wastes for a given mining project. It could bring major advances in ore extraction, mineral processing, and integrated waste management. However, mineralogical analyses can be very tedious, when done manually. Consequently, automated mineralogy was developed during the last three decades to improve the rapidity of mineralogical characterization, so that mineralogical information can be routinely obtained. Nowadays, the systems commonly used are based on expensive equipment including scanning electron microscopes (SEM) with energy dispersive X-ray analyzers (EDX). Optical Microscopy (OM) is neglected, although this route can provide reliable and quick results, yet cheaper. In this study, the possibility of using optical microscopy in reflected light mode to automatically characterize opaque minerals is explored. The identification and quantification of six common sulfides from polymetallic ores (arsenopyrite, chalcopyrite, galena, pyrite, pyrrhotite, and sphalerite) were automatically accomplished on a polished section by optical microscopy. Six spectral images were acquired for multispectral image analysis. Five of them were acquired under a white light source, equipped with four different excitation filters (436 nm, 480 nm, 605 nm, and 650 nm). The sixth image was acquired under an UV-light source at 365 nm, after modifying the optical pathway to detect the reflectance of the minerals in the UV-spectrum without changing the acquiring camera. Two image analysis software solutions were then tested to automatically classify and quantify the six sulfide minerals. The classification was systematically done on the acquired multispectral images by grey thresholding with the Clemex Vision PE® software. The GOCAD® software used principal component analysis (PCA) analysis and a supervised K-means clustering method to classify the minerals. Then, the obtained results were compared to the SEM-EDS quantification, considered here as the reference. The differences between the computed surface areas were mainly due to the artifacts of the polished sections preparation. The two software solutions present promising results and could be fully exploited to proceed to other mineralogical analyses, such as mineral liberation, mineral associations, textures identification, and particle size distribution. This study is an initial step towards differentiate and identify sulfides via an optical route arguably reliable and cheaper to characterize mine products.

Quantum Dot-labeled Tags Improve Minimal Detection Limit of CA125 in Ovarian Cancer Cells and Tissues

In recent years, a lot of attention has been paid to quantum dot (QD) nanoparticles as fluorescent sensors for sensitive and accurate detection of cancer biomarkers. Here, using a homemade specific monoclonal antibody against CA125 and QD525- or FITC-labeled probes, expression of this marker in an ovarian cancer cell line and cancer tissues were traced and optical properties of fluorophores were compared qualitatively and quantitatively. Our results clearly showed that besides lower background and exceptionally higher photobleaching resistance, QD525 exhibited higher fluorescent intensity for both ovarian cancer cell and tissues at different exposure times (p<0.0001) and excitation filter sets (p<0.0001) exemplified by significantly higher staining index (p<0.016). More importantly, the FITC-labeled probe detected antigen-antibody complex at minimum concentration of 0.3 mg/mL of anti-CA125, while reactivity limit decreased to 0.078 mg/mL of anti-CA125 when QD525-labeled probe was applied showing four times higher reactivity level of QD525 probe compared to the same probe labeled with FITC. Based on our results, it seems that QDs are inimitable tags for sensitive detection and localization of ovarian cancer micrometastasis and molecular demarcation of cancer tissues in surgical practice, which subsequently figure out accurate therapeutic approaches.

Novel electrophoretic acetonitrile-based stacking for sensitive monitoring of the antiepileptic drug perampanel in human serum

Perampanel is a novel antiepileptic drug used in paediatric patients. Existing methods that determine serum perampanel are of limited practicability. We developed a novel capillary electrophoresis (CE) method using a new version of acetonitrile stacking for on-line sample pre-concentration, and fluorescence detection (FD). CE separations were performed in a fused-silica capillary where the electroosmotic flow was reduced by coating the inner surface using a INST coating solution. The optimised background electrolyte composition was 50 mM chloroacetic acid with addition of 0.5% m/v polyvinylalcohol (pH 2.15) and separation was driven by application of positive voltage + 30 kV. Serum samples (25 μL) treated by the addition of acetonitrile in a ratio of 1:3 v/v were each injected into the capillary at a large volume that corresponded to the length (129 mm) of the sample zone (hydrodynamic pressure impulse 6000 mbars). Acetonitrile stacking is based on the forcing the sample zone out of the capillary with simultaneous application of the separation voltage. Under such conditions, the enhancing factor achieves the value 57 for peak area compared to the small sample injection length (3.2 mm, hydrodynamic pressure impulse 150 mbar.s). A fluorescence detector with a broad excitation filter (240–400 nm) and an emission filter (495 nm) was used for visualisation of the native fluorescence of perampanel. The calibration dependence of the method was linear (in the range of 10–1000 ng mL−1), with adequate accuracy (99.8–103.3 %) and precision (13.1%). LOD and LOQ for perampanel were 2.9 ng mL-1 and 9.5 ng mL−1, respectively. Clinical applicability was validated using serum samples from patients treated with perampanel and the results corresponded with reference LC–MS/MS values. Our method offers a promising alternative for determining serum perampanel with several advantages. In particular, the low quantity of serum (25 μL) required means that testing can be performed on samples obtained for monitoring other antiepileptic medications, and thus reduces the test-burden on paediatric patients.

