1. These slides are on the PURDUE CYTOMETRY WEB SITE
Functional assays - Principles and Methods
Purdue University Cytometry Laboratories
Presented at the Polish Society for Cytometry Meeting, Gdansk, Poland, October 18, 1998
Presentation given to the Polish Society for Cytometry meeting, Gdansk, 18 October, 1998.
Material on this site is copyrighted by J.Paul Robinson, however, all the material may be freely used for non-commercial educational use. Examples include lectures, tutorials and lecture notes. If slides and material are used, appropriate acknowledgement should be as follows:
Taken/modified from material prepared by J.Paul Robinson, Purdue University, West Lafayette, Indiana.
For contact purposes:
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Purdue University, West Lafayette, In. Ph (765)
Fax (765)
Date Published to the WEB 25 October, 1998
J Paul Robinson
These slides are on the PURDUE CYTOMETRY WEB SITE

2. Poland taken from Comsat C1 satellite
October 15, you can clearly see Warsaw in the center. Top center-left is Gdansk.
This image was taken on October 15, 1998 from the Comsat C1 satelite and represents Poland at Night! In the center is Warsaw, with Krakow at the lower left. Top left is Gdansk. The next slide shows the exact same view taken during daylight.
The sight address is

3. Gdansk
Warsaw
Krakow

4. Gdansk

5. The goals of this presentation are:
To identify the nature of functional assays in Cytometry
To expand on how they operate
To discuss the advantages and disadvantages of each
To discuss the application of these assays
The techniques of flow cytometry are ideal for evaluation of functional properties of live cells. One unique aspect of flow cytometry is that it allows the measurement of particular aspects of a single cell, regardless of the composition of the suspension or the heterogeneity of the cell population. Additionally, because light scatter is by nature of flow technology an integral measurement, it does not negatively interfere with fluorescence measurements as would occur using bulk fluorometry. Thus, flow cytometry can use scatter to identify a population, fluorescence properties to identify a response, and time to evaluate the kinetics of this response. Examples of this system would include the measurement of the oxidative burst of phagocytic cells, changes in calcium flux, alterations in membrane potential of activated cells or efflux pumps eliminating a drug or dye.

6. Kinetics Principle of Time Measurements
Live cells can be measured as easily as dead cells
You only need a small number of cells in a changing environment
End point assays can describe the activity of the cell
An important principle is the ability of some dyes to be incorporated into live cells without impacting their short-term function (some may be toxic eventually). There are several specific dyes of interest, depending upon the function to be determined. Dyes, which can respond to the presence of strong oxidants, can be used to identify the production of these oxidants - an example is H2O2. There are several fluorescent indicators of H2O2 activity such as DCFH-DA or dihydrorhodamine Another useful dye for strong oxidants is Hydroethidine which can detect the superoxide radical O2-. All of these dyes can be used to detect the production of these reactive oxygen species in live cells in real time.

7. Cellular Functions Ionic Flux Determinations Membrane Potential
Cell Viability
Phagocytosis
Organelle Function
mitochondria, ER
endosomes, Golgi
Oxidative Reactions
Superoxide
Hydrogen Peroxide
Nitric Oxide
Glutathione levels
Ionic Flux Determinations
Calcium
Intracellular pH
Membrane Potential
Membrane Polarization
Lipid Peroxidation
Another kinetic measurement of importance is the change in calcium flux that is apparent in activated cells. The initial steps of signal transduction following receptor-ligand interaction involve activation of phospholipase c and membrane bound glycophotidyl inositol. This leads to the release of inositol phosphates and fatty acids which trigger activation of protein kinase C and subsequent flux of calcium across the plasma membrane. Thus a direct cellular response can be measured if alterations of calcium concentration can be monitored.

