Emission Filter Research Articles

Dye-Specific Wavelength Offsets to Resolve Spectrally Overlapping and Co-Localized Two-Photon Induced Fluorescence.

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitter's detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon-Dye-Specific Excitation-Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.

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.

RETRACTED ARTICLE: An alternative high-throughput staining method for detection of neutral lipids in green microalgae for biodiesel applications

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO4). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure.

Lymph node mapping using quantum dot-labeled polymersomes.

The present study was designed to investigate whether poly-ion complex hollow vesicles (polymersomes), based on chemically-modified chitosan, are appropriate for lymph node mapping in the context of their application in the development of theranostic nanosized drug delivery systems (nano-DDS). The experiments were performed on Balb/c nude mice (colon cancer-grafted). The mice were subjected to anesthesia and quantum dot (QD(705))-labeled polymersomes (d-120 nm) were injected intravenously via the tail vein. The optical imaging was carried out on Maestro EX Imaging System (excitation filter: 435-480 nm; emission filter: 700 nm). A strong fluorescent signal, corresponding to QD(705) fluorescence, was detected in the lymph nodes, as well as in the tumor. A very weak fluorescent signal was found in the liver area. The half-life of QD(705)-labelled polymersomes was 6 ± 2 hours in the bloodstream and 11 ± 3 hours in the lymph nodes. The data suggest that polymersomes are very promising carriers for lymph node mapping using QD as a contrast agent. They are useful matrix for development of nano-formulations with theranostic capabilities.

New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging.

To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti:Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.

Simultaneous Sodium and Calcium Imaging from Dendrites and Axons.

Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca2+]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca2+]i and [Na+]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca2+]i and [Na+]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).

Second Harmonic Generation Imaging Analysis of Collagen Arrangement in Human Cornea.

To describe the horizontal arrangement of human corneal collagen bundles by using second harmonic generation (SHG) imaging. Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane. Collagen bundles in the corneal lamellae demonstrated a complex layout merging and splitting within a single lamellar plane. The patterns were significantly different in the superficial and limbal cornea when compared with deep and central regions. Collagen bundles were smaller in the superficial layer and larger in deep lamellae. By using SHG imaging, the horizontal arrangement of corneal collagen bundles was elucidated at different depths and focal regions of the human cornea.

Image based quantitative reader for Lateral flow immunofluorescence assay.

Fluorescence Lateral flow immunoassays (LFIA) have wide range of applications in point-of-care testing (POCT). An integrated, motion-free, accurate, reliable reader that performs automated quantitative analysis of LFIA is essential for POCT diagnosis. We demonstrate an image based quantitative method to read the lateral flow immunofluorescence test strips. The developed reader uses line laser diode module to illuminate the LFIA test strip having fluorescent dye. Fluorescence light coming from the region of interest (ROI) of the LFIA test strip was filtered using an emission filter and imaged using a camera following which images were processed in computer. A dedicated control program was developed that automated the entire process including illumination of the test strip using laser diode, capturing the ROI of the test strip, processing and analyzing the images and displaying of results. Reproducibility of the reader has been evaluated using few reference cartridges and HbA1c (Glycated haemoglobin) test cartridges. The proposed system can be upgraded to a compact reader for widespread testing of LFIA test strips.

Combining fluorescence and bioluminescence microscopy.

ABSTRACTBioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin—a substrate of luciferase in promoter assays of gene expression in cultured cells—confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492–506 nm and 524–578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol‐12‐myristate‐13‐acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech. 78:715–722, 2015. © 2015 Wiley Periodicals, Inc.

Comparative study reveals better far-red fluorescent protein for whole body imaging.

Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.

HPTLC with Fluorescence Densitometry for Simultaneous Determination of Some Angiotensin II Receptor Blockers in Tablets and Plasma

A selective, accurate, precise, and highly sensitive HPTLC assay with reflectance/fluorescence densitometry was developed to separate and quantify some angiotensin II receptor blockers (ARBs), namely, losartan potassium, irbesartan, valsartan, and olmesartan medoxomil, in tablets and plasma. Separation of the target ARBs was performed on precoated silica gel HPTLC plates developed using chloroform-glacial acetic acid (7.5 + 2.5, v/v) mobile phase. The drugs were adequately resolved. After the exposure of the chromatograms to concentrated hydrochloric acid vapor for 10 min, the scanner was set in the reflectance/fluorescence mode with 255 nm excitation wavelength and >400 nm emission filter. The linear regression analysis data for the calibration curves of all studied drugs produced a good linear relationship with correlation coefficients ranging from 0.9991 to 0.9999 over the concentration range of 2-20 ng/band. LOD and LOQ values of all studied drugs ranged from 0.6 to 0.8 and from 1.7 to 2.4 ng/band, respectively. The developed method was successfully applied for analysis of the target ARBs in tablets and spiked human plasma samples with good precision and accuracy.