Simultaneous Optogenetics and Cellular Resolution Calcium Imaging During Active Behavior Using a Miniaturized Microscope.

The ability to precisely monitor and manipulate neural circuits is essential to understand the brain. Advancements over the last decade in optical techniques such as calcium imaging and optogenetics have empowered researchers to gain insight into brain function by systematically manipulating or monitoring defined neural circuits. Combining these cutting-edge techniques enables a more direct mechanism for ascribing neural dynamics to behavior. Here, we developed a miniaturized integrated microscope that allows for simultaneous optogenetic manipulation and cellular-resolution calcium imaging within the same field of view in freely behaving mice. The integrated microscope has two LEDs, one filtered with a 435–460 nm excitation filter for imaging green calcium indicators, and a second LED filtered with a 590–650 nm excitation filter for optogenetic modulation of red-shifted opsins. We developed and tested this technology to minimize biological and optical crosstalk. We observed insignificant amounts of biological and optical crosstalk with regards to the optogenetic LED affecting calcium imaging. We observed some amounts of residual crosstalk of the imaging light on optogenetic manipulation. Despite residual crosstalk, we have demonstrated the utility of this technology by probing the causal relationship between basolateral amygdala (BLA) -to- nucleus accumbens (NAc) circuit function, behavior, and network dynamics. Using this integrated microscope we were able to observe both a significant behavioral and cellular calcium response of the optogenetic modulation on the BLA-to-NAc circuit. This integrated strategy will allow for routine investigation of the causality of circuit manipulation on cellular-resolution network dynamics and behavior.

Antioxidant activity of mangaba (Hancornia speciosa) and acerola (Malpighia emarginata) fruit pulp and residues

Antioxidants are compounds which protect the biological system against the damaging effects of processes or reactions that can cause excessive oxidation. Fruits contain various antioxidants whose activities have been well proven in reduction of degenerative diseases. The aim of the study was to determine the total antioxidant activity by free radical capture method DPPH (2,2-diphenyl-1-picryl hydrazyl), TEAC (Trolox Equivalent Antioxidant Capacity) and ORAC (Oxygen Radical Absorbance Capacity) and compare the levels of total phenolic compounds in ethanol, methanolic and aqueous extracts of pulp and residue of mangaba and acerola fruits. The flour was obtained by the residue of the peel and seeds of the fruits after pulping, the extracts were obtained by ethanol, methanol and water using ultrasound. The DPPH and TEAC tests were performed with a spectrophotometer at 517 and 750 nm, respectively, while ORAC was carried out with the aid of fluorimeter, using 520 nm emission and 485 nm excitation filters. The results showed better antioxidant activity with the aqueous extracts in all methods, but the TEAC method had the highest antioxidant activity for acerola pulp and residue (429 µM Trolox g-1 and 90.89 µM Trolox g-1, respectively), followed by mangaba pulp and residue (201 µM Trolox g-1 and 61.87 µM Trolox g-1, respectively). In addition, the highest content of phenolic compounds was found in the residue of acerola (31 mg gallic acid 100 g-1), followed by the mangaba pulp (21 mg gallic acid 100 g-1).

A feasibility study of an integrated NIR/gamma/visible imaging system for endoscopic sentinel lymph node mapping.