8. What do we measure? Fluorescence TIME
The principle of kinetic measurements when applied to flow cytometry, provides a powerful tool for functional assays. In the above example, a changing fluorescence signal is plotted with time (x axis). Therefore we could be looking at an increasing signal (such as when measuring increasing H2O2 production with DCF) or a decreasing signal (Such as with paranaric acid - lipid peroxidation assay).
TIME

9. Fluorescent Indicators
How the assays work:
Superoxide: Utilizes hydroethidine the sodium borohydride reduced derivative of EB
Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent DCF
Glutathione: In human samples measured using 40 M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time.
Nitric Oxide: DCFH-DA can also be used as an indicator for nitric oxide in a manner similar to H2O2

10. Organelle Function Mitochondria Rhodamine 123 Endosomes Ceramides
Golgi BODIPY-Ceramide
Endoplasmic Reticulum DiOC6(3) Carbocyanine

11. DCFH-DA DCFH DCF Fluorescent Hydrolysis Oxidation H2O2 DCFH-DA
COOH H Cl O
O-C-CH3
CH3-C-O OH HO
Fluorescent
Hydrolysis
Oxidation
2’,7’-dichlorofluorescin
2’,7’-dichlorofluorescin diacetate
2’,7’-dichlorofluorescein
Cellular Esterases
H2O2
DCFH-DA
DCFH
DCF
H O
2 2
Lymphocytes
Monocytes
Neutrophils
log FITC Fluorescence .1
1000
100 10 1 20 40 60
counts
PMA-stimulated PMN
Control 80
Dichlorofluorescin diacetate is hydrolysed by cellular esterases to dichlorofluorescin (DCFH) and is then oxidized to a fluorescent product dichlorofluorescein (DCF) primarily bu H2O2. Dihydrorhodamine (DHR 123) is by far the most-used probe for measurement of intracellular H2O2. DHR 123 is oxidized directly to rhodamine 123, which is excitable at 488 and emits at 515 nm in the same emission range as DCF and FITC (Rothe et al., 1988). Publications describe its use in human neutrophils, human eosinophils, HL60 cells, rat mast cells, guinea pig neutrophils, cultured chondrocytes, rat brain, rat renal proximal tubular cells, mesangial cells and L929 cells. Combinations of DHR123 with surface receptor analysis cell viability using propidium iodide, and calcium indicators demonstrate how the probe can be used for simultaneous measurements.
DHR 123 enters the cells as a freely permeable dye which is converted to rhodamine 123 and subsequently localized in the mitochondria. The conversion from the non- fluorescent to the fluorescent molecule is entirely dependent upon oxidation products and does not require enzymatic catalysis. Once oxidized, the probe is identical to rhodamine 123, a common laser dye. One significant advantage of the DHR probe is that the oxidation product, rhodamine 123, remains essentially within the cell, unlike the oxidation product DCF, which has a strong tendency to leak from cells and requires careful controls to monitor leakage.

12. Hydroethidine HE EB O2- O2- H2O2 Example: Neutrophil Oxidative Burst
CH2CH3
NH2
H2N H
O2-
Br- N
CH2CH3
NH2
H2N +
Phagocytic Vacuole
NADPH Oxidase
NADPH O2 HE
O2-
Hydroethidine (HE) operates effectively as a probe for measurement of O2-. The dye enters cells freely and is dehydrogenated to ethidium bromide. A major advantage of this probe is its ability to distinguish between O2- and H2O2. Studies have been performed using neutrophils and endothelial cells as well as HL60 cells and macrophages. The probe has been used extensively with NK cell and as a vital dye for identification of proliferation and hypoxic cells in tumors. Fluorescence emission occurs at around 600 nm.
NADP
DCF
SOD
O2-
H2O2
DCF
H2O2
OH-
Example: Neutrophil Oxidative Burst

13. Both these images are cells stained to measure for H2O2 production.
Chondrocytes
Neutrophil
On the left is shown chondrocytes loaded with Hydroethidine undergoing an oxidative reaction to produce O2- (red areas - note the fluoresnceec). On the right is a neutrophil which has been fully oxidized after loading with DCFH-DA and activated by PMA (100 nm). An intense green fluorescence is evident.