The Quality of In Vivo Upconversion Fluorescence Signals Inside Different Anatomic Structures.

Fluorescence imaging is a broadly interesting and rapidly growing strategy for non-invasive clinical applications. However, because of interference from light scattering, absorbance, and tissue autofluorescence, the images can exhibit low sensitivity and poor quality. Upconversion fluorescence imaging, which is based on the use of near-infrared (NIR) light for excitation, has recently been introduced as an improved approach to minimize the effects of light scattering and tissue autofluorescence. This strategy is promising for ultrasensitive and deep tissue imaging applications. However, the emitted upconversion fluorescence signals are primarily in the visible range and are likely to be absorbed and scattered by tissues. Therefore, different anatomic structures could impose various effects on the quality of the images. In this study, we used upconversion-core/silica-shell nanoprobes to evaluate the quality of upconversion fluorescence at different anatomic locations in athymic nude mice. The nanoprobe contained an upconversion core, which was green (β-NaYF4:Yb3+/Ho3+) or red (β-NaYF4:Yb3+/Er3+), and a nonporous silica shell to allow for multicolor imaging. High-quality upconversion fluorescence signals were detected with signal-to-noise ratios of up to 170 at tissue depths of up to - 1.0 cm when a 980 nm laser excitation source and a bandpass emission filter were used. The presence of dense tissue structures along the imaging path reduced the signal intensity and imaging quality, and nanoprobes with longer-wavelength emission spectra were therefore preferable. This study offers a detailed analysis of the quality of upconversion signals in vivo inside different anatomic structures. Such information could be essential for the analysis of upconversion fluorescence images in any in vivo biodiagnostic and microbial tracking applications.

Seleção de traçadores para estudos de eficiência das aplicações de produtos fitossanitários

Para avaliar a deposição e as perdas de calda nas aplicações de produtos fitossanitários, na maioria dos casos são utilizados traçadores. Entretanto, não há consenso sobre qual é o traçador mais adequado para cada tipo de aplicação. O objetivo deste trabalho foi avaliar o uso de alguns compostos químicos empregados como traçadores, buscando selecionar os mais adequados aos estudos de deposição e perda de produtos fitossanitários. Os traçadores utilizados foram: azul brilhante, blankophor, fluoresceína sódica, rodamina B e sulfoflavina brilhante; as concentrações usadas foram: 1.000, 20, 15, 50 e 1.000 ppm, respectivamente. A quantificação do azul brilhante foi feita por espectrofotometria, e a dos demais, por espectrofluorimetria. Os filtros de excitação e emissão do fluorímetro variaram de acordo com o traçador. No espectrofotômetro, utilizou-se o comprimento de onda de 630 nm. As características avaliadas nas soluções contendo os traçadores foram: tensão superficial, pH, viscosidade, extração em alvos de papel filtrante e estabilidade à luz solar e à agitação. Os dados foram submetidos à análise de variância e, constatada diferença significativa, aplicou-se teste de médias e regressão. Entre as características físico-químicas, somente a viscosidade não foi influenciada pelos traçadores. A maior eficiência de extração em alvo de papel foi obtida pela sulfoflavina brilhante, que, juntamente com o azul brilhante, demonstrou ser o traçador mais estável à luz solar. O blankophor foi o traçador que teve menor estabilidade à agitação da calda. A sulfoflavina brilhante mostrou ser o traçador mais adequado aos estudos de deposição de calda e deriva nas aplicações de produtos fitossanitários. Entre os traçadores de baixo custo e maior disponibilidade no Brasil, o corante fluorescente rodamina B mostrou ser a melhor opção ao se utilizar como alvo papel filtrante.

