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This cookie is set by GDPR Cookie Consent plugin. While we encourage open and honest conversation, we reserve the right to edit or remove comments that contain offensive, obscene or profane language. Well to well variation in thermal cycler fluorescence? Topics covered include:click o. The multiple photochemical reaction pathways responsible for photobleaching have been investigated and described in considerable detail. Nevertheless, a fluorescence microplate reader with better sensitivity will deliver data with a better statistic, less variability among replicates, and a larger delta (distance) between responding and not-responding samples. It might well be that a luminescence device, designed to detect very weak levels of luminescence is blinded (i.e., saturated) by stronger light. The Infinite 200 PRO is an easy-to-use multimode plate reader family that offers affordable high performance detection solutions empowered by monochromator- or filter based technologies. It heps in delivering localised language version. It also helps in not showing the cookie consent box upon re-entry to the website. Thus, I would expect that a typical fluorescence reader should also be able to measure luminescence. This may be something to explore since I imagine gel imagers are more widespread that plate readers. Molecules that transition to the excited state lose energy due to heat or collisions with other molecules and return to the ground state. As opposed to absorbance, fluorescence detection is not an absolute measurement. For each conjugate, three data sets, representing different fields of view, were averaged and then normalized to the same initial fluorescence intensity value to facilitate comparison. Moreover, our multi-mode microplate readers are developed to provide optimum performance for years. The monomer-to-excimer ratio (371.5/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. The hardware for measuring luminescence and fluorescence is different. and Brown, C.M., Live-cell microscopy: Tips and tools,. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation photons. Anecdotally, all of the fluorescence capable plate readers that I have used have also been luminescence capable (BMG and Tecan models). Some of these cookies are essential for our website to work. The purpose of this introduction is to briefly outline fluorescence principles and techniques for newcomers to the field. With this type of indicator, the ratio of the optical signals (S1 and S2 inFigure 7) can be used to monitor the association equilibrium and to calculate ion concentrations. LVF Monochromators have filter-like performance and make the CLARIOstar Plus the most sensitive monochromator-based microplate reader on the market. The following two tabs change content below. Fluorescence scanners, including microarray readers, resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms. Fluorescence instruments are primarily of four types, each providing distinctly different information: Other types of instrumentation that use fluorescence detection include capillary electrophoresis apparatus, DNA sequencers and microfluidic devices. When a compound is irradiated with light, electrons in the energetically stable ground state use the energy of the light to transition to energetically high and unstable excited states. TheEnhanced Dynamic Rangefeature on theCLARIOstarPlus and VANTAstar offers the largest possible dynamic range on the market (8 concentration decades), and makes it possible to automatically measure assays with very bright and dim signals in the same measurement no adjustment required. Can I infer that Schrdinger's cat is dead without opening the box, if I wait a thousand years? Fluorescent probes enable researchers to detect particular components of complex biomolecular assemblies, such as live cells, with exquisite sensitivity and selectivity. Multi-user software packageAll our instruments come with a multi-user software package that can be installed on as many computers as users requires, without the need to purchase licenses. The cells were then fixed in 1% formaldehyde, washed and wet-mounted. Filter-based readers require a specialized filter for each wavelength. For these reasons, unless you need to perform spectral scans, filter-based readers would be the preferred choice. In addition, monochromators have the capability to acquire excitation or emission scans. Go to "set up" to enter your experimental settings (see window below) like for example wavelength, type of measurements etc. This emission light is captured by another series of mirrors, grating and slits to limit the emission to a desired wavelength, which then enters a detector for signal readout. Figure 12. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the relative extent to which these processes occur. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching, see below), the same fluorophore can be repeatedly excited and detected. GloMax plate readers are pre-loaded with a range of excitation and emission filter sets for the most common fluorophores. In general, a filter-based reader will provide more sensitive detection and can be used with luminescence or multiplex assays at a lower cost. Especially for fast responses, the event you are looking for may already be gone before you detect it. Fluorophores such as BCECF and carboxy SNARF-1 that have strongly pH-dependent absorption and fluorescence characteristics can be used as physiological pH indicators. This gives you the chance to keep your options open even if you dont use the full scope of your microplate reader right at the bat. Our technical services scientists are available to help at Promega.com. For Research Use Only. Maybe we can exchange ideas at some point? The process responsible for the fluorescence of fluorescent probes and other fluorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown inFigure 2. Measuring protein concentration, Bradford vs. Nanodrop? This cookie is set by doubleclick.net. The cookies store information anonymously and assign a randomly generated number to identify unique visitors. FRET is a dual dye fluorescence detection assay based on energy transfer between a donor and an acceptor. For quantification purposes, fluorescence plate readers generally offer greater sensitivity than, Filters or monochromators can be employed for this purpose. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Did an AI-enabled drone attack the human operator in a simulation environment? Figure 1. An X,Y plot of wavelength versus absorbance of a chromophore or fluorophore. This ensures the desired wavelength is selected to excite the fluorophore. First a 10 mM stock solution of the substrate pNPA in DMSO is prepared. SensitivityFor quantification purposes, fluorescence plate readers generally offer greater sensitivity than absorbance readers. Search Filter-based readers work well with these assays because the filter wheels allow quick switching between two wavelengthswithin a fraction of a second. Other fluorescence-based detection modes such asfluorescence polarisationortime-resolved fluorescenceandTR-FRETcan only be detected occasionally and with poor sensitivity, or not at all by single-mode fluorescence microplate readers. Excerpts and links may be used, provided that full and clear credit is given to Promega Corporation with appropriate and specific direction to the original content. The NovaFluor spectrometer uses an X-Y stage, pulsed laser, PMT, and digitizer for direct waveform recording (DWR) of fluorescence lifetime decays. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Learn how your comment data is processed. Excitation (EX) in overlapping absorption bands A1 and A2 produces two fluorescent species with spectra E1 and E2. Flexible Microplate Format A low level of polarization indicates that small fluorescent molecules move freely in the sample. The linear range of the assay under these conditions is from 100 to 20,000 cells per 100 L sample. The three most common are: Fluorescence spectra may be strongly dependent on solvent. Our instruments are developed, produced and tested in Germany and are built to be extremely robust and reliable. Would sending audio fragments over a phone call be considered a form of cryptology? Can I image Coomassie and GFP in gels at the same time with a fluorescence scanner? The purpose of the cookie is to determine if the user's browser supports cookies. Once the fluorophore is excited, it emits light at a different, longer wavelength. In some casesfor example the Ca2+indicators fura-2 and indo-1 (Fluorescent Ca2+ Indicators Excited with UV LightSection 19.2) and the pH indicators BCECF and SNARF (Probes Useful at Near-Neutral pHSection 20.2)the free and ion-bound forms of fluorescent ion indicators have different emission or excitation spectra. Fascinating --- I didn't realize there was so much variety in luciferase. This characteristic is most often observed with fluorophores that have large excited-state dipole moments, resulting in fluorescence spectral shifts to longer wavelengths in polar solvents. Principle of enzyme detection via the disruption of intramolecular self-quenching. However, the tradeoff is low sensitivity and high cost. The six new tailored configurations provide excellence in ELISA assays, nucleic acid quantifications, reporter assay technologies, and . Powerful and most sensitive HTS plate reader, Most flexible plate