14. Some examples of rapidly changing antigen expression systems
Endothelial
Adhesion Molecules
Neutrophil Counter Ligand
P-selectin (CD62P)
s-Lex (CD15s)
E-selectin (CD62E)
s-Lex, CD66, L-selectin, b2 integrins
Neutrophil
Adhesion Molecules
Endothelial Counter Ligand
L-selectin (CD62L)
s-Lex (CD15s)
CD11a/CD18
ICAM-1 (CD54), ICAM-2 (CD102)
CD11b/CD18
ICAM-1 (CD54), [iC3b, fibrinogen, factor X]
CD11c/CD18
?, [iC3b, fibrinogen]

15. a c b d TNFa IL-1 BACTERIAL INFECTION Neutrophils Endothelial Cells
E-selectin & P-selectin
ICAM-1
L-selectin &CHO ligands
(e.g. sLex)
CD11b
The circulating neutrophil (a) and the initiation of rolling (b) as molecular tethers are formed between selectin and CHO ligands on neutrophils and endothelial cells. If an adequate number of tethers are formed, the neutrophil completely decelerates and with chemotactic stimulation of the neutrophil, L-selectin is rapidly shed while other receptors like E-selectin, CD11b and ICAM-1 are up-regulated by cytokines and other inflammatory mediators (c). Firm neutrophil/endothelial cell adhesion is mediated by CD11b and ICAM-1 and is followed by emigration of the neutrophil through the endothelium (d).

16. M ? (NOO-) Mmembrane damage - -
H2O2
O2-
NO.
OH- - - M
VCAM + +
NO. + + +
ROS +
NFkB + + +
E-selectin
ICAM-1
TNFa
Bradykinin
P-selectin
Known and unknown interactions between neutrophils and endothelial cells. Nitric oxide (NO.) and reactive oxygen species (ROS) are produced by both neutrophils and endothelial cells thus the interaction between these reactive species becomes very complicated.
CD11b
Mmembrane damage
conjugated dienes
+ stimulatory effect
- inhibitory effect

17. Oxidative Reactions Superoxide Hydroethidine
Hydrogen Peroxide Dichlorofluorescein
Glutathione levels Monobromobimane
Nitric Oxide Dichlorofluorescein

18. Rat Pulmonary Artery Endothelial Cells Oxidization via H2O2
Periodicity of Fluorescence
Meridian UltimaTM Analysis
Purdue University Cytometry Laboratories

19. Macrovascular Endothelial Cells in Culture
Time (minutes) 60

20. Confocal System
Culture System

21. top view 1 2 3 4 5 6 7 8 Step 1: Cell Culture Step 2: Cell Wash
Step 3: Transfer to Lab-Tek plates 7 8
side view
170 M coverslip
Lab-Tek
Step 4: Addition of DCFH-DA, Indo-1, or HE
stimulant/inhibitor added
37o heated stage
oil immersion
objective
confocal microscope

22. Hydrogen peroxide measurements with DCFH-DA1 2 3 4 5
Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.
525 nm
Step 6B: Export data from measured
regions to Microsoft Excel
200
400
600
800
1000
1200
1400
1600
1800
2000
500
1500
2500
3000
Time in seconds
cell 1
cell 2
cell 3
cell 4
cell 5
base to Delta Graph
Step 7B: Export data from Excel data
% change (DCF fluorescence)

23. Superoxide measured with hydroethidine
cell 1
cell 2
cell 3
cell 4
cell 5
Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.
-200
200
400
600
800
1000
1200
1400
1600
1800
cell 1
cell 2
cell 3
cell 4
cell 5
Time in seconds
Export data from measured
regions to Microsoft Excel
Export data from Excel data
base to Delta Graph
%change (DCF fluorescence)

24. Metamyelocyte Promyelocyte Myelocyte Neutrophil
Padma Narayanan figures hl60.ppt

25. Change in Mean DCF Fluorescence36 30
0 ng/ml PMA
8 ng/ml PMA 24
50 ng/ml PMA 18
Change in Mean DCF Fluorescence d1 e 12 c1 b1 d 6 c a a b
0 HOURS
24 HOURS
48 HOURS
72 HOURS
96 HOURS

26. Diphenyleneiodonium chloride [mM]36
0 HOURS PMA (8 ng/ml) 30
0 HOURS PMA (50 ng/ml)
96 HOURS PMA (8 ng/ml) 24
96 HOURS PMA (50 ng/ml) 18
Change in Mean DCF Fluorescence 12 6
0.1 1 5 10 20 50
Diphenyleneiodonium chloride [mM]