Delivery of size-controlled long-circulating polymersomes in solid tumours, visualized by quantum dots and optical imaging in vivo

The present study was designed to investigate whether poly-ion complex hollow vesicles (polymersomes), based on chemically modified chitosan, are appropriate for passive tumour targeting in the context of their application as drug carriers. The experiments were performed on colon cancer-grafted mice. The mice were subjected to anaesthesia and injected intravenously with water-soluble nanoparticles: (1) QD705-labelled polymersomes (average size ∼120 nm; size distribution ∼10%) or (2) native QD705. The optical imaging was carried out on Maestro EX 2.10 In Vivo Imaging System (excitation filter 435–480 nm; emission filter 700 nm, longpass). In the case of QD705, the fluorescence appeared in the tumour area within 1 min after injection and disappeared completely within 60 min. A strong fluorescent signal was detected in the liver on the 30th minute. The visualization of tumour using QD705 was based only on angiogenesis. In the case of QD705-labelled polymersomes, the fluorescence appeared in the tumour area immediately after injection with excellent visualization of blood vessels in the whole body. A strong fluorescent signal was detected in the tumour area within 16 hours. This indicated that QD705-labelled polymersomes were delivered predominantly into the tumour due to their long circulation in the bloodstream and enhanced permeability and retention effect. A very weak fluorescent signal was found in the liver area. The data suggest that size-controlled long-circulating polymersomes are very promising carriers for drug delivery in solid tumours, including delivery of small nanoparticles and contrast substances.

Fluorescence detection and identification of eight sulphonamides using capillary electrophoresis on released excipients in lake water

A simple and sensitive capillary electrophoresis method with fluorescence detection was developed for the determination of sulphanilamide, sulphamerazine, sulphacetamide and sulphanilic acid, sulphathiazole, Sulphisomidine, sulphadoxine and sulphadiazine in lake water. The sulphonamides were extracted from lake water, derivatized with fluorescamine and determination of sulphonamide was achieved using 20mM borate buffer of pH 9.5 at an applied voltage of 25kV. Detection was performed using UG-11 excitation filter of 405nm and 495nm emission filters. A fast, simple and sensitive method with limit of detection in the range 0.89–1.43nmolL−1 for all the eight sulphonamides with good recoveries of 80–110% is seen. Inter-day and intra-day validation of the separation method shows fairly good results. The detection and quantification limits for this newly developed method are too low to determine drug residues in lake water.

Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E, Tocopherol Succinate and Tocopherol Phosphate

Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met(80)) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c(+/+) cells than in cytochrome c(-/-) cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity.

BI-24 * COLLAGEN PLAYS A ROLE IN GLIOBLASTOMA TUMOR INVASION AND PATIENT SURVIVAL

BACKGROUND: Glioblastoma (GBM) is the most malignant brain cancer. Collagen is minimally expressed in normal brain parenchyma; however, previous reports show upregulated collagen gene expression in GBM. We perform the first direct GBM collagen visualization, identify clinically relevant collagen signatures in focal and invasive GBMs and link them to differential patient survival. METHODS: Customized second harmonic generation (SHG) multiphoton workstation with excitation wavelength tuned to 890nm and a 445± 25nm bandpass emission filter was used to detect collagen in a clinically-annotated human GBM tissue microarray (TMA) for 111 patients. Human GBM cancer stem cell lines that form focal or invasive xenografts were implanted into mice brains. Symptomatic mice with verified xenografts were euthanized, brains excised, and stained with picrosirius red. Quantitative characterization of collagen fibers in xenografts and TMA images was performed with custom software. RESULTS: Collagen was directly visualized in GBMs with SHG and picrosirius red. In focal GBM xenografts, collagen was observed at tumor-brain boundaries, appearing to encapsulate the tumors. For invasive GBM xenografts, collagen was intercalated with tumor cells. Quantitative collagen analysis showed significant differences for mean angle between fibers for focal (60.3856 ± 2.2113) and invasive (70.2529 ± 1.6211) GBM xenografts. GBM TMA specimens were segregated into two collagen groups: fibrillar and immature. Patients with fibrillar GBM collagen had a statistically significantly better median survival (69.6 weeks) compared to immature GBM collagen patients (30.4 weeks). The mean angle between fibers was significantly different for GBMs with fibrillar (84.37 ± 7.298) versus immature (113.2 ± 11.60) collagen. CONCLUSIONS: We demonstrate that collagen can be directly visualized in GBMs, and is different in focal vs. invasive tumors. Our data suggest collagen is a potential survival marker: patients with fibrillar collagen have improved survival. Therefore, we hypothesize that collagen may influence tumor cell invasion by acting as a barrier, and collagen disruption may promote tumor invasion.