reader for assay development, Flexible microplate reader with simplified workflows, Upgradable single and multi-mode microplate reader series, Basic information about fluorescence plate readers. Excimer formation by pyrene in ethanol. Fluorescence intensity detection can be performed on the PHERAstarFSX, CLARIOstarPlus, VANTAstar and FLUOstarOmega plate reader. The fluorophore then emits the light at a different wavelength selected by the emission filter, and the emitted fluorescence enters a detector for signal readout. Consequently, fluorescence plate readers measure the light signal emitted by a sample in Relative Fluorescent Units (RFU). On the other hand, searching online, I am swamped by manufacturers eager to sell me specialized luminescence readers that emphasize their sensitivity, which leads me to think that maybe typical fluorescence plate readers can't measure luminescence. . Stay connected and never miss another tip, update or insight from our team of science bloggers. Filters are usually more sensitive, whereas monochromators offer more flexibility. Unfortunately, it is not easy to find single dyes with the requisite combination of a large extinction coefficient for absorption and a large Stokes shift. Still, it is better to consult a real expert before spending money on a new piece of equipment. The ACU provides the ideal environment for any application with living cells. Learn more about our easy-to-use GloMax plate readers with high sensitivity and broad dynamic range! This technique has major applications in flow cytometry,DNA sequencing,fluorescencein situhybridizationand fluorescence microscopy. This cookie is used by vimeo to collect tracking information. Fluorescence microplate readers usually contain a light source, an optical system for the selection of excitation light (i.e., filters and/or monochromators), a second optical system for the selection of emission light, and a detector, usually one or multiple photomultiplier tubes (PMTs). A high level of polarization indicates that fluorescent is attached to a larger molecular complex. Do you know if luciferase is flash or glow? *Our online Fluorescence SpectraViewer provides an interactive utility for plotting and comparing fluorescence excitation and emission spectra for over 250 fluorophores (Using the Fluorescence SpectraViewerNote 23.1).Generally true for single fluorophore species in homogeneous solutions but not in more complex heterogeneous samples.Multiplex detection refers to the process of simultaneously labeling a specimen with two or more fluorescent probes to allow correlation of multiple structural or functional features. Signal distortion caused by autofluorescence of cells, tissues and biological fluids is most readily minimized by using probes that can be excited at >500 nm. A photon of energy hEM is emitted, returning the fluorophore to its ground state S0. Monochromator-based readers are generally less sensitive because they only transmit a small fraction of light to the sample. Taraska, J.W. From a first principles perspective, any fluorescence plate reader ought to be able to measure luminescence as well - just don't turn on the excitation light source. Filter-based plate readers can also provide flexibility in selecting desired excitation and emission wavelengths by using customized filters sets across any desired wavelength of the optical detector. Regarding your idea for normalizing luminescence, I also have strong interest in that and we have already started working on that. What is more relevant to your case, the detectors in a fluorescence device might be insufficiently sensitive for practical purposes of luminescence study. Reagent injectorsWhen looking for a fluorescence plate reader, you should consider the nature of the assay you want to run. Your results may vary. The absorption spectrum can therefore be used as a surrogate excitation spectrum data set. Absorbance measurements are performed through the plate. This cookie is set by the provider Media.net. Environmental controlIf your research requires long-time live cell based assays and you do not want to shuffle your microplate back and forth between fluorescence microplate reader and incubator, make sure that your reader of choice has the capability to control its internal gas environment.The Omega series, the VANTAstar and the CLARIOstar Plus can be equipped with our Atmospheric Control Unit (ACU). and Fortina, P., Analytical ancestry: "Firsts" in fluorescent labeling of nucleosides, nucleotides, and nucleic acids,. Probes that have a high fluorescence quantum yield when bound to a particular target but are otherwise effectively nonfluorescent yield extremely low reagent background signals. All-round service and supportWe strive to provide you with the very best customer service. 16 years 7 months 15 days 6 hours 22 minutes. Regardless of the application, compatibility of these four elements is essential for optimizing fluorescence detection. So to summarise, while luminescence can be measured by plate readers which can measure fluorescence, the sensitivity may be too low. For enzymatic kinetic assays or real-time cell-based responses, the use of reagent injectors can be beneficial. The intensity of the fluorescent signal is usually relative to other measurements or to a refence measurement taken by an instrument. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength (Figure 4). The preceding discussion has introduced some general principles to consider when selecting a fluorescent probe. During excitation, the light passes through a narrow slit, directed by a series of mirrors and diffraction grating and then passes through a second narrow slit prior to reaching the sample. Antifade reagents such as Molecular ProbesSlowFade and ProLong reagents (Fluorescence Microscopy Accessories and Reference StandardsSection 23.1) can also be applied to reduce photobleaching; however, they are usually incompatible with live cells. You just have to check your platereader model if it is capable of measuring luminescence.So you don't need a seperate platereader just for luminescence, but you need one, which has the luminescence addon installed. The ACU independently regulates both O2and CO2gas levels within the microplate reader chamber. Some assays require multiple wavelengths to be measured in a very short timeframe, such as multiplex assays that detect two different fluorescent wavelengths, or both fluorescence and luminescence signals. In other cases, it can perform complex analyses. Fluorescence quenching can be defined as a bimolecular process that reduces the fluorescence quantum yield without changing the fluorescence emission spectrum (Table 1); it can result from transient excited-state interactions (collisional quenching) or from formation of nonfluorescent ground-state species. If quenching is caused by collisional interactions, as is usually the case, information on the proximity of the fluorophore and quencher and their mutual diffusion rate can be derived. . An X,Y plot of excitation wavelength versus number of fluorescence photons generated by a fluorophore. This cookie is set by Youtube. Alternatively, a less photolabile fluorophore may be substituted in the experiment. Is there a faster algorithm for max(ctz(x), ctz(y))? This cookie is used to calculate unique devices accessing the website. However, filters can be tailored to work with specific assays to achieve maximum performance. How does fluorescence plate reader work? Simultaneous Dual Emission(SDE) detection is available on the PHERAstar FSX and is an option on the LUMIstar Omega and FLUOstar Omega. Antibodies labeled with more than four to six fluorophores per protein may exhibit reduced specificity and reduced binding affinity. First, the source light is filtered through an excitation filter that determines the excitation wavelength. 40 L of substrate (solved in DMSO) at different concentrations is added using the onboard injectors. Product Manager, Promega Corporation. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form. Fluorescence plate readers are used for different assays and applications in various fields, such as academic life science research, drug discovery and screening, clinical laboratories, synthetic biology and food or water quality monitoring. A priori what you say about the fluorescence and luminescence readers is correct, even wikipedia states this directly: However, it is worth comparing the fluorescence and the luminescence devices sensitivity and saturation threshold. For some readers, the software only controls the measurement process and provides a raw assay data output. How can I convert plate reader measurements of Pichia pastoris OD to cells per ml? The emission system of the plate reader uses polarizing filters to analyze the polarity of the emitted light. Turn on the plate reader 2. Luminescence detection is optically simpler than fluorescence detection as it does not require a light source or specific optics for excitation. Infinite possibilities. Optical filters isolate quantitative emission signals S1 and S2. Molecular ProbesAlexa Fluor 488 dye is an important fluorescein substitute that provides significantly greater photostability than fluorescein (Figure 8, Figure 9, ), yet is compatible with standard fluorescein optical filters. Dynamic rangeIn fluorescence intensity detection, samples often cover a vast range of concentrations. Next-generation DNA sequencing methods,, Patton, W.F., "A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics,". It does not store any personal data. Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum. This cookie is installed by Google Analytics. EC values at the absorption maximum wavelength are listed in the Section Data Tables throughout the Molecular Probes Handbook. Very dim samples may be undiscernible from the blanks. Reactions which cause flash luminescence produce a short burst of light (which could last seconds to minutes) which is typically brighter than that produced by glow luminescence. Molecular Probes Qdot nanocrystals have even larger extinction coefficients (>2 106 cm-1M-1), particularly in the blue visible and ultraviolet wavelength regions (Qdot NanocrystalsSection 6.6). LVF Monochromators can be used in fluorescence intensity and luminescence. What are all the times Gandalf was either late or early? Combined withshaking and temperature incubation, the ACU provides a real walk-away solution for any cell-based assay from standard cell growth to hypoxic assays. Four essential elements of fluorescence detection systems can be identified from the preceding discussion: 1) an excitation light source (Figure 5), 2) a fluorophore, 3) wavelength filters to isolate emission photons from excitation photons (Figure 5), 4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal. The difference in energy or wavelength represented by (hEX hEM) is called the Stokes shift. This cookie is set by GDPR Cookie Consent plugin. This cookies is set by Youtube and is used to track the views of embedded videos. Fluorescence plate readers are usually intended for fluorescence intensity and FRET detection. Fluorescence detection sensitivity is severely compromised by background signals, which may originate from endogenous sample constituents (referred to as autofluorescence) or from unbound or nonspecifically bound probes (referred to as reagent background). This tutorial describes the systems that make up a flow cytometer, and how those systems work together to collect information on individual cells as they pass through the flow cytometer's laser beam. This means you could miss a crucial result. Do you want to use a fluorescence plate reader in your lab? When you need to change methods or fluorophores, it takes only a few software commands to adjust the system. Your data quality and accuracy is not guaranteed by virtue of following the instructions in this video. As well as specific association with their targets, the probes must have distinctive spectroscopic properties that can be discriminated by the detection instrument.EC (units: cm-1 M-1) is defined by the Beer-Lambert law A=ECcl, where A = absorbance, c = molar concentration, l = optical pathlength. Agilent BioTek absorbance readers provide great performance and ease of use for many applications. Just make sure that your fluorescence microplate reader of choice is also capable of reading the microplate format you plan on using. Comparison of the relative fluorescence of goat antimouse IgG antibody conjugates of Rhodamine Red-X succinimidyl ester (R6160, ) and Lissamine rhodamine B sulfonyl chloride (). For Research Use Only. Instrument does not work. For example, coupling a single fluorescein label to a protein typically reduces fluorescein's QY ~60% but only decreases its EC by ~10%. To learn more about our approach to Privacy we invite you to, Eight Considerations for Getting the Best Data from Your Luminescent Assays, Three Factors That Can Hurt Your Assay Results, How to Get Real-Time Kinetic Data With GloMax Microplate Readers, Studying the Genetic Basis of Type 2 Diabetes with An Automated Maxwell Workflow, MISpheroID: A Knowledgebase to Improve Reproducibility in Spheroid Research. Kobayashi, H., Ogawa, M., Alford, R., Choyke, P.L. When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the relationship becomes nonlinear and measurements may be distorted by artifacts such as self-absorption and the inner-filter effect. and Conchello, J.A., Fluorescence microscopy,, Ntziachristos, V., "Fluorescence molecular imaging,", Patterson, G., Davidson, M., Manley, S. and Lippincott-Schwartz, J., Superresolution imaging using single-molecule localization,, Svoboda, K. and Yasuda, R., Principles of two-photon excitation microscopy and its applications to neuroscience,, Tsien, R.Y., " Imagining imaging's future,". Unauthorized use and/or duplication of this material without express and written permission from this Promega Corporation is strictly prohibited. For polyatomic molecules in solution, the discrete electronic transitions represented by hEX and hEM in Figure 2 are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively (Figure 3, Table 1). This knowledge gets visible in the results that our instruments deliver - the only factor that counts in your lab! The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitivity of fluorescence detection techniques. Molecular Probes tutorial seriesIntroduction to fluorescence. In each well 190 L phosphate buffer (50 mM, pH 7.4) and 10 L enzyme preparation is pipetted. Fluorescence output per fluorophore (brightness) is proportional to the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield. Also, increased labeling of proteins or membranes ultimately leads to precipitation of the protein or gross changes in membrane permeability. To subscribe to this RSS feed, copy and paste this URL into your RSS reader. Can you be arrested for not paying a vendor like a taxi driver or gas station? The range of these parameters among organic dye and autofluorescent protein fluorophores is approximately 5000 to 200,000 cm-1M-1 for EC and 0.05 to 1.0 for QY. Our fluorescence plate readers equipped with reagent injectors can simultaneously inject and detect the signal of a well, and then move to the next one. Filters are usually more sensitive, whereas monochromators offer more flexibility. Fluorescein and hydroxycoumarins (umbelliferones) are further examples of this type of fluorophore. (https://www.biotek.com/products/detection-flash-luminescence-technology.html) These may or may not be included in a plate reader dedicated to fluorescent readings. Absorption and emission efficiencies are most usefully quantified in terms of the molar extinction coefficient (EC) for absorption and the quantum yield (QY) for fluorescence. First, the energy of S1' is partially dissipated, yielding a relaxed singlet excited state (S1) from which fluorescence emission originates. Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein. Figure 6. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Sulen und Kartuschen fr die Chromatographie, Kunststoffartikel und Zubehr fr das Labor, Spektroskopie, Element- und Isotopenanalyse, Alle Themen fr Hilfe und Support anzeigen, Status und Nachverfolgung von Bestellungen, Chapter 1Fluorophores and Their Amine-Reactive Derivatives, Chapter 3Click Chemistry and other Functional Group Modifications, Chapter 5Crosslinking and Photoactivatable Reagents, Chapter 6Ultrasensitive Detection Technology, Chapter 7Antibodies, Avidins and Lectins, Chapter 8Nucleic Acid Detection and Analysis, Chapter 11Probes for Cytoskeletal Proteins, Chapter 13Probes for Lipids and Membranes, Chapter 14Fluorescent Tracers of Cell Morphology and Fluid Flow, Chapter 15Assays for Cell Viability, Proliferation and Function, Chapter 16Probes for Endocytosis, Receptors and Ion Channels, Chapter 17Probes for Signal Transduction, Chapter 18Probes for Reactive Oxygen Species, Including Nitric Oxide, Chapter 19Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions, Chapter 21Indicators for Na+, K+, Cl and Miscellaneous Ions, Chapter 23Antifades and Other Tools for Fluorescence Applications, Fluorophores and Their Amine-Reactive DerivativesChapter 1, Click Chemistry and Other Functional Group ModificationsChapter 3, Crosslinking and Photoactivatable ReagentsChapter 5, Ultrasensitive Detection TechnologyChapter 6, Antibodies, Avidins and LectinsChapter 7, Nucleic Acid Detection and AnalysisChapter 8, Probes for Cytoskeletal ProteinsChapter 11, Probes for Lipids and MembranesChapter 13, Fluorescent Tracers of Cell Morphology and Fluid FlowChapter 14, Assays for Cell Viability, Proliferation and FunctionChapter 15, Probes for Endocytosis, Receptors and Ion ChannelsChapter 16, Probes for Signal TransductionChapter 17, Probes for Reactive Oxygen Species, Including Nitric OxideChapter 18, Indicators for Ca2+, Mg2+, Zn2+ and Other Metal IonsChapter19, Indicators for Na+, K+, Cl and Miscellaneous IonsChapter 21, Antifades and Other Tools for Fluorescence ApplicationsChapter 23, The Molecular Probes Handbook, 11th Edition Download, Environmental Sensitivity of Fluorescence, Fluorescence Resonance Energy Transfer (FRET)Note 1.2, Using the Fluorescence SpectraViewerNote 23.1, Fluorescence Microscopy Accessories and Reference StandardsSection 23.1, Flow Cytometry Reference StandardsSection 23.2, Fluorescent Ca2+ Indicators Excited with UV LightSection 19.2, Probes Useful at Near-Neutral pHSection 20.2, Loading and Calibration of Intracellular Ion IndicatorsNote 19.1, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity MatricesSection 7.6, Detecting Peptidases and ProteasesSection 10.4, Monitoring Protein-Folding Processes with Environment-Sensitive DyesNote 9.1, Detecting Chloride, Phosphate, Nitrite and Other AnionsSection 21.2, Anti-Dye and Anti-Hapten AntibodiesSection 7.4, Alexa Fluor Dyes Spanning the Visible and Infrared SpectrumSection 1.3.