27. Change in Mean Channel Fluorescence
HL-60 cells 20
DCF Fluorescence
EB Fluorescence 15 10
Change in Mean Channel Fluorescence 5
Passage 28
Passage 60

28. Phagocytosis How the assay works:
Uptake of Fluorescent labeled particles
Determination of intracellular or extracellular state of particles
How the assay works:
Particles or cells are labeled with a fluorescent probe
The cells and particles are mixed so phagocytosis takes place
The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles
The remaining fluorescence
represents internal particles
FITC-Labeled Bacteria
The Phagocytic process can be divided up into a number of clearly defined stages broadly defined as attachment or particle binding, ingestion and destruction. Unless phagocytes are able to bind to the microbe, phagocytosis will not take place. By utilizing both opsonized and non-opsonized organisms, both opsonic capacity and phagocytosis can be measured at the same time. Thus it is important to determine whether abnormal phagocytosis is due to a failure in the opsonization process or a defect in the ingestion capability of the phagocyte. Since the main cell receptors for phagocytosis are C3b (CR1) and FcR (Fc portion of IgG) it is also possible to evaluate these functional receptors as discussed earlier.
Phagocytosis of bacteria has been extensively developed for flow cytometry primarily by Bassoe et al. Immune complexes can also be measured in methods similar to those for bacteria. Flow cytometry may have an advantage over other methods because of the relatively small number of cells required and less preparative procedures for isolating leukocytes.

29. Trypan Blue FITC-Labeled Bacteria
In the above figure, a neutrophil is seen phagocytosing a FITC-labelled organism and then external organisms are quenched using trypan blue.
One useful method described uses fluorescein heat-killed Candida albicans, and after phagocytosis is complete, ethidium bromide (EB) (50 ug/ml) is added. Analysis using ultraviolet (UV) excitation with red and green emission reveals green internalized organisms, while surface attached, but non-internalized organisms are red. This procedure utilizes the phenomenon of resonance energy transfer between FITC and ethidium bromide. EB does not penetrate the cell membrane so only the external organisms are affected by the providing discrimination between internal and external organisms. This assay can be performed on clinical analyzers with simple laser configurations.
Flow cytometry can also be used for evaluating neutrophil function in the phagocytosis of fluorescent-labeled viruses. FITC-labeled Herpes simplex viruses (HSV) have been shown to be phagocytosed by human neutrophils and both internalization and surface binding were determined by flow cytometry. Surface bound virus fluorescence was quenched using a trypan blue quenching procedure.
Pinocytosis can also be a useful measure of cell function and several well-defined assays have been developed for flow cytometry. fMLP stimulated pinocytosis studies have demonstrated a linkage between the initial phase of pinocytosis and the characteristic shape changes observed in activated neutrophils. This assay uses FITC-dextran and is relatively simple to establish. A number of concomitant effects such as pH changes, kinetics of the responses, temperature and ionic concentration effects can be evaluated in this manner.

30. pH Sensitive Indicators
Probe Excitation Emission
SNARF
BCECF /620
440/
[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

31. Applications Probe Ratioing Calcium Flux (Indo-1)
pH indicators (BCECF, SNARF)
Molecule-probe Excitation Emission
Calcium - Indo nm 405, >460 nm
Calcium- Fluo nm 525 nm
Calcium - Fura nm >500 nm
Calcium - Calcium Green 488 nm 515 nm
Magnesium - Mag-Indo nm 405, >460 nm
Phospholipase A- Acyl Pyrene 351 nm 405, >460 nm

