Статті в журналах: "Fluorescence imaging systems"

1

Farrell, Joyce, Zheng Lyu, Zhenyi Liu, Henryk Blasinski, Zhihao Xu, Jian Rong, Feng Xiao, and Brian Wandell. "Soft-prototyping imaging systems for oral cancer screening." Electronic Imaging 2020, no. 7 (January 26, 2020): 212–1. http://dx.doi.org/10.2352/issn.2470-1173.2020.7.iss-212.

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We are using image systems simulation technology to design a digital camera for measuring fluorescent signals; a first application is oral cancer screening. We validate the simulations by creating a camera model that accurately predicts measured RGB values for any spectral radiance. Then we use the excitationemission spectra for different biological fluorophores to predict measurements of fluorescence of oral mucosal tissue under several different illuminations. The simulations and measurements are useful for (a) designing cameras that measure tissue fluorescence and (b) clarifying which fluorophores may be diagnostic in identifying precancerous tissue.

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2

Yan, Yuling, M. Emma Marriott, Chutima Petchprayoon, and Gerard Marriott. "Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems." Biochemical Journal 433, no. 3 (January 14, 2011): 411–22. http://dx.doi.org/10.1042/bj20100992.

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Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

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3

Etrych, Tomáš, Olga Janoušková, and Petr Chytil. "Fluorescence Imaging as a Tool in Preclinical Evaluation of Polymer-Based Nano-DDS Systems Intended for Cancer Treatment." Pharmaceutics 11, no. 9 (September 12, 2019): 471. http://dx.doi.org/10.3390/pharmaceutics11090471.

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Targeted drug delivery using nano-sized carrier systems with targeting functions to malignant and inflammatory tissue and tailored controlled drug release inside targeted tissues or cells has been and is still intensively studied. A detailed understanding of the correlation between the pharmacokinetic properties and structure of the nano-sized carrier is crucial for the successful transition of targeted drug delivery nanomedicines into clinical practice. In preclinical research in particular, fluorescence imaging has become one of the most commonly used powerful imaging tools. Increasing numbers of suitable fluorescent dyes that are excitable in the visible to near-infrared (NIR) wavelengths of the spectrum and the non-invasive nature of the method have significantly expanded the applicability of fluorescence imaging. This chapter summarizes non-invasive fluorescence-based imaging methods and discusses their potential advantages and limitations in the field of drug delivery, especially in anticancer therapy. This chapter focuses on fluorescent imaging from the cellular level up to the highly sophisticated three-dimensional imaging modality at a systemic level. Moreover, we describe the possibility for simultaneous treatment and imaging using fluorescence theranostics and the combination of different imaging techniques, e.g., fluorescence imaging with computed tomography.

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4

Royon, Arnaud, and Noël Converset. "Quality Control of Fluorescence Imaging Systems." Optik & Photonik 12, no. 2 (April 2017): 22–25. http://dx.doi.org/10.1002/opph.201700005.

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5

Georgiev, Nikolai I., Ventsislav V. Bakov, Kameliya K. Anichina, and Vladimir B. Bojinov. "Fluorescent Probes as a Tool in Diagnostic and Drug Delivery Systems." Pharmaceuticals 16, no. 3 (March 1, 2023): 381. http://dx.doi.org/10.3390/ph16030381.

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Over the last few years, the development of fluorescent probes has received considerable attention. Fluorescence signaling allows noninvasive and harmless real-time imaging with great spectral resolution in living objects, which is extremely useful for modern biomedical applications. This review presents the basic photophysical principles and strategies for the rational design of fluorescent probes as visualization agents in medical diagnosis and drug delivery systems. Common photophysical phenomena, such as Intramolecular Charge Transfer (ICT), Twisted Intramolecular Charge Transfer (TICT), Photoinduced Electron Transfer (PET), Excited-State Intramolecular Proton Transfer (ESIPT), Fluorescent Resonance Energy Transfer (FRET), and Aggregation-Induced Emission (AIE), are described as platforms for fluorescence sensing and imaging in vivo and in vitro. The presented examples are focused on the visualization of pH, biologically important cations and anions, reactive oxygen species (ROS), viscosity, biomolecules, and enzymes that find application for diagnostic purposes. The general strategies regarding fluorescence probes as molecular logic devices and fluorescence–drug conjugates for theranostic and drug delivery systems are discussed. This work could be of help for researchers working in the field of fluorescence sensing compounds, molecular logic gates, and drug delivery.

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6

Fenton, James M., and Antony R. Crofts. "Computer aided fluorescence imaging of photosynthetic systems." Photosynthesis Research 26, no. 1 (October 1990): 59–66. http://dx.doi.org/10.1007/bf00048977.

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7

Pawlowski, Michal E., and Yiran Yang. "Achromatization method for multichannel fluorescence imaging systems." Optical Engineering 58, no. 01 (January 22, 2019): 1. http://dx.doi.org/10.1117/1.oe.58.1.015106.

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8

Kudryavtsev, Volodymyr, Suren Felekyan, Anna K. Woźniak, Marcelle König, Carl Sandhagen, Ralf Kühnemuth, Claus A. M. Seidel, and Filipp Oesterhelt. "Monitoring dynamic systems with multiparameter fluorescence imaging." Analytical and Bioanalytical Chemistry 387, no. 1 (December 12, 2006): 71–82. http://dx.doi.org/10.1007/s00216-006-0917-0.