The aim of this study is to integrate NIR, gamma, and visible imaging tools into a single endoscopic system to overcome the limitation of NIR using gamma imaging and to demonstrate the feasibility of endoscopic NIR/gamma/visible fusion imaging for sentinel lymph node (SLN) mapping with a small animal. The endoscopic NIR/gamma/visible imaging system consists of a tungsten pinhole collimator, a plastic focusing lens, a BGO crystal (11×11×2mm3 ), a fiber-optic taper (front=11×11mm2 , end=4×4mm2 ), a 122-cm long endoscopic fiber bundle, an NIR emission filter, a relay lens, and a CCD camera. A custom-made Derenzo-like phantom filled with a mixture of 99m Tc and indocyanine green (ICG) was used to assess the spatial resolution of the NIR and gamma images. The ICG fluorophore was excited using a light-emitting diode (LED) with an excitation filter (723-758nm), and the emitted fluorescence photons were detected with an emission filter (780-820nm) for a duration of 100ms. Subsequently, the 99m Tc distribution in the phantom was imaged for 3min. The feasibility of invivo SLN mapping with a mouse was investigated by injecting a mixture of 99m Tc-antimony sulfur colloid (12 MBq) and ICG (0.1 mL) into the right paw of the mouse (C57/B6) subcutaneously. After one hour, NIR, gamma, and visible images were acquired sequentially. Subsequently, the dissected SLN was imaged in the same way as the invivo SLN mapping. The NIR, gamma, and visible images of the Derenzo-like phantom can be obtained with the proposed endoscopic imaging system. The NIR/gamma/visible fusion image of the SLN showed a good correlation among the NIR, gamma, and visible images both for the invivo and exvivo imaging. We demonstrated the feasibility of the integrated NIR/gamma/visible imaging system using a single endoscopic fiber bundle. In future, we plan to investigate miniaturization of the endoscope head and simultaneous NIR/gamma/visible imaging with dichroic mirrors and three CCD cameras.

Red and Green Fluorescence from Oral Biofilms.

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

Murine fundus fluorescein angiography: An alternative approach using a handheld camera

In today’s modern pharmacologic approach to treating sight-threatening retinal vascular disorders, there is an increasing demand for a compact, mobile, lightweight and cost-effective fluorescein fundus camera to document the effects of antiangiogenic drugs on laser-induced choroidal neovascularization (CNV) in mice and other experimental animals. We have adapted the use of the Kowa Genesis Df Camera to perform Fundus Fluorescein Angiography (FFA) in mice. The 1 kg, 28 cm high camera has built-in barrier and exciter filters to allow digital FFA recording to a Compact Flash memory card. Furthermore, this handheld unit has a steady Indirect Lens Holder that firmly attaches to the main unit, that securely holds a 90 diopter lens in position, in order to facilitate appropriate focus and stability, for photographing the delicate central murine fundus. This easily portable fundus fluorescein camera can effectively record exceptional central retinal vascular detail in murine laser-induced CNV, while readily allowing the investigator to adjust the camera’s position according to the variable head and eye movements that can randomly occur while the mouse is optimally anesthetized. This movable image recording device, with efficiencies of space, time, cost, energy and personnel, has enabled us to accurately document the alterations in the central choroidal and retinal vasculature following induction of CNV, implemented by argon-green laser photocoagulation and disruption of Bruch’s Membrane, in the experimental murine model of exudative macular degeneration.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

A Novel, Real-Time, In Vivo Mouse Retinal Imaging System

To develop an efficient, low-cost instrument for robust real-time imaging of the mouse retina in vivo, and assess system capabilities by evaluating various animal models. Following multiple disappointing attempts to visualize the mouse retina during a subretinal injection using commercially available systems, we identified the key limitation to be inadequate illumination due to off axis illumination and poor optical train optimization. Therefore, we designed a paraxial illumination system for Greenough-type stereo dissecting microscope incorporating an optimized optical launch and an efficiently coupled fiber optic delivery system. Excitation and emission filters control spectral bandwidth. A color coupled-charged device (CCD) camera is coupled to the microscope for image capture. Although, field of view (FOV) is constrained by the small pupil aperture, the high optical power of the mouse eye, and the long working distance (needed for surgical manipulations), these limitations can be compensated by eye positioning in order to observe the entire retina. The retinal imaging system delivers an adjustable narrow beam to the dilated pupil with minimal vignetting. The optic nerve, vasculature, and posterior pole are crisply visualized and the entire retina can be observed through eye positioning. Normal and degenerative retinal phenotypes can be followed over time. Subretinal or intraocular injection procedures are followed in real time. Real-time, intravenous fluorescein angiography for the live mouse has been achieved. A novel device is established for real-time viewing and image capture of the small animal retina during subretinal injections for preclinical gene therapy studies.