Abstract 4312: Seamless fluorescence guided surgery for pancreatic cancer is facilitated by a hand held electrocautery pencil with excitation light source

Abstract Fluorescent labeling of tumors with the goal of fluorescence guided surgical resection (FGS) continues to be perfected and investigated. In addition to a bright fluorophore that specifically and selectively labels tumor tissue, a specific excitation light source that can be turned on as needed by the surgeon without having to take their hands off the dissecting instruments would be the most ideal system. The optimal method for tissue excitation would therefore require the excitation light source to be on the surgeon's dissecting or cutting instrument that could be easily turned on or off as needed and in direct line with the labeled tumor tissue. In designing the excitation light source we decided to use the NIR window (600-1000 nm) due to it's favorable imaging qualities that would allow for fluorophore excitation without the necessity of eliminating white light from the field and with little to no tissue autofluorescence. An electrocautery pencil was fitted with LEDs at the base of the tip with an emitted signal between 633-660nm. The complementary dye selected for labeling was DyLight 650 (Ex: 652 nm, Em: 672 nm). Nude mice were implanted with patient-derived orthotopic xenograft (PDOX) pancreatic tumors and allowed 4 weeks for engraftment. Anti-CA 19-9 antibody was then conjugated to DyLight 650 and injected intravenously 24 hours prior to FGS. After 24 hours the mice underwent surgical exploration for an attempt at FGS with the new device. A Canon EOS 60D camera fitted with a 672 nm emission filter connected to a 19 inch monitor was selected as the fluorescence image-capturing device. The mice were grounded appropriately to a surgical monopolar generator selected to power to the electrocautery pencil. After initial incision and tumor exposure under bright light the tumor was illuminated by the excitation LEDs by simply pushing forward on the cautery activation button on the device. The fluorescently labeled image of the tumor was easily seen and confirmed on the monitor in real-time as the surgery proceeded. The tumor was brightly visible with good background contrast. Using this method had the benefit of allowing tumor resection with electrocautery using both ambient light and the fluorescent image simultaneously to confirm a complete resection. In this respect, critical surrounding structures are visible with the ambient light, helping to avoid inadvertent tissue injury. Coupling an excitation LED light source to an electrocautery pencil allows for good imaging of pre-labeled tumors during resection without mounting an external excitation light source. The set-up is very compact, efficient, easy to sterilize and works very efficiently during surgical resection without the need to eliminate background light. This is highly advantageous and allows simultaneous visualization of the surgical field in white light and the labeled tumor via an imaging device connected to a monitor. Citation Format: Ali A. Maawy, Yukihiko Hiroshima, George A. Luiken, Yong Zhang, Richard P. Fleenor, Robert M. Hoffman, Michael Bouvet. Seamless fluorescence guided surgery for pancreatic cancer is facilitated by a hand held electrocautery pencil with excitation light source. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4312. doi:10.1158/1538-7445.AM2014-4312

Non-invasive assessment of cutaneous wound healing using fluorescent imaging.

Optical imaging is a very important technique in the biomedical sciences. The purpose of this study was to develop an in vivo optical system for fluorescent imaging and molecular imaging applications using quantum dots (QDs). The in vivo optical system was composed of modular parts, including a light source, light guide, excitation filter wheel, excitation filters, emission filter wheel, emission filters, liquid crystal tunable filter (LCTF), macro lens, dark chamber, and a cooled charged-coupled device (CCD) camera for recording images. Filters were selected based on the excitation and absorption spectra of QDs to allow spectral separation and optimization of the acquired image. In contrast with conventional systems, our system allows selection of the emission bandwidth. The system was tested in an in vivo study using a wound-healing model in nude mice. The healing process was examined after injection of fibroblasts and keratinocytes labeled with two different sets of QDs. The different QD probes were readily detected and distinguished using our system. An in vivo optical system is a very useful tool for the detection of genes, proteins, and small-molecule drugs inside living animals, and this imaging modality can also be adopted for real-time visualization of cancer cell metastasis in live animals.