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9

Lo, Shih-Jie, Chen-Meng Kuan, Min-Wei Hung, Yun Fu, J. Yeh, Da-Jeng Yao, and Chao-Min Cheng. "A Simple Imaging Device for Fluorescence-Relevant Applications." Micromachines 9, no. 8 (August 20, 2018): 418. http://dx.doi.org/10.3390/mi9080418.

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This article unveiled the development of an inexpensive, lightweight, easy-to-use, and portable fluorescence imaging device for paper-based analytical applications. We used commercial fluorescent dyes, as proof of concept, to verify the feasibility of our fluorescence imaging device for bioanalysis. This approach may provide an alternative method for nucleotide detection and semen analysis, using a miniaturized fluorescence reader that is more compact and portable than conventional analytical equipment.

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10

Wang, Li, Mingguang Ren, Zihong Li, Lixuan Dai, and Weiying Lin. "A ratiometric two-photon fluorescent probe for the rapid detection of HClO in living systems." Analytical Methods 11, no. 12 (2019): 1580–84. http://dx.doi.org/10.1039/c9ay00205g.

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11

Bettiol, Andrew A., Zhaohong Mi, Sudheer Kumar Vanga, Ce-belle Chen, Ye Tao, and Frank Watt. "Ion beam induced fluorescence imaging in biological systems." Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 348 (April 2015): 131–36. http://dx.doi.org/10.1016/j.nimb.2014.11.120.

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12

Yang, Andrew Wootae, Sang Uk Cho, Myung Yung Jeong, and Hak Soo Choi. "NIR Fluorescence Imaging Systems with Optical Packaging Technology." Journal of the Microelectronics and Packaging Society 21, no. 4 (December 30, 2014): 25–31. http://dx.doi.org/10.6117/kmeps.2014.21.4.025.

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13

CHEN, CHAO-WEI, TIFFANY R. BLACKWELL, RENEE NAPHAS, PAUL T. WINNARD, VENU RAMAN, KRISTINE GLUNDE, and YU CHEN. "DEVELOPMENT OF NEEDLE-BASED MICROENDOSCOPY FOR FLUORESCENCE MOLECULAR IMAGING OF BREAST TUMOR MODELS." Journal of Innovative Optical Health Sciences 02, no. 04 (October 2009): 343–52. http://dx.doi.org/10.1142/s1793545809000747.

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Fluorescence molecular imaging enables the visualization of basic molecular processes such as gene expression, enzyme activity, and disease-specific molecular interactions in vivo using targeted contrast agents, and therefore, is being developed for early detection and in situ characterization of breast cancers. Recent advances in developing near-infrared fluorescent imaging contrast agents have enabled the specific labeling of human breast cancer cells in mouse model systems. In synergy with contrast agent development, this paper describes a needle-based fluorescence molecular imaging device that has the strong potential to be translated into clinical breast biopsy procedures. This microendoscopy probe is based on a gradient-index (GRIN) lens interfaced with a laser scanning microscope. Specifications of the imaging performance, including the field-of-view, transverse resolution, and focus tracking characteristics were calibrated. Orthotopic MDA-MB-231 breast cancer xenografts stably expressing the tdTomato red fluorescent protein (RFP) were used to detect the tumor cells in this tumor model as a proof of principle study. With further development, this technology, in conjunction with the development of clinically applicable, injectable fluorescent molecular imaging agents, promises to perform fluorescence molecular imaging of breast cancers in vivo for breast biopsy guidance.

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14

Carr, Jessica A., Daniel Franke, Justin R. Caram, Collin F. Perkinson, Mari Saif, Vasileios Askoxylakis, Meenal Datta, et al. "Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green." Proceedings of the National Academy of Sciences 115, no. 17 (April 6, 2018): 4465–70. http://dx.doi.org/10.1073/pnas.1718917115.

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Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave IR (SWIR; 1,000–2,000 nm) promises higher contrast, sensitivity, and penetration depths compared with conventional visible and near-IR (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, partially due to the absence of US Food and Drug Administration (FDA)-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Even though their emission spectra peak in the NIR, these dyes outperform commercial SWIR fluorophores and can be imaged in the SWIR, even beyond 1,500 nm. We show real-time fluorescence imaging using ICG at clinically relevant doses, including intravital microscopy, noninvasive imaging in blood and lymph vessels, and imaging of hepatobiliary clearance, and show increased contrast compared with NIR fluorescence imaging. Furthermore, we show tumor-targeted SWIR imaging with IRDye 800CW-labeled trastuzumab, an NIR dye being tested in multiple clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide-based SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications. Indeed, our findings suggest that emerging SWIR-fluorescent in vivo contrast agents should be benchmarked against the SWIR emission of ICG in blood.

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15

Zhang, Miao, Zhang, Liu, Gong, Li, Cui, et al. "Development of a Surface Plasmon Resonance and Fluorescence Imaging System for Biochemical Sensing." Micromachines 10, no. 7 (July 1, 2019): 442. http://dx.doi.org/10.3390/mi10070442.

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Surface plasmon resonance (SPR) biosensors are an extremely sensitive optical technique used to detect the changes in refractive index occurring at the sensor interface. Fluorescence involves the emission of light by a substance that has absorbed light or other electromagnetic radiation, and the parameters of the absorbed and emitted radiation are used to identify the presence and the amount of specific molecules in a specimen. SPR biosensors and fluorescence analysis are both effective methods for real-time detection. The combination of these technologies would improve the quantitative detection sensitivity of fluorescence analysis and the specificity of SPR detection. We designed and developed an SPR and fluorescence synchronous detection system. The SPR module was based on two kinds of modulation methods, and the fluorescence module was capable of switching between four wavelengths. The fluorescence microspheres and A549 cells of different concentration were both detected by the SPR and fluorescence method synchronously in real time. The fluorescent signal and the optical signal of the SPR were shown to correlate. The correlation coefficient for fluorescent microspheres detection reached up to 0.9866. The system could be used in cell analysis and molecule diagnosis in the future.