32. Ionic Flux Determinations
Calcium Indo-1
Intracellular pH BCECF
How the assay works:
Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner
The emission wavelength decreases as the probe binds available calcium
Time (Seconds) 36 72
108
144
180
Stimulation
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 50
100
150
200
Ratio: intensity of 460nm / 405nm signals
Time (seconds)
Flow Cytometry Image Analysis
Since calcium plays a critical role in cell function it is important to be able to determine the extent to which chemical interactions affect the redistribution of this divalent cation. There are a number of dyes useful for these measurements such as Indo-1, fura-2 and fluo-3. Indo-1 is an excellent dye for flow cytometric measurement of free intracellular calcium but it does require a UV light source. This dye has the ability to undergo a fluorescent wavelength emission shift when bound to calcium. Indo-1 is introduced to the cells as an acetoxymethyl ester which undergoes enzymatic hydrolysis in cells to yield free dye.
Flow cytometry has proved to be a valuable resource in the evaluation of the role of calcium in neutrophil function. A major spectral change can be measured when indicators of Ca2+ penetrate cells and are excited at 357 nm (ultra-violet excitation). Cells are loaded with Indo-1 (final concentration 3 um) for 15 min at 37oC and then immediately run on the flow cytometer to obtain fluorescence histograms at two emission wavelengths; 395 nm (bound Ca2+) and 525 nm (non-bound calcium). The Ca2+ concentration of cells can be determined independently of dye concentration by evaluating the ratios of the two fluorescent emissions. Thus a high 395/525 nm ratio would indicate bound Ca2+. Ionomycin is used as a positive control for measurement of calcium flux. Ionomycin (3-5 mM) will cause an increase in the BOUND (long wavelength) fluorescence signal (i.e. increase in BOUND [Ca2+] inside the cell).

33. Calcium ratios with Indo-1
Changes in the fluorescence were measured using the Bio-Rad calcium ratioing software. The same region in each wave length was measured and the relative change in each region was recorded and exported to a spread sheet for analysis.. 1 1 2 2 3 3
460 nm
405/35 nm
Export data from measured regions to Microsoft Excel
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
cell 1
cell 2
cell 3
Ratio: intensity1 (460nm) / intensity2 (405/35nm) 50
100
150
200
base to Delta Graph
Export data from Excel data
This measurement is a very rapid event which can be observed on the flow cytometer in real time given appropriate instrumentation. Accurate determination of intracellular calcium concentration can be made if aliquots of Indo-1 loaded cells are placed in solutions of various known calcium concentrations and treated with an ionophore such as Ionomycin. The properties of Indo-1 are well-described in the literature. Observations of the real time alteration in [Ca2+] can be performed using list mode on the flow cytometer.

34. Probes for Ions INDO-1 Ex350 Em405/480 QUIN-2 Ex350 Em490
Fluo-3 Ex488 Em525
Fura -2 Ex330/360 Em510

35. Membrane Potential Cyanine Probes Oxanol Probes How the assay works:
Carbocyanine dyes released into the surrounding media as cells depolarize
Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs
Time (sec)
Green Fluorescence
PMA Added
fMLP Added
Repolarized Cells
This is an example of a membrane potential assay where neutrophils were loaded with a cyanine probe and stimulated with PMA (left)or fMLP (right). Interestingly on the right, the cells were also 24 hours old and it is apparent that while normally when activated by fMLP, nearly all the neutrophils will repolarize within a few minutes. However, because these cells are 24 hours old, they fail to repolarize indicating a loss of functional integrity. This is despite the fact that these cells appear to produce normal amounts of respiratory burst and also appear to phagocytose normally. Clearly, the membrane potential is one of the earlier functions to suffer from “old age”!!
Green Fluorescence
Depolarized Cells
300
150
Time (sec)

36. Lipid Peroxidation How the assay works:
Probe: 5 M cis-paranaric acid (Molecular Probes)
How the assay works:
Cis-paranaric acid is a naturally fluorescent fatty acid which has 4 conjugated double bonds which become targeted by lipid peroxidation reactions with a subsequent loss of fluorescence
The Paranaric acids are the closest structural analogs of intrinsic membrane lipids among current fluorescent probes. Cis-paranaric acid, a naturally fluorescent fatty acid, loses its fluorescence over time when the four conjugated double bonds of the backbone chain become the target of lipid peroxidation reactions.

