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Figure 1 Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence. The labeled stages 1, 2 and 3 are explained in the adjoining text.
S 0
Energy
S 1 ′
S 1
hνEM
hνEX 1 3
Fluorescent probes enable researchers to detect particular com- ponents of complex biomolecular assemblies, such as live cells, with exquisite sensitivity and selectivity. The purpose of this introduction is to briefly outline fluorescence principles and techniques for newcomers to the field.
The Fluorescence Process
Fluorescence is the result of a three-stage process that occurs in certain molecules (generally polyaromatic hydrocarbons or hetero- cycles) called fluorophores or fluorescent dyes. A fluorescent probe is a fluorophore designed to respond to a specific stimulus or to localize within a specific region of a biological specimen. The process respon- sible for the fluorescence of fluorescent probes and other fluorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown in Figure 1.
Stage 1: Excitation A photon of energy hνEX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creat- ing an excited electronic singlet state (S 1 ´). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction.
Stage 2: Excited-State Lifetime The excited state exists for a finite time (typically 1–10 nano- seconds). During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important con- sequences. First, the energy of S 1 ´ is partially dissipated, yielding a re- laxed singlet excited state (S 1 ) from which fluorescence emission origi- nates. Second, not all the molecules initially excited by absorption (Stage
- return to the ground state (S 0 ) by fluorescence emission. Other process- es such as collisional quenching, fluorescence resonance energy trans- fer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) and intersystem crossing may also depopulate S 1. The fluorescence
Introduction to Fluorescence Techniques
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 mea- sure of the relative extent to which these processes occur.
Stage 3: Fluorescence Emission A photon of energy hνEM is emitted, returning the fluorophore to its ground state S 0. Due to energy dissipation during the excited-state life- time, the energy of this photon is lower, and therefore of longer wave- length, than the excitation photon hνEX. The difference in energy or wavelength represented by (hνEX – hνEM ) is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low back- ground, isolated from excitation photons. In contrast, absorption spec- trophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength.
Fluorescence Spectra The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching), the same fluorophore can be repeatedly ex- cited and detected. The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitiv- ity of fluorescence detection techniques. For polyatomic molecules in solution, the discrete electronic transitions represented by hνEX and hνEM in Figure 1 are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively (Table 1). The bandwidths of these spectra are parameters of particular importance for applications in which two or more differ- ent fluorophores are simultaneously detected. The fluorescence excita- tion spectrum of a single fluorophore species in dilute solution is usu- ally identical to its absorption spectrum. The absorption spectrum can therefore be used as a surrogate excitation spectrum data set. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime, as illustrated in Figure 1. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength (Figure 2).
Figure 2 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 inten- sity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum.
Excitation spectrum
Emission spectrum
EX 3
EX 1
EX 2
EM 3
EM 1
EM 2
Wavelength
Fluorescence excitation Fluorescence emission
Fluorescence Detection
Fluorescence Instrumentation Four essential elements of fluorescence detection systems can be identified from the preceding discussion: 1) an excitation source, 2) a fluorophore, 3) wavelength filters to isolate emission photons from excitation photons, 4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal. Regardless of the application, compatibility of these four elements is essential for optimizing fluorescence detection. Fluorescence instruments are primarily of four types, each provid- ing distinctly different information:
- Spectrofluorometers and microplate readers measure the average properties of bulk (μL to mL) samples.
- Fluorescence microscopes resolve fluorescence as a function of spa- tial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm diameter).
- Fluorescence scanners, including microarray readers, resolve fluo- rescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms.
- Flow cytometers measure fluorescence per cell in a flowing stream, allowing subpopulations within a large sample to be identified and quantitated.
Other types of instrumentation that use fluorescence detection include capillary electrophoresis apparatus, DNA sequencers 1 and microfluidic devices. 2,3^ Each type of instrument produces different
Table 1 Spectroscopic proterties of fluorescent dyes.
Property Definition Significance Fluorescence excitation spectrum *
An X,Y plot of excitation wavelength versus number of fluorescence photons generated by a fluorophore.
Optimum instrument setup should deliver excitation light as close to the peak of the excitation spectrum of the fluorophore as possible. Absorption spectrum An X,Y plot of wavelength versus absorbance of a chromophore or fluorophore.
To a first approximation, the absorption spectrum of a fluorophore is equivalent to the fluorescence excitation spectrum.† To the extent that this approximation holds, the absorption spectrum can be used as a surrogate for the fluorescence excitation spectrum. Fluorescence emission spectrum *
An X,Y plot of emission wavelength versus number of fluorescence photons generated by a fluorophore.
Fluorescence emission spectral discrimination is the most straightforward basis for multiplex detection ‡ and for resolving probe fluorescence from background autofluorescence. Extinction coefficient (EC) Capacity for light absorption at a specific wavelength.§ Fluorescence output per fluorophore (“brightness”) is proportional to the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield. Fluorescence quantum yield (QY)
Number of fluorescence photons emitted per excitation photon absorbed.
See “Extinction coefficient.”
Quenching Loss of fluorescence signal due to short-range interactions between the fluorophore and the local molecular environment, including other fluorophores (self-quenching).
Loss of fluorescence is reversible to the extent that the causative molecular interactions can be controlled.**
Photobleaching Destruction of the excited fluorophore due to photosensitized generation of reactive oxygen species (ROS), particularly singlet oxygen ( 1 O 2 ).
Loss of fluorescence signal is irreversible if the bleached fluorophore population is not replenished (e.g., via diffusion). Extent of photobleaching is dependent on the duration and intensity of exposure to excitation light. ***** Our online Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) provides an interactive utility for plotting and comparing fluorescence excitation and emission spectra for over 250 fluorophores (Using the Fluorescence SpectraViewer—Note 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. 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 = EC•c•l, where A = absorbance, c = molar concentration, l = optical pathlength. EC values at the absorption maximum wavelength are listed in the Section Data Tables throughout The Molecular Probes® Handbook. ****** In the case of self-quenching, this can be accomplished by disruption of fluorophore compartmentalization, denaturation or fragmentation of biopolymer conjugates, or functionalization of the fluorophore to produce increased electrostatic repulsion and water solubility.
measurement artifacts and makes different demands on the fluores- cent probe. For example, although photobleaching is often a significant problem in fluorescence microscopy, it is not a major impediment in flow cytometry because the dwell time of individual cells in the excita- tion beam is short.
Fluorescence Signals Fluorescence intensity is quantitatively dependent on the same pa- rameters as absorbance—defined by the Beer–Lambert law as the prod- uct of the molar extinction coefficient, optical path length and solute concentration—as well as on the fluorescence quantum yield of the dye and the excitation source intensity and fluorescence collection efficien- cy of the instrument (Table 1). In dilute solutions or suspensions, fluo- rescence intensity is linearly proportional to these parameters. When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the rela- tionship becomes nonlinear and measurements may be distorted by artifacts such as self-absorption and the inner-filter effect. 4, Because fluorescence quantitation is dependent on the instrument, fluorescent reference standards are essential for calibrating measure- ments made at different times or using different instrument configura- tions. 6–8^ To meet these requirements, we offer high-precision fluores- cent microsphere reference standards for fluorescence microscopy and flow cytometry and a set of ready-made fluorescent standard solutions for spectrofluorometry (Section 23.1, Section 23.2). A spectrofluorometer is extremely flexible, providing continuous ranges of excitation and emission wavelengths. Laser-scanning micro- scopes and flow cytometers, however, require probes that are excitable at a single fixed wavelength. In contemporary instruments, the excita- tion source is usually the 488 nm spectral line of the argon-ion laser. As
reduced. Detection sensitivity is enhanced by low-light detection devices such as CCD cam- eras, as well as by high–numerical aperture objectives and the widest bandpass emission filters compatible with satisfactory signal isolation. Alternatively, a less photolabile fluorophore may be substituted in the experiment. 26 Alexa Fluor® 488 dye is an important fluorescein substitute that provides significantly greater photostability than fluorescein (Figure 4, Figure 5, Figure 6), yet is compatible with standard fluorescein optical filters. Antifade reagents such as SlowFade® and ProLong® reagents (Section 23.1) can also be applied to reduce photobleaching; however, they are usually incompatible with live cells. In general, it is difficult to predict the necessity for and effectiveness of such countermeasures because photobleaching rates are dependent to some extent on the fluorophore’s environment. 27–
Signal Amplification The most straightforward way to enhance fluorescence signals is to increase the number of fluorophores available for detection. 30 Fluorescent signals can be amplified using 1) avidin–bio- tin or antibody–hapten secondary detection techniques, 2) enzyme-labeled secondary detection reagents in conjunction with fluorogenic substrates 31–33^ or 3) probes that contain multiple fluo- rophores such as phycobiliproteins or FluoSpheres® fluorescent microspheres. Our most sensitive reagents and methods for signal amplification are discussed in Chapter 6. Simply increasing the probe concentration can be counterproductive and often produces marked changes in the probe’s chemical and optical characteristics. It is important to note that the effective intracellular concentration of probes loaded by bulk permeabilization methods (Loading and Calibration of Intracellular Ion Indicators—Note 19.1) is usually much higher (>10-fold) than the extracellular incubation concentration. Also, increased labeling of proteins or mem- branes ultimately leads to precipitation of the protein or gross changes in membrane permeabil- ity. Antibodies labeled with more than four to six fluorophores per protein may exhibit reduced specificity and reduced binding affinity. Furthermore, at high degrees of substitution, the extra fluorescence obtained per added fluorophore typically decreases due to self-quenching (Figure 7).
Environmental Sensitivity of Fluorescence
Fluorescence spectra and quantum yields are generally more dependent on the environ- ment than absorption spectra and extinction coefficients. For example, coupling a single fluo- rescein label to a protein typically reduces fluorescein’s QY ~60% but only decreases its EC by ~10%. Interactions either between two adjacent fluorophores or between a fluorophore and other species in the surrounding environment can produce environment-sensitive fluorescence. Figure 5 Photobleaching resistance of the green-fluores- cent Alexa Fluor® 488, Oregon Green® 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor® 488 (S11223; S32354), Oregon Green® 488 (S6368) or fluorescein (S869) streptavidin. The cells were then fixed in 1% formaldehyde, washed and wet- mounted. 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. Scan du- rations were approximately 5 minutes, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.
% Fluorescence intensity
of
first scan
100
80
60
40
20
0 1 2 3
Oregon Green®^488
Fluorescein
Alexa Fluor®^488
4 5 6 7 8 9 10 Scan repetition
Figure 6 Comparison of the photobleaching rates of the Alexa Fluor® 488 and Alexa Fluor® 546 dyes and the well-known fluorescein and Cy®3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with (top series) Alexa Fluor® 488 phalloidin (A12379) and mouse monoclonal anti–α-tubulin antibody (A11126) in combination with Alexa Fluor® 546 goat anti–mouse IgG antibody (A11003) or (bottom series) fluorescein phalloidin (F432) and the anti– α-tubulin antibody in combination with a commercially available Cy®3 goat anti–mouse IgG antibody. The pseudocolored images were taken at 30-second intervals (0, 30, 90 and 210 seconds of exposure from left to right). The images were acquired with bandpass filter sets appropriate for fluorescein and rhodamine.
Figure 4 Comparison of photostability of green-fluorescent antibody conjugates. The following fluorescent goat anti– mouse IgG antibody conjugates were used to detect mouse anti–human IgG antibody labeling of human anti-nuclear antibodies in HEp-2 cells on prefixed test slides (INOVA Diagnostics Corp.): Alexa Fluor® 488 (A11001, s), Oregon Green® 514 (O6383, j), BODIPY® FL (B2752, n), Oregon Green® 488 (O6380, h) or fluorescein (F2761, d). Samples were continuously illuminated and viewed on a fluorescence microscope using a fluorescein longpass filter set. Images were acquired every 5 seconds. For each conjugate, three data sets, representing different fields of view, were aver- aged and then normalized to the same initial fluorescence intensity value to facilitate comparison.
0
100
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Fluorescence (% of initial)
Fluorophore–Fluorophore Interactions Fluorescence quenching can be defined as a bimolecular process that reduces the fluores- cence 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. Self-quenching is the quenching of one fluorophore by an- other; 34 it therefore tends to occur when high loading concentrations or labeling densities are used (Figure 7, Figure 8). DQ™ substrates (Section 10.4) are heavily labeled and therefore highly quenched biopolymers that exhibit dramatic fluorescence enhancement upon enzymatic cleav- age 35 (Figure 9). Fluorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) is a strongly distance-dependent excited-state interaction in which emission of one fluorophore is coupled to the excitation of another. Some excited fluorophores interact to form excimers, which are excited-state dimers that exhibit altered emission spectra. Excimer formation by the polyaromatic hydrocarbon pyrene is described in Section 13.2 (Figure 10). Because they all depend on the interaction of adjacent fluorophores, self-quenching, FRET and excimer formation can be exploited for monitoring a wide array of molecular assembly or fragmentation processes such as membrane fusion (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3), nucleic acid hybridization, ligand–receptor binding and polypeptide hydrolysis.
Other Environmental Factors Many other environmental factors exert influences on fluorescence properties. The three most common are:
- Solvent polarity (solvent in this context includes interior regions of cells, proteins, mem- branes and other biomolecular structures)
- Proximity and concentrations of quenching species
- pH of the aqueous medium
Fluorescence spectra may be strongly dependent on solvent. This characteristic is most often observed with fluorophores that have large excited-state dipole moments, resulting in fluores- cence spectral shifts to longer wavelengths in polar solvents. Representative fluorophores include the aminonaphthalenes such as prodan, badan (Figure 11) and dansyl, which are effective probes of environmental polarity in, for example, a protein’s interior. 36 Binding of a probe to its target can dramatically affect its fluorescence quantum yield (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Probes
Figure 7 Comparison of relative fluorescence as a func- tion of the number of fluorophores attached per protein for goat anti–mouse IgG antibody conjugates prepared using Oregon Green® 514 carboxylic acid succinimidyl ester (O6139, j), Oregon Green® 488 carboxylic acid suc- cinimidyl ester (O6147, d), fluorescein-5-EX succinimidyl ester (F6130, s) and fluorescein isothiocyanate (FITC; F143, F1906, F1907; h). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugat- ed dye relative to that of the free dye and multiplying by the number of fluorophores per protein.
0 2 4 6 8 10 12 14 16
Fluorescein-EX FITC
OregonGreen ® 488
Fluorophores/protein (mol:mol)
Conjugate
fluorescence
OregonGreen® 514
Figure 8 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Rhodamine Red™-X succinimidyl ester (R6160, d) and Lissamine rhodamine B sulfonyl chloride (L20, L1908; s). 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 pro- tein. Higher numbers of fluorophores attached per protein are attainable with Rhodamine Red™-X dye due to the lesser tendency of this dye to induce protein precipitation.
Fluorophores/protein (mol:mol)
Rhodamine Red™-X
Lissamine™ rhodamine B
Conjugate
fl
uorescence
0 1 2 3 4 5
Figure 10 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normal- ization. Spectra are as follows: 1 ) 2 mM pyrene, purged with argon to remove oxygen; 2 ) 2 mM pyrene, air-equilibrated; 3 ) 0.5 mM pyrene (argon-purged); and 4 ) 2 μM pyrene (argon- purged). The monomer-to-excimer ratio (371.5/470 nm) is de- pendent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.
Fluorescence emission
Wavelength (nm)
350 400 450 500 550 600
1
2
3
4
Figure 11 Fluorescence emission spectra of the 2-mercapto- ethanol adduct of badan (B6057) in: 1 ) toluene, 2 ) chloroform, 3 ) acetonitrile, 4 ) ethanol, 5 ) methanol and 6 ) water. Each solu- tion contains the same concentration of the adduct. Excitation of all samples is at 380 nm.
Fluorescence emission
Wavelength (nm)
(^400 500 )
1
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450 550
Figure 9 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hy- drolysis of the heavily labeled and almost totally quenched substrates provided in the EnzChek® Assay Kits relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.
Intramolecularly quenched substrate
Enzyme
Fluorescent cleavage products
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Fluorescence Microscopy Frigault, M.M., Lacoste, J., Swift, J.L. and Brown, C.M., “Live-cell mi- croscopy: Tips and tools,” J Cell Sci (2009) 122:753–767. Fujimoto, J.G. and Farkas, D., Eds., Biomedical Optical Imaging, Oxford University Press (2009). Goldman, R.D., Swedlow, J.R. and Spector, D.L., Eds., Live Cell Imaging: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (2009). Hell, S.W., “Microscopy and its focal switch,” Nat Methods (2009) 6:24–32. Herman, B., Fluorescence Microscopy, Second Edition, BIOS Scientific Publishers (1998). Hibbs, A.R., Confocal Microscopy for Biologists, Springer (2004). Huang, B., Bates, M. and Zhuang, X., “Super-resolution fluorescence microscopy,” Annu Rev Biochem (2009) 78:993–1016. Inoué, S. and Spring, K.R., Video Microscopy, Second Edition, Plenum Publishing (1997). Lichtman, J.W. and Conchello, J.A., “Fluorescence microscopy,” Nat Methods (2005) 2:910–919. Masters, B.R., Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging, SPIE Press (2006). Matsumoto, B., Ed., Cell Biological Applications of Confocal Microscopy, Second Edition (Methods in Cell Biology, Volume 70), Academic Press (2003). Murphy, D.B., Fundamentals of Light Microscopy and Electronic Imaging, John Wiley and Sons (2001). Ntziachristos, V., "Fluorescence molecular imaging," Annu Rev Biomed Eng (2006) 8:1–32. Patterson, G., Davidson, M., Manley, S. and Lippincott-Schwartz, J., “Superresolution imaging using single-molecule localization,” Annu Rev Phys Chem (2010) 61:345–367.
Pawley, J.B., Ed., Handbook of Biological Confocal Microscopy, Third Edition, Springer (2006). Periasamy, A., Ed., Methods in Cellular Imaging, Oxford University Press (2001). Rosenthal, E. and Zinn, K.R., Eds., Optical Imaging of Cancer: Clinical Applications, Springer (2009). Spector, D.L. and Goldman, R.D., Basic Methods in Microscopy, Cold Spring Harbor Laboratory Press (2005). Svoboda, K. and Yasuda, R., “Principles of two-photon excitation mi- croscopy and its applications to neuroscience,” Neuron (2006) 50:823–839. Tsien, R.Y., " Imagining imaging’s future," Nat Rev Mol Cell Biol (2003) 4:SS16–SS21. Yuste, R. and Konnerth, A., Eds., Imaging in Neuroscience and Development: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2005).
Flow Cytometry Darzynkiewicz, Z., Crissman, H.A. and Robinson, J.P., Eds., Cytometry, Third Edition Parts A and B (Methods in Cell Biology, Volumes 63 and 64), Academic Press (2001). Givan, A.L., Flow Cytometry: First Principles, Second Edition, John Wiley and Sons (2001). Herzenberg, L.A., Parks, D., Sahaf, B., Perez, O., Roederer, M. and Herzenberg, L.A., "The history and future of the fluorescence ac- tivated cell sorter and flow cytometry: A view from Stanford," Clin Chem (2002) 48:1819–1827. Herzenberg, L.A., Tung, J., Moore, W.A., Herzenberg, L.A. and Parks, D.R., “Interpreting flow cytometry data: A guide for the perplexed,” Nat Immunol (2006) 7:681–685. Preffer F. and Dombkowski, D. “Advances in complex multiparameter flow cytometry technology: Applications in stem cell research,” Cytometry B (2009) 76:295–314. Shapiro, H.M., "Optical measurement in cytometry: Light scattering, extinction, absorption and fluorescence," Meth Cell Biol (2001) 63:107–129. Shapiro, H.M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss (2003). Sklar, L.A., Ed., Flow Cytometry for Biotechnology, Oxford University Press (2005).
Other Fluorescence Measurement Techniques Dorak, M.T., Ed., Real-Time PCR, Taylor and Francis (2006). Gore, M., Ed., Spectrophotometry and Spectrofluorimetry: A Practical Approach, Second Edition, Oxford University Press (2000). Mardis, E.R. “Next-generation DNA sequencing methods,” Annu Rev Genomics Hum Genet (2008) 9:387–402. Patton, W.F., "A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics," Electrophoresis (2000) 21:1123–1144. Shimomura, O., Bioluminescence: Chemical Principles and Methods, World Scientific Publishing (2006).
Fluorophores and Fluorescent Probes, continued