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16

Sajedi, Salar, Hamid Sabet, and Hak Soo Choi. "Intraoperative biophotonic imaging systems for image-guided interventions." Nanophotonics 8, no. 1 (December 14, 2018): 99–116. http://dx.doi.org/10.1515/nanoph-2018-0134.

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AbstractBiophotonic imaging has revolutionized the operation room by providing surgeons intraoperative image-guidance to diagnose tumors more efficiently and to resect tumors with real-time image navigation. Among many medical imaging modalities, near-infrared (NIR) light is ideal for image-guided surgery because it penetrates relatively deeply into living tissue, while nuclear imaging provides quantitative and unlimited depth information. It is therefore ideal to develop an integrated imaging system by combining NIR fluorescence and gamma-positron imaging to provide surgeons with highly sensitive and quantitative detection of diseases, such as cancer, in real-time without changing the look of the surgical field. The focus of this review is to provide recent progress in intraoperative biophotonic imaging systems, NIR fluorescence imaging and intraoperative nuclear imaging devices, and their future perspectives for image-guided interventions.

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17

Gonçalves, Raquel C. R., Efres Belmonte-Reche, João Pina, Milene Costa da Silva, Sónia C. S. Pinto, Juan Gallo, Susana P. G. Costa, and M. Manuela M. Raposo. "Bioimaging of Lysosomes with a BODIPY pH-Dependent Fluorescent Probe." Molecules 27, no. 22 (November 20, 2022): 8065. http://dx.doi.org/10.3390/molecules27228065.

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Fluorescence-based probes represent a powerful tool for noninvasive imaging of living systems in real time and with a high temporal and spatial resolution. Amongst several known fluorophores, 3-difluoroborodipyrromethene (BODIPY) derivatives have become a cornerstone for innovative fluorescent labelling applications, mainly due to their advantageous features including their facile synthesis, structural versatility and exceptional photophysical properties. In this context, we report a BODIPY-based fluorescent probe for imaging of lysosomes in living cells. The BODIPY derivative displayed a remarkable fluorescence enhancement at low pH values with a pKa* of 3.1. In vitro studies by confocal microscopy in HeLa cells demonstrated that the compound was able to permeate cell membrane and selectively label lysosome whilst remaining innocuous to the cell culture at the maximum concentration tested. Herein, the BODIPY derivative holds the promise of investigating lysosomal dynamics and function in living cells through fluorescence imaging.

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18

Joosten, Johanna J., Paul R. Bloemen, Richard M. van den Elzen, Jeffrey Dalli, Ronan A. Cahill, Mark I. van Berge Henegouwen, Roel Hompes, and Daniel M. de Bruin. "Investigating and Compensating for Periphery-Center Effect among Commercial Near Infrared Imaging Systems Using an Indocyanine Green Phantom." Applied Sciences 13, no. 4 (February 4, 2023): 2042. http://dx.doi.org/10.3390/app13042042.

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Near infrared imaging (NIR) camera systems have been clinically deployed to visualize intravenous injected indocyanine green (ICG) spreading through the vascular bed, thereby creating the ability to assess tissue perfusion. While standardization is key to make fluorescence angiography (FA) comparable and reproducible, optical characteristics like field illumination homogeneity are often not considered. Therefore the aim of this study is to investigate light distribution and the center-periphery effect among five different NIR imaging devices in an indocyanine green phantom. A 13 × 13 cm fluorescence phantom was created by diluting ICG in Intralipid (representing 0.1 mg/kg dose in an 80 kg reference male), to evaluate the overall spatial collection efficiency with fluorescent modalities of five different NIR camera systems using a 0-degree laparoscope. The fluorescence signal from the phantom was quantified at a fixed distance of 16 cm using tailor-made software in Python. The results showed considerable variability in regard to light distribution among the five camera systems, especially toward the periphery of the field of view. In conclusion, NIR signal distribution varies between different systems and within the same displayed image. The fluorescence intensity diminishes peripherally away from the center of the field of view. These optical phenomena need to be considered when clinically interpreting the signal and in the development of computational fluorescence quantification.

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Zhao, Xinyu, Shuqing He, and Mei Chee Tan. "Advancements in infrared imaging platforms: complementary imaging systems and contrast agents." Journal of Materials Chemistry B 5, no. 23 (2017): 4266–75. http://dx.doi.org/10.1039/c7tb00123a.

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Recent advancements in the design of complementary infrared (IR) fluorescence imaging systems and IR-emitting contrast agents are highlighted. The ability to maximize the full performance of any IR imaging platform relies on the thorough understanding of the requirements of the imaging system and physical characteristics of the complementary contrast agents.

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20

Bowman, Adam J., Cheng Huang, Mark J. Schnitzer, and Mark A. Kasevich. "Wide-field fluorescence lifetime imaging of neuron spiking and subthreshold activity in vivo." Science 380, no. 6651 (June 23, 2023): 1270–75. http://dx.doi.org/10.1126/science.adf9725.

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The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult Drosophila . Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

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Plamont, Marie-Aude, Emmanuelle Billon-Denis, Sylvie Maurin, Carole Gauron, Frederico M. Pimenta, Christian G. Specht, Jian Shi, et al. "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo." Proceedings of the National Academy of Sciences 113, no. 3 (December 28, 2015): 497–502. http://dx.doi.org/10.1073/pnas.1513094113.

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This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

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Ishizawa, Takeaki, Peter McCulloch, Laurents Stassen, Jacqueline van den Bos, Jean-Marc Regimbeau, Jeanne Dembinski, Sylke Schneider-Koriath, et al. "Assessing the development status of intraoperative fluorescence imaging for anatomy visualisation, using the IDEAL framework." BMJ Surgery, Interventions, & Health Technologies 4, no. 1 (November 2022): e000156. http://dx.doi.org/10.1136/bmjsit-2022-000156.

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ObjectivesIntraoperative fluorescence imaging is currently used in a variety of surgical fields for four main purposes: visualising anatomy, assessing tissue perfusion, identifying/localising cancer and mapping lymphatic systems. To establish evidence-based guidance for research and practice, understanding the state of research on fluorescence imaging in different surgical fields is needed. We evaluated the evidence on fluorescence imaging used to visualise anatomical structures using the IDEAL framework, a framework designed to describe the stages of innovation in surgery and other interventional procedures.DesignIDEAL staging based on a thorough literature review.SettingAll publications on intraoperative fluorescence imaging for visualising anatomical structures reported in PubMed through 2020 were identified for five surgical procedures: cholangiography, hepatic segmentation, lung segmentation, ureterography and parathyroid identification.Main outcome measuresThe IDEAL stage of research evidence was determined for each of the five procedures using a previously described approach.Results225 articles (8427 cases) were selected for analysis. Current status of research evidence on fluorescence imaging was rated IDEAL stage 2a for ureterography and lung segmentation, IDEAL 2b for hepatic segmentation and IDEAL stage 3 for cholangiography and parathyroid identification. Enhanced tissue identification rates using fluorescence imaging relative to conventional white-light imaging have been documented for all five procedures by comparative studies including randomised controlled trials for cholangiography and parathyroid identification. Advantages of anatomy visualisation with fluorescence imaging for improving short-term and long-term postoperative outcomes also were demonstrated, especially for hepatobiliary surgery and (para)thyroidectomy. No adverse reactions associated with fluorescent agents were reported.ConclusionsIntraoperative fluorescence imaging can be used safely to enhance the identification of anatomical structures, which may lead to improved postoperative outcomes. Overviewing current research knowledge using the IDEAL framework aids in designing further studies to develop fluorescence imaging techniques into an essential intraoperative navigation tool in each surgical field.

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Zhang, Qimei, Anna M. Grabowska, Philip A. Clarke, and Stephen P. Morgan. "Numerical Simulation of a Scanning Illumination System for Deep Tissue Fluorescence Imaging." Journal of Imaging 5, no. 11 (October 24, 2019): 83. http://dx.doi.org/10.3390/jimaging5110083.

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The spatial resolution and light detected in fluorescence imaging for small animals are limited by light scattering, absorption and autofluorescence. To address this, novel near-infrared fluorescent contrast agents and imaging configurations have been investigated. In this paper, the influence of the light wavelength and imaging configurations (full-field illumination system and scanning system) on fluorescence imaging are compared quantitatively. The surface radiance for both systems is calculated by modifying the simulation tool Near-Infrared Fluorescence and Spectral Tomography. Fluorescent targets are embedded within a scattering medium at different positions. The surface radiance and spatial resolution are obtained for emission wavelengths between 620 nm and 1000 nm. It was found that the spatial resolution of the scanning system is independent of the tissue optical properties, whereas for full-field illumination, the spatial resolution degrades at longer wavelength. The full width at half maximum obtained by the scanning system is 25% lower than that obtained by the full-field illumination system when the targets are located in the middle of the phantom. The results indicate that although imaging at near-infrared wavelength can achieve a higher surface radiance, it may produce worse spatial resolution.

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Dempster, John, and David Wokosin. "Fluorescence imaging systems: A quick overview of the technology." Physiology News, Autumn 2002 (September 1, 2002): 12–14. http://dx.doi.org/10.36866/pn.48.12.

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Kim, Hong Rae, Hyun Min Lee, Heon Yoo, Seung Hoon Lee, and Kwang Gi Kim. "Review of Neurosurgical Fluorescence Imaging Systems for Clinical Application." Journal of the Optical Society of Korea 20, no. 2 (April 25, 2016): 305–13. http://dx.doi.org/10.3807/josk.2016.20.2.305.

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Kim, Taehoon, Connor O'Brien, Hak Soo Choi, and Myung Yung Jeong. "Fluorescence molecular imaging systems for intraoperative image-guided surgery." Applied Spectroscopy Reviews 53, no. 2-4 (May 25, 2017): 349–59. http://dx.doi.org/10.1080/05704928.2017.1323311.

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Christenson, Mark. "Advances in Detector Systems for Imaging Single Molecule Fluorescence." Single Molecules 1, no. 2 (June 2000): 177–79. http://dx.doi.org/10.1002/1438-5171(200006)1:2<177::aid-simo177>3.0.co;2-4.

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Li, Zheng, Xiaodong Chen, Liqiang Ren, Jie Song, Yuhua Li, Bin Zheng, and Hong Liu. "Simultaneous Dual-Color Fluorescence Microscope: A Characterization Study." Analytical Cellular Pathology 36, no. 5-6 (2013): 163–72. http://dx.doi.org/10.1155/2013/143785.

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Background: High spatial resolution and geometric accuracy is crucial for chromosomal analysis of clinical cytogenetic applications. High resolution and rapid simultaneous acquisition of multiple fluorescent wavelengths can be achieved by utilizing concurrent imaging with multiple detectors. However, such class of microscopic systems functions differently from traditional fluorescence microscopes.Objective: To develop a practical characterization framework to assess and optimize the performance of a high resolution and dual-color fluorescence microscope designed for clinical chromosomal analysis.Methods: A dual-band microscopic imaging system utilizes a dichroic mirror, two sets of specially selected optical filters, and two detectors to simultaneously acquire two fluorescent wavelengths. The system’s geometric distortion, linearity, the modulation transfer function, and the dual detectors’ alignment were characterized.Results: Experiment results show that the geometric distortion at lens periphery is less than 1%. Both fluorescent channels show linear signal responses, but there exists discrepancy between the two due to the detectors’ non-uniform response ratio to different wavelengths. In terms of the spatial resolution, the two contrast transfer function curves trend agreeably with the spatial frequency. The alignment measurement allows quantitatively assessing the cameras' alignment. A result image of adjusted alignment is demonstrated to show the reduced discrepancy by using the alignment measurement method.Conclusions: In this paper, we present a system characterization study and its methods for a specially designed imaging system for clinical cytogenetic applications. The presented characterization methods are not only unique to this dual-color imaging system but also applicable to evaluation and optimization of other similar multi-color microscopic image systems for improving their clinical utilities for future cytogenetic applications.

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Mao, Shiqi, Yachen Ying, Xiaotian Wu, Christopher J. Krueger, and Antony K. Chen. "CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci." Nucleic Acids Research 47, no. 20 (August 31, 2019): e131-e131. http://dx.doi.org/10.1093/nar/gkz752.

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Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic imaging systems predominantly rely on fluorescent protein reporters, which lack the optical properties essential for sensitive dynamic imaging. Here, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) that can undergo fluorescence resonance energy transfer (FRET) and demonstrated that the resulting system, CRISPR/dual-FRET MB, enables dynamic imaging of non-repetitive genomic loci with only three unique sgRNAs.

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Soleimaninejad, Hamid, Kenneth P. Ghiggino, Trevor A. Smith, and Matthew F. Paige. "Fluorescence anisotropy imaging of a polydiacetylene photopolymer film." Canadian Journal of Chemistry 97, no. 6 (June 2019): 422–29. http://dx.doi.org/10.1139/cjc-2018-0360.

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UV-illumination of phase-separated surfactant films prepared from mixtures of photopolymerizable 10,12-pentacosadiynoic acid and perfluorotetradecanoic acid results in the formation of fluorescent polydiacetylene fibers and aggregates. In this work, the orientation of polymer strands that comprise the resulting photopolymer structures has been probed using fluorescence anisotropy imaging in combination with defocused single-molecule fluorescence imaging. Imaging experiments indicate the presence of significant fiber-to-fiber heterogeneity, as well as anisotropy within each fiber (or aggregate), with both of these properties changing as a function of film preparation conditions. This anisotropy can be attributed to various alignments of the constituent polymer strands that comprise the larger fibers and aggregates. Intriguingly, when using defocused imaging, fiber images consisted of a series of discrete “doughnut” fluorescence emission patterns, which exhibited intermittent on–off blinking behavior; both of these properties are characteristic of individual emission transition dipoles (single molecules). Further, all of the individual emission transition dipoles had a uniform orientation with respect to the axis of the fiber, indicating a common orientation of discrete emitters in the larger polymer fiber. The implications of these results for future studies of the electronic properties of conjugated polymers in larger macroscopic systems are noted.

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31

Lukk, Tiit, Richard E. Gillilan, Doletha M. E. Szebenyi, and Warren R. Zipfel. "A visible-light-excited fluorescence method for imaging protein crystals without added dyes." Journal of Applied Crystallography 49, no. 1 (February 1, 2016): 234–40. http://dx.doi.org/10.1107/s160057671502419x.

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Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.

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32

Tarver, Crissy L., and Marc Pusey. "A low-cost method for visible fluorescence imaging." Acta Crystallographica Section F Structural Biology Communications 73, no. 12 (November 10, 2017): 657–63. http://dx.doi.org/10.1107/s2053230x17015941.

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A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

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33

Meng, Fangfang, Yong Liu, Xiaoqiang Yu, and Weiying Lin. "A dual-site two-photon fluorescent probe for visualizing mitochondrial aminothiols in living cells and mouse liver tissues." New Journal of Chemistry 40, no. 9 (2016): 7399–406. http://dx.doi.org/10.1039/c6nj00330c.

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In this work, we developed a dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging mitochondrial RSH in living systems. In particular, probe CA-TPP was capable of using TP fluorescence to track mitochondrial RSH over a long period for the first time.

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34

Curtis, Angharad, Kang Li, Mohammed Ali, and Nigel Copner. "The Optical Properties of Indocyanine Green suspended in Solution as Observed under Near Infrared LED and LASER Light Conditions." International journal of Science and Engineering Applications 10, no. 5 (April 28, 2021): 080–89. http://dx.doi.org/10.7753/ijsea1005.1005.

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The use of Indocyanine Green (ICG) as a fluorescent marker at Near Infrared (NIR) excitation wavelengths is well established in clinical imaging. Typical systems comprise multiple LED sources for optimal imaging which can result in unnecessary energy transfer to patients and contribute to tissue damage. An experimental setup comprising a 780 nm excitation channel generating up to 10 mW of optical power is used in order to determine if there is potential to exploit the optical properties of ICG, in order to reduce the total excitation power through pulsing. We demonstrate in this work that a single 1.6 Megapixel CMOS camera with quantum efficiency of less than 30% is appropriate to capture both fluorescent and non-fluorescent landmarks at NIR wavelengths. Experimental results verify that all ICG solutions tested yielded detectable fluorescence and that degradation of fluorescence intensity over time is multifaceted.

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35

Wang, Tian, Yingying Chen, Bo Wang, Xiaofan Gao, and Mingfu Wu. "Recent Progress in Second Near-Infrared (NIR-II) Fluorescence Imaging in Cancer." Biomolecules 12, no. 8 (July 28, 2022): 1044. http://dx.doi.org/10.3390/biom12081044.

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Cancer continues to be one of the leading causes of death worldwide, and its incidence is on the rise. Although cancer diagnosis and therapy have advanced significantly in recent decades, it is still a challenge to achieve the accurate identification and localization of cancer and to complete tumor elimination with a maximum preservation of normal tissue. Recently, second near-infrared region (NIR-II, 1000–1700 nm) fluorescence has shown great application potential in cancer theranostics due to its inherent advantages, such as great penetration capacity, minimal tissue absorption and scattering, and low autofluorescence. With the development of fluorescence imaging systems and fluorescent probes, tumor detection, margin definition, and individualized therapy can be achieved quickly, enabling an increasingly accurate diagnosis and treatment of cancer. Herein, this review introduces the role of NIR-II fluorescence imaging in cancer diagnosis and summarizes the representative applications of NIR-II image-guided treatment in cancer therapy. Ultimately, we discuss the present challenges and future perspectives on fluorescence imaging in the field of cancer theranostics and put forward our opinions on how to improve the accuracy and efficiency of cancer diagnosis and therapeutics.

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36

Zhang, Weichun, Martín Caldarola, Xuxing Lu, Biswajit Pradhan, and Michel Orrit. "Single-molecule fluorescence enhancement of a near-infrared dye by gold nanorods using DNA transient binding." Physical Chemistry Chemical Physics 20, no. 31 (2018): 20468–75. http://dx.doi.org/10.1039/c8cp03114b.

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Fluorescence enhancement by plasmonic nanostructures enables the optical detection of single molecules with weak fluorescence, extending the scope of molecular fluorescence imaging to new materials and systems.

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37

Reja, Shahi Imam, Yuichiro Hori, Takuya Kamikawa, Kohei Yamasaki, Miyako Nishiura, Steven D. Bull, and Kazuya Kikuchi. "An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems." Chemical Science 13, no. 5 (2022): 1419–27. http://dx.doi.org/10.1039/d1sc06274c.

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38

Zhao, Jintao, Tao Ma, Bingbing Chang, and Jianguo Fang. "Recent Progress on NIR Fluorescent Probes for Enzymes." Molecules 27, no. 18 (September 12, 2022): 5922. http://dx.doi.org/10.3390/molecules27185922.

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The majority of diseases’ biomarkers are enzymes, and the regulation of enzymes is fundamental but crucial. Biological system disorders and diseases can result from abnormal enzymatic activity. Given the biological significance of enzymes, researchers have devised a plethora of tools to map the activity of particular enzymes in order to gain insight regarding their function and distribution. Near-infrared (NIR) fluorescence imaging studies on enzymes may help to better understand their roles in living systems due to their natural imaging advantages. We review the NIR fluorescent probe design strategies that have been attempted by researchers to develop NIR fluorescent sensors of enzymes, and these works have provided deep and intuitive insights into the study of enzymes in biological systems. The recent enzyme-activated NIR fluorescent probes and their applications in imaging are summarized, and the prospects and challenges of developing enzyme-activated NIR fluorescent probes are discussed.

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39

Powless, Amy, Sandra Prieto, Madison Gramling, Jingyi Chen, and Timothy Muldoon. "A Light-Sheet-Based Imaging Spectrometer to Characterize Acridine Orange Fluorescence within Leukocytes." Diagnostics 10, no. 12 (December 12, 2020): 1082. http://dx.doi.org/10.3390/diagnostics10121082.

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Low-cost imaging systems that utilize exogenous fluorescent dyes, such as acridine orange (AO), have recently been developed for use as point-of-care (POC) blood analyzers. AO-based fluorescence imaging exploits variations in emission wavelength within different cell types to enumerate and classify leukocyte subpopulations from whole blood specimens. This approach to leukocyte classification relies on accurate and reproducible colorimetric features, which have previously been demonstrated to be highly dependent on the cell staining protocols (such as specific AO concentration, timing, and pH). We have developed a light-sheet-based fluorescence imaging spectrometer, featuring a spectral resolution of 9 nm, with an automated spectral extraction algorithm as an investigative tool to study the spectral features from AO-stained leukocytes. Whole blood specimens were collected from human subjects, stained with AO using POC methods, and leukocyte spectra were acquired on a cell-by-cell basis. The post-processing method involves three steps: image segmentation to isolate individual cells in each spectral image; image quality control to exclude cells with low emission intensity, out-of-focus cells, and cellular debris; and the extraction of spectra for each cell. An increase in AO concentration was determined to contribute to the red-shift in AO-fluorescence, while varied pH values did not cause a change in fluorescence. In relation to the spectra of AO-stained leukocytes, there were corresponding red-shift trends associated with dye accumulation within acidic vesicles and at increasing incubation periods. The system presented here could guide future development of POC systems reliant on AO fluorescence and colorimetric features to identify leukocyte subpopulations in whole blood specimens.

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40

Nakai, Nori, Keisuke Sato, Tomomi Tani, Kenta Saito, Fumiya Sato, and Sumio Terada. "Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy." Microscopy 68, no. 5 (July 2, 2019): 359–68. http://dx.doi.org/10.1093/jmicro/dfz022.

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Abstract Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

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41

Conrad, Christian, Annelie Wünsche, Tze Heng Tan, Jutta Bulkescher, Frank Sieckmann, Fatima Verissimo, Arthur Edelstein, et al. "Micropilot: automation of fluorescence microscopy–based imaging for systems biology." Nature Methods 8, no. 3 (January 23, 2011): 246–49. http://dx.doi.org/10.1038/nmeth.1558.

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42

Dedecker, Peter, Gary C. H. Mo, and Jin Zhang. "Widely Accessible Method for Superresolution Fluorescence Imaging of Living Systems." Biophysical Journal 104, no. 2 (January 2013): 535a. http://dx.doi.org/10.1016/j.bpj.2012.11.2960.

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43

Dedecker, P., G. C. H. Mo, T. Dertinger, and J. Zhang. "Widely accessible method for superresolution fluorescence imaging of living systems." Proceedings of the National Academy of Sciences 109, no. 27 (June 18, 2012): 10909–14. http://dx.doi.org/10.1073/pnas.1204917109.

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44

LEE, H., E. J. CHO, H. SON, H. O. KIM, Y. HONG, S. H. YANG, S. J. YOON, et al. "PODOPLANIN-TARGETABLE MR/OPTIC DUAL-MODE NANOCOMPOSITES FOR GLIOBLASTOMA MULTIFORME IN MOUSE BRAIN CANCER." Digest Journal of Nanomaterials and Biostructures 15, no. 3 (September 2020): 841–47. http://dx.doi.org/10.15251/djnb.2020.153.841.

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It is very desirable to design dual-mode nanocomposites for diagnostic applications via flexible strategies. Herein, we proposed to synthesize one type of multifunctional magnetic resonance (MR)/optic nanocomposites based on manganese ferrite magnetic nanoparticles (MFNPs) modified by fluorescent podoplanin (PDPN) antibodies. MFNPs were synthesized using a thermal decomposition method and were modified by the fluorescent PDPN antibodies on their surfaces. The fluorescent PDPN antibody-conjugated MFNPs enabled strong MR imaging and fluorescence imaging due to the antibodies that targeted brain tumors. These results demonstrate that MR/optic nanocomposites have potential as precision diagnostic systems for brain cancer.

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45

Hamm, Christopher W., Sarah F. Winburn, and Matthew T. Cabeen. "Using Fluorescence in Biotechnology Instruction to Visualize Antibiotic Resistance & DNA." American Biology Teacher 83, no. 6 (August 1, 2021): 395–401. http://dx.doi.org/10.1525/abt.2021.83.6.395.

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Fluorescence technology has many useful applications for both research and teaching, among them the detection of fluorescence in live transgenic organisms and of DNA in agarose gels. However, dedicated fluorescence imaging systems can be expensive and complex. We describe a simple apparatus for non-microscopic fluorescence imaging using affordable and readily available parts. We describe three activities of increasing complexity that utilize fluorescence illumination and teach principles of fluorescence. At the high school level, our more advanced activities can be used for lessons addressing NGSS performance expectations in HS-LS3 and HS-LS4.

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46

Lee, Seung Hyun, Yu Hua Quan, Min Sub Kim, Ki Hyeok Kwon, Byeong Hyeon Choi, Hyun Koo Kim, and Beop-Min Kim. "Design and Testing of Augmented Reality-Based Fluorescence Imaging Goggle for Intraoperative Imaging-Guided Surgery." Diagnostics 11, no. 6 (May 21, 2021): 927. http://dx.doi.org/10.3390/diagnostics11060927.

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The different pathways between the position of a near-infrared camera and the user’s eye limit the use of existing near-infrared fluorescence imaging systems for tumor margin assessments. By utilizing an optical system that precisely matches the near-infrared fluorescence image and the optical path of visible light, we developed an augmented reality (AR)-based fluorescence imaging system that provides users with a fluorescence image that matches the real-field, without requiring any additional algorithms. Commercial smart glasses, dichroic beam splitters, mirrors, and custom near-infrared cameras were employed to develop the proposed system, and each mount was designed and utilized. After its performance was assessed in the laboratory, preclinical experiments involving tumor detection and lung lobectomy in mice and rabbits by using indocyanine green (ICG) were conducted. The results showed that the proposed system provided a stable image of fluorescence that matched the actual site. In addition, preclinical experiments confirmed that the proposed system could be used to detect tumors using ICG and evaluate lung lobectomies. The AR-based intraoperative smart goggle system could detect fluorescence images for tumor margin assessments in animal models, without disrupting the surgical workflow in an operating room. Additionally, it was confirmed that, even when the system itself was distorted when worn, the fluorescence image consistently matched the actual site.

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47

Campbell, Benjamin C., Elisa M. Nabel, Mitchell H. Murdock, Cristina Lao-Peregrin, Pantelis Tsoulfas, Murray G. Blackmore, Francis S. Lee, Conor Liston, Hirofumi Morishita, and Gregory A. Petsko. "mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging." Proceedings of the National Academy of Sciences 117, no. 48 (November 18, 2020): 30710–21. http://dx.doi.org/10.1073/pnas.2000942117.

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Although ubiquitous in biological studies, the enhanced green and yellow fluorescent proteins (EGFP and EYFP) were not specifically optimized for neuroscience, and their underwhelming brightness and slow expression in brain tissue limits the fidelity of dendritic spine analysis and other indispensable techniques for studying neurodevelopment and plasticity. We hypothesized that EGFP’s low solubility in mammalian systems must limit the total fluorescence output of whole cells, and that improving folding efficiency could therefore translate into greater brightness of expressing neurons. By introducing rationally selected combinations of folding-enhancing mutations into GFP templates and screening for brightness and expression rate in human cells, we developed mGreenLantern, a fluorescent protein having up to sixfold greater brightness in cells than EGFP. mGreenLantern illuminates neurons in the mouse brain within 72 h, dramatically reducing lag time between viral transduction and imaging, while its high brightness improves detection of neuronal morphology using widefield, confocal, and two-photon microscopy. When virally expressed to projection neurons in vivo, mGreenLantern fluorescence developed four times faster than EYFP and highlighted long-range processes that were poorly detectable in EYFP-labeled cells. Additionally, mGreenLantern retains strong fluorescence after tissue clearing and expansion microscopy, thereby facilitating superresolution and whole-brain imaging without immunohistochemistry. mGreenLantern can directly replace EGFP/EYFP in diverse systems due to its compatibility with GFP filter sets, recognition by EGFP antibodies, and excellent performance in mouse, human, and bacterial cells. Our screening and rational engineering approach is broadly applicable and suggests that greater potential of fluorescent proteins, including biosensors, could be unlocked using a similar strategy.

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48

Huber, Rudolf M., F. Gamarra, H. Hautmann, K. Häußinger, S. Wagner, M. Castro, and R. Baumgartner. "5-Aminolaevulinic Acid (ALA) for the Fluorescence Detection of Bronchial Tumors." Diagnostic and Therapeutic Endoscopy 5, no. 2 (January 1, 1999): 113–18. http://dx.doi.org/10.1155/dte.5.113.

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At the moment only early detection of lung cancer offers a good prognosis for the patients. Conventional white light endoscopy is mostly insufficient for early diagnosis. Therefore we developed a system of fluorescence diagnosis using 5-aminolaevulinic acid (ALA) exogeneously applied. As precursor of the heme synthesis it is metabolized to protoporphyrin IX – a red fluorescent substance. Therefore protoporphyrin IX accumulates in tumorous and premalignant tissue, and can be directly visualized by fluorescence bronchoscopy. Excitation with blue light (380–435 nm) causes a red fluorescence, which can be detected after filtering most of the blue component with the naked eye or a camera system. After earlier work with laser systems and cold light sources we now use the system D-Light AF for the fluorescence diagnosis using ALA-induced protoporphyrin IX fluorescence.

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49

Shin, Jun Geun, and Jonghyun Eom. "Double-Clad Optical Fiber-Based Multi-Contrast Noncontact Photoacoustic and Fluorescence Imaging System." Electronics 10, no. 23 (December 2, 2021): 3008. http://dx.doi.org/10.3390/electronics10233008.

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A noncontact photoacoustic and fluorescence dual-modality imaging system is proposed, which integrates a fiber-based fluorescence imaging system with noncontact photoacoustic imaging using a specially fabricated double-cladding fiber (DCF) coupler and a DCF lens. The performance of the DCF coupler and lens was evaluated, and the feasibility of this new imaging system was demonstrated using simple tubing phantoms with black ink and fluorophore. Our imaging results demonstrated that the multimodal imaging technique can simultaneously acquire photoacoustic and fluorescence images without coming into contact with the sample. Consequently, the developed method is the first noncontact scheme among multimodal imaging systems that is integrated with a photoacoustic imaging system, which can provide varied and complementary information about the sample.

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50

Waks Serra, María V., Dirk Grosenick, Rainer Macdonald, Juan A. Pomarico, and Daniela I. Iriarte. "A systematic study on fluorescence contrast in near infrared diffuse transmittance imaging with indocyanine green." Journal of Near Infrared Spectroscopy 27, no. 5 (June 25, 2019): 333–44. http://dx.doi.org/10.1177/0967033519857733.

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Near infrared fluorescence imaging is a sensitive, noninvasive technique for diagnostic applications in biomedical optics. The main purpose of this work is thus to explore how to improve the contrast of images obtained by near infrared light using a fluorescent extrinsic agent. Among different fluorophores, indocyanine green has been mostly studied because it is approved for use in humans. In this work, simulations and experimental studies on phantoms (systems that optically emulate biological tissues) are used to systematically investigate the influence of the increased intrinsic tissue absorption by adding indocyanine green. The experiments reproduce the situation of fluorescence imaging of carcinomas in the human breast, where the natural absorption due to neovascularization is increased by the injection of this fluorophore. Assuming measurements in transmission geometry, the breast is modeled by a homogeneous background medium containing a tumor-like inclusion (or lesion) with two- or threefold increased absorption. Fluorescence contrast is simulated over a broad range of dye concentrations using diffusion theory. Selected concentrations ratios are applied in experimental studies with laser excitation of indocyanine green fluorescence and with a charge-coupled device camera for fluorescence detection. Both simulations and experiments show that the intrinsic absorption of the inclusion strongly reduces the number of detected fluorescence photons and that the fluorescence contrast can be canceled or become even negative. It was found that for typical optical properties and geometrical conditions, in fluorescence imaging of breast cancer, a dye ratio of about 10:1 (lesion:background) is required to turn from negative to positive fluorescence contrast. Since such a high ratio is difficult to attain, raw fluorescence images need to be normalized by the intrinsic lesion absorption (without indocyanine green (ICG)) to enhance the presence of the dye in the lesion.

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