37. Lipid Peroxidation Paranaric Acid
Over time - Paranaric acid loses its fluorescence as the double bonds are destroyed
Cartoon of the curve that would be derived from the data at left.
Thanks to its extensive unsaturation, paranaric acid is quite susceptible to oxidation. It is this property that is utilized in measuring lipid peroxidation. Because a 325 nm UV excitation source is required, the use of paranaric acid is restricted to spectrofluorometry and flow cytometers with helium-cadmium lasers.
Note in the above data, the left figure is reproduced from the reference listed, however the data on the right is simply a cartoon representation of the the mean kinetic change that would be observed from the data on the left.
Data taken from Hedley, et al, Cytometry, 13: , 1992
TIME (Seconds)
TIME (Seconds)
Data on left taken from Hedley, et al, Cytometry, 13: , 1992

38. “Caged” Photoactivatable Probes
Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at nm
Available Probes
Ca++: Nitr-5
Ca++ - buffering: Diazo-2
IP3
cAMP
cGMP
ATP
ATP--S
See next slide for explanations.

39. Release of “Caged” Compounds
UV Beam
Culture dish
Release of “Cage”
“Caged” compounds are reactive molecules that have been protected from their environments using chemical configurations that are essentially non reactive without intervention. In this case the intervention is the intense UV laser beam(355 nm). This breaks the “cage” releasing the active compound into the cell or medium. The advantage of this technique is that it can be performed very specifically in certain cells in a tissue culture disk, while still allowing cells at a remote area to be used as a control population (this may be impacted by the nature of the activation process of course).

40. Caged Nitric Oxide study
Regions were selectively excited using UV light to release the cage nitric oxide. Images of the excited and adjacent control region were then collected.
UV excited
Control Region
Time (seconds) after UV FLASH 20 40 60 80
100
120
140
160 50
150
200
250
Fluorescence Emission at 515 nm
Export data from measured
regions to Microsoft Excel
dbase to Delta Graph
Export data from Excel data

41. FRAP %F Recovery of fluorescence Intense laser Beam
Bleaches Fluorescence
Time
Recovery of fluorescence
Fluorescence recovery after photobleaching allows the monitoring of cell-cell communication. As the cell fluorescence is bleached by an intense laser beam, a plot of time versus fluorescence is created to trace the relative change in fluorescence intensity.
Zero time
10 seconds
30 seconds

42. Conclusions & Summary Functional Studies In Cytometry
Oxygen radicals
Nitrogen radicals
Antioxidants
Cell viability
Organelle function
Lipid peroxidation
Membrane potential
Calcium fluxes
pH changes
These examples are but a few of the functional assays now available in linking cell biology with flow cytometry. The major advantage flow cytometry offers is the ability to identify single cell fluorescence so that heterogeneic populations can be identified with statistical accuracy. Flow cytometry should be considered a viable alternative approach when cells are available in suspension (or can be removed from tissue or cell cultures appropriately), when one needs to understand a functional component of the cell, an appropriate fluorescent probe is available, heterogeneity may exist requiring “electronic” separation of cells (e.g. blood) and of course, you have a flow cytometer available!

43. Acknowledgements Kathy Ragheb Gretchen Lawler Steve Kelley
Monica Shively
Dave Whittinghill
Stephanie Sincock
Karen Cornell
Karin Kooreman
Nian-Yu Li
Padma Narayanan (Smith Kline)
Wayne Carter (Pfizer)
Here are some of the team from Purdue University plus some of our colleagues and collaborators! We always seem to have a good time when we discuss science!

44. Specific Organelle Probes
Probe Site Excitation Emission
BODIPY Golgi
NBD Golgi
DPH Lipid
TMA-DPH Lipid
Rhodamine 123 Mitochondria
DiO Lipid
diI-Cn-(5) Lipid
diO-Cn-(3) Lipid
These fluorescent probes are some of those available to evaluate cell function. The excitation and emission optima are shown as well as the preferred site of binding for each probe.
This lecture was entitled:
Functional assays - Principles and Methods
and was presented at the 4th Polish Cytometry Society Congress in Gdansk, Poland, 18 October, 1998.
J. Paul Robinson
Professor of Immunopharmacology
Purdue University
West Lafayette, USA
Material copyright by J.Paul Robinson. See first slide notes for use information. Date Published to the WEB 25 October, 1998
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammonium