FLUOROPHORES

Structure, Properties and Applications

Contents
Green fluorescent protein..................................................................................................................... 5 APPLICATIONS ................................................................................................................................ 6 1. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells ........................... 6

2. Novel assays for proteases using green uorescent protein-tagged substrate immobilized on a membrane disk ............................................................................................................................. 6 3. Engineered GFP as a vital reporter in plants........................................................................... 7

4. In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation .......................................................................................................................... 7 5. Nanoscale resolution in GFP-based microscopy..................................................................... 8

Acridine orange ..................................................................................................................................... 8 APPLICATIONS ................................................................................................................................ 8 1. Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis ............................ 8

2. Acridine Orange for malaria diagnosis: its diagnostic performance, its promotion and implementation in Tanzania, and the implications for malaria control........................................... 9 3. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. ... 9

4. In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells .................................................................................................................. 9 5. Photodynamic therapy with acridine orange in musculoskeletal sarcomas .......................... 10

DAPI ..................................................................................................................................................... 10 APPLICATIONS .............................................................................................................................. 11 1. 2. 3. Applications of DAPI cytochemistry to neurobiology. .......................................................... 11 Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging ...................... 11 Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using DAPI ................ 11

4. FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene ..................................................................................................................................... 12 5. Chromosomal Variations Within Aneuploid Cancer Lines .................................................. 12

Fluorescein ........................................................................................................................................... 13 APPLICATIONS .............................................................................................................................. 13 1. 2. 3. Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish. 13 A novel fluorescein derivative as a colorimetric chemosensor for detecting copper(II) ion.14 Use of Fluoroscein-EDTA System in Photogalvanic Cell for Solar Energy Conversion .......... 14

4. A simple spectrophotometric streptavidinbiotin binding assay utilizing biotin-4fluorescein..................................................................................................................................... 14 5. One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells ..................................................................... 15 Cyanines ............................................................................................................................................... 15 APPLICATIONS .............................................................................................................................. 16 1. Detection of Oligosaccharides Labeled with Cyanine Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.................................................................................... 16 2. Cyanine dyes and their applications as luminescence quenching compounds...................... 17

3. Cyanine dyes as Labelling Reagents for Detection of Biological and other Materials by Luminescence Methods................................................................................................................. 17 4. Application of near-infrared absorbing heptamethine cyanine dyes as sensitizers for zinc oxide solar cell. ............................................................................................................................. 17 Rhodamine ........................................................................................................................................... 18 APPLICATIONS .............................................................................................................................. 18 1. 2. Localization of mitochondria in living cells with rhodamine 123 ........................................ 18 Rhodamine immunohistofluorescence applied to plant tissue .............................................. 19

3. A RhodamineHydroxamic Acid-Based Fluorescent Probe for Hypochlorous Acid and Its Applications to Biological Imagings............................................................................................. 19 Novel Application of Fluorophore ..................................................................................................... 20 References ............................................................................................................................................ 22

This page has been intentionally left blank.

Green fluorescent protein

The native green fluorescent protein (GFP), first so named by Morin and Hastings (1971), from the jellyfish Aequorea victoria contains 238 amino acids. Residues 65-67 (Ser-Tyr-Gly) in the GFP sequence spontaneously form the fluorescent chromophore phydroxybenzylideneimidazolinone

Figure 1. The fluorescence chromophore formed by amino acid residues 65-67 (Ser-Tyr-Gly) in the primary structure of GFP

The excitation spectrum of GFP fluorescence has a dominant maximum at about 400 nm and a significantly smaller maximum at about 470 nm, while the emission spectrum has a sharp maximum at about 505 nm and a shoulder around 540 nm.

Figure 2. Fluorescence excitation (full-line curve) and emission (dashed curve) spectra of native GFP from Aequorea victoria

The crystal structure of GFP is an eleven-stranded - barrel, threaded by an -helix, running up along the axis of the cylinder. The chromophore is in the -helix, very close to the centre of the can-like cylinder. A very large part of the primary structure of the protein is used to construct the -barrel and the threading -helix.

Figure 3. GFP molecules, one fully and one with the side of the beta barrel cut away to reveal the chromophore

APPLICATIONS 1. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells

George H. Patterson and Jennifer Lippincott-Schwartz (2002). Science 297(5588): 1873-1877

A photoactivatable variant of theAequorea victoria green fluorescent protein (GFP) has been reported that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, the photoactivatable GFP has been used both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange. 2. Novel assays for proteases using green uorescent protein-tagged substrate immobilized on a membrane disk
Takashi Aoki, Shirou Tsuchida, Taemi Yahara, Naoya Hamaue (2008). Analytical Biochemistry 378 (2): 132-137

Green uorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for uorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated Cleave-Checker on which GFPSpAB (domain B in staphylococcal protein A) is immobilized. The SpAB region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpAB region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a

highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. 3. Engineered GFP as a vital reporter in plants
Wan-ling Chiu, Yasuo Niwa, Weike Zeng, Takanori Hirano, Hirokazu Kobayashi, Jen Sheen (1996). Current Biology 6 (3): 325-330

It has been reported that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants. It has been shown that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation. Also, this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants. The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. 4. In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation
Hiroyuki Kishimoto, Ming Zhao, Katsuhiro Hayashi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara, Sheldon Penman and Robert M. Hoffman (2009). PNAS 106 (34): 14514-14517

Cancer surgery requires the complete and precise identification of malignant tissue margins including the smallest disseminated lesions. Internal green fluorescent protein (GFP) fluorescence can intensely illuminate even single cells but requires GFP sequence transcription within the cell. Introducing and selectively activating the GFP gene in malignant tissue in vivo is made possible by the development of OBP-401, a telomerasedependent, replication-competent adenovirus expressing GFP. This potentially powerful adjunct to surgical navigation was demonstrated in 2 nude mouse models that represent difficult surgical challengesthe resection of widely disseminated cancer. HCT-116, a model of intraperitoneal disseminated human colon cancer, was labeled by virus injection into the peritoneal cavity. A549, a model of pleural dissemination of human lung cancer, was labeled by virus administered into the pleural cavity. Only the malignant tissue fluoresced brightly in both models. In the intraperitoneal model of disseminated cancer, fluorescence-guided

surgery enabled resection of all tumor nodules labeled with GFP by OBP-401. The data in this report suggest that adenoviral-GFP labeling tumors in patients can enable fluorescenceguided surgical navigation. 5. Nanoscale resolution in GFP-based microscopy
Katrin I Willig, Robert R Kellner, Rebecca Medda, Birka Hein, Stefan Jakobs & Stefan W Hell (2006). Nature Methods 3(9): 721-723

Attainment of subdiffraction resolution using stimulated emission depletion (STED) microscopy with GFP labeled samples has been reported. The B70 nm lateral resolution attained in this study is demonstrated by imaging GFP-labeled viruses and the endoplasmic reticulum (ER) of a mammalian cell. The results mark the advent of nanoscale biological microscopy with genetically encoded markers.

Acridine orange
Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. It is cell-permeable and interacts with DNA and RNA by intercalation or electrostatic attractions respectively. When bound to DNA, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). Like fluorescein, it is also useful as a non-specific stain for backlighting conventionally stained cells on the surface of a solid sample of tissue (fluorescence backlighted staining). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red).

Figure 4. Acridine orange molecule

APPLICATIONS 1. Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Wetzel, and Douglas R. Green (2006). Cold Spring Harb Protoc Finucane,Thomas Brunner, Ella Bossy-

Acridine orange will also enter acidic compartments such as lysosomes and become protonated and sequestered. In these low pH conditions, the dye will emit orange light when excited by blue light. Thus, acridine orange can be used to identify engulfed apoptotic cells,

because it will fluoresce upon engulfment. The dye is often used in epifluorescence microscopy. Acridine orange can be used in conjunction with ethidium bromide to differentiate between viable, apoptotic and necrotic cells. Additionally, Acridine orange may be used on blood samples to fluoresce bacterial DNA, aiding in clinical diagnosis of bacterial infection once serum and debris have been filtered. 2. Acridine Orange for malaria diagnosis: its diagnostic performance, its promotion and implementation in Tanzania, and the implications for malaria control.
J. Keiser, J. Utzinger, Z. Premji, Y. Yamagata and B. H. Singer (2002). Annals of Tropical Medicine & Parasitology 96 (7): 643654.

One hundred years ago, Giemsas stain was employed for the first time for malaria diagnosis. Giemsa staining continues to be the method of choice in most malarious countries, although, in the recent past, several alternatives have been developed that exhibit some advantages. Considerable progress has been made with fluorescent dyes, particularly with Acridine Orange (AO). AO shows a good diagnostic performance, with sensitivities of 81.3%100% and specificities of 86.4%100%. However, sensitivities decrease with lower parasite densities, and species differentiation may occasionally be difficult. The most notable advantage of the AO method over Giemsa staining is its promptness; results are readily available within 310 min, whereas Giemsa staining may take 45 min or even longer. This is an important advantage for the organization of health services and the provision of effective treatment of malaria cases 3. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence.
Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S(1984). Fertil Steri. 42(1):87-91.

A practical test for evaluating the fertility of a male subject was developed. Twenty-eight donors whose semen had induced at least one pregnancy resulting in a normal delivery and 61 patients attending our infertility clinic were studied. Semen smears stained with acridine orange were read on a fluorescence microscope; sperm heads appeared either green (fertile) or red (nonfertile). The concept of an "effective sperm count" which is obtained by multiplying the percentage of green-fluorescing sperm by the actual sperm count was introduced. Of the fertile subjects, 27 of 28 (96.4%) exhibited an effective sperm count of greater than or equal to 50 million/ml, while 60 of 61 (98.3%) infertile patients fell below this value. The percent green correlates with neither actual sperm count nor motility, indicating that this test measures a new parameter of male fertility. 4. In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells
Tolga ava (2008). Food and Chemical Toxicology 46(1): 352-358

The genotoxic effects of mercury chloride and lead acetate were evaluated in vivo using the micronucleus (MN) assay on acridine-orange (AO) stained peripheral blood erythrocytes, gill

10

and fin epithelial cells of Carassius auratus auratus. Fish were exposed to three different concentrations of mercury chloride (MC) (1 g/, 5 g/L and 10 g/L) and lead acetate (LA) (10 g/L, 50 g/L and 100 g/L) for 2, 4 and 6 days. A single dose of 5 mg/L cyclophosphamide was used as a positive control. In addition to micronuclei, nuclear buds (NBs) were assessed in the erythrocytes. The ratio of polychromatic and normochromatic erythrocytes (PCE/NCE) in peripheral blood was also evaluated to assess cytotoxicity. MN frequencies in all three tissues were elevated in fish exposed to both LA and MC. However, NBs showed different sensitivity to metal treatments. MN frequencies in both control and treated fish were highest in gill cells and generally lower in erythrocytes and fin cells. PCE/NCE rations decreased in relation to MC and LA treatments. The results of this study indicate that LA and MC have genotoxic and cytotoxic damage in fish and confirmed that AO staining is a suitable technique for in vivo MN test in fish. 5. Photodynamic therapy with acridine orange in musculoskeletal sarcomas
T Matsubara, K Kusuzaki, A Matsumine (2010). Journal of Bone and Joint Surgery 92-B(6): 760-762

Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. Alternative treatments for musculoskeletal sarcoma has been studied since 1990, and a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells has been recently established. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

DAPI
The blue-fluorescent DAPI or 4',6-diamidino-2-phenylindole nucleic acid stain preferentially stains dsDNA. It appears to associate with AT clusters in the minor groove. Binding of DAPI to dsDNA produces a ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. DAPI can pass through an intact cell membrane therefore it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower. DAPI also binds RNA, however in a different binding modeone thought to involve AU-selective intercalation. The DAPI/RNA complex exhibits a longer-wavelength fluorescence emission maximum than the DAPI/dsDNA complex (~500 nm versus ~460 nm) and a quantum yield that is only about 20% as high.

11

Figure 5. DAPI molecule

When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore for fluorescence microscopy DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA. APPLICATIONS 1. Applications of DAPI cytochemistry to neurobiology.
Sanna PP, Jirikowski GF, Lewandowski GA, Bloom FE (1992). Biotech Histochem. 67(6):346-50.

DAPI can be employed as a fluorescent chromatin counterstain on sections immunofluorescent-stained using rhodamine and on tissues enzymatically stained using betagalactosidase. DAPI also allows easy identification of mitotic figures and can be used to supplement cytochemical studies involving cell division in the nervous system.

2. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

TN Siegel, DR Hekstra (2008). Molecular and Biochemical Parasitology 160(2): 171-174

The use of quantitative DAPI imaging was reported to determine the cell-cycle stage of individual procyclic cells. Using this approach, it was found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. This finding was confirmed by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.

3. Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using DAPI

Anna N. Kulakova, Darragh Hobbs (2011). Environ. Sci. Technol. 45 (18): 77997803

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in

12

microbial cultures and environmental samples. A direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification has been reported. This increased the efficiency of polyP detection by 2855% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling. 4. FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene
Konrad Ocalewicz, Jose C. Mota-Velasco, Rafael Campos-Ramos and David J. Penman (2009). Chromosome Research 17(6): 773-782

Bivalent 1 of the synaptonemal complex (SC) in XY male Oreochromis niloticus shows an unpaired terminal region in early pachytene. This appears to be related to recombination suppression around a sex determination locus. To allow more detailed analysis of this, and unpaired regions in the karyotype of other Oreochromis species, techniques were developed for FISH on SC preparations, combined with DAPI staining. DAPI staining identified presumptive centromeres in SC bivalents, which appeared to correspond to the positions observed in the mitotic karyotype (the kinetochores could be identified only sporadically in silver-stained EM SC images). Furthermore, two BAC clones containing Dmo (dmrt4) and OniY227 markers that hybridize to known positions in chromosome pair 1 in mitotic spreads (near the centromere, Flpter 0.25, and the putative sex-determination locus, Flpter 0.57, respectively) were used as FISH probes on SCs to verify that the presumptive centromere identified by DAPI staining was located in the expected position. Visualization of both the centromere and FISH signals on bivalent 1 allowed the unpaired region to be positioned at Flpter 0.80 to 1.00, demonstrating that the unpaired region is located in the distal part of the long arm(s). 5. Chromosomal Variations Within Aneuploid Cancer Lines
Takahiro Isaka, Andrea L. Nestor, Tadahiro Takada and David C. Allison (2003), J Histochem Cytochem 51(10): 1343-1353

Aneuploid cancers exhibit a wide spectrum of clinical aggressiveness, possibly because of varying chromosome compositions. To test this, karyotypes from the diploid CCD-34Lu fibroblast and the aneuploid A549 and SUIT-2 cancer lines underwent fluorescence in situ hybridization (FISH) and DAPI counterstaining. The number of DAPI-stained and FISHidentified chromosomes, 122, X,Y, as well as structural abnormalities, were counted and compared using the 2, MannWhitney rank sum test and the Levene' equality of variance. Virtually all of the evaluable diploid CCD-34Lu karyotypes had 46 chromosomes with two normal-appearing homologues. The aneuploid chromosome numbers per karyotype were highly variable, averaging 62 and 72 for the A549 and SUIT-2 lines, respectively. However, the A549 chromosome numbers were more narrowly distributed than the SUIT-2 karyotype chromosome numbers. Furthermore, 25% of the A549 chromosomes had structural

13

abnormalities compared to only 7% of the SUIT-2 chromosomes. The chromosomal compositions of the aneuploid A549 and SUIT-2 cancer lines are widely divergent, suggesting that diverse genetic alterations, rather than chance, may govern the chromosome makeups of aneuploid cancers.

Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.

Figure 6.Fluorescein molecule

Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and emission maximum of 521 nm (in water). The major derivatives are fluorescein isothiocyanate (FITC) and, in oligonucleotide synthesis, 6-FAM phosphoramidite. Another derivative is a cell-permeable fluorogenic probe 2, 7-Dichlorodihydrofluorescin diacetate (DCFH-DA). Fluorescein has a pKa of 6.4, and its ionization equilibrium leads to pHdependent absorption and emission over the range of 5 to 9. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns, which allows for pH determination from nonintensity based measurements. APPLICATIONS 1. Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish.
Noga E. J., Udomkusonsri, P. (2002). Vet Pathol 39 (6): 726731

Exposure of fish to as little as 0.10 mg fluorescein per milliliter of water for 3 minutes was sufficient to identify experimentally induced lesions, even pinpoint ulcerations. Such lesions were not visible to the naked eye but were clearly demarcated with fluorescein treatment. Examination of fish that appeared clinically normal often revealed the presence of focal ulcerations, which might have been a consequence of damage during capture, but it also might suggest that skin ulceration may be common even in clinically normal fish. Exposure of either nonulcerated or experimentally ulcerated hybrid striped bass to an excessively high

14

concentration of fluorescein had no apparent effect on health or survival. The study suggest that fluorescein may be a highly useful tool for rapid health screening in fish populations. 2. A novel fluorescein derivative as a colorimetric chemosensor for detecting copper(II) ion.
Tianrong Li, Zhengyin Yang, Yong Li, Zengchen Liu, Gaofei Qi, Baodui Wang (2011). Dyes and Pigments 88 (1): 103108

A novel fluorescein derivative, synthesized by the reaction of fluorescein hydrazide and 1phenyl-3-methyl-4-benzoyl-5-pyrazolone, was evaluated as a chemoselective metal ion sensor. Addition of Cu2+ to an aqueous solution of the fluorescein derivative resulted in a rapid color change from colorless to deep yellow together with a distinctive change in UV vis absorption spectrum. However, other common alkali-, alkaline earth-, transition- and rare earth metal ions induced no or minimal spectral changes. The stoichiometry of the reaction and association constant of the fluorescein derivative with Cu2+ are described. Experimental results indicate that the fluorescein derivative could provide a rapid, selective and sensitive response to Cu2+, and could be used as a potential Cu2+ colorimetric chemosensor in aqueous solution. 3. Use of Fluoroscein-EDTA System in Photogalvanic Cell for Solar Energy Conversion
S. Madhwani, R. Ameta, J. Vardia, P. B. Punjabi & V. K. Sharma (2007). Energy Sources 29(8): 721-729

Fluoroscein has been used as a photosensitizer in photogalvanic cell for solar energy conversion. EDTA was used as an electron donor. The photopotential and photocurrent generated by this cell were 418 mV and 42 A, respectively. The effect of various parameters like pH, light intensity, diffusion length, reductant concentration, dye concentration, etc. on the electrical output of the cell has been studied. The current voltage (iV) characteristics of the cell has also been observed and a tentative mechanism for the generation of photocurrent has been proposed. Performance of the cell was determined in dark at its power point. 4. A simple spectrophotometric streptavidinbiotin binding assay utilizing biotin-4fluorescein
Mark J. Waner , David P. Mascotti (2008). Journal of Biochemical and Biophysical Methods70(6): 873-877

A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the sensitivity is not substantially lower than other comparable techniques, this assay allows one added flexibility in working range and instrumentation, since the same stock solutions may be used for this new photometric assay or the fluorescence assay for which this ligand was first developed.

15

5. One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells
Ying Zhang, Zhongfu Wanga, Xiaorui Zhang, Wenxia Zhou, Linjuan Huang (2011), Carbohydrate Research 346(14):2156-64

A simple and efficient procedure for the fluorescent labeling of saccharides is a prerequisite step for imaging the transport of polysaccharides in living cells. A one-pot strategy for the fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide (FTSC) had been reported, which introduces the thiosemicarbazide group of FTSC to the aldehyde group at the reducing end of saccharides to form stable amino derivatives via reductive amination. The Glc-FTSC derivative was characterized by HPLC-MS, HRESIMS and NMR spectroscopy. Saccharides were quantitatively labeled with FTSC at 75C for 1 h under optimum reaction conditions. Fluorescence studies illustrated that the conjugation of FTSC to saccharides did not change its florescence properties ((ex)=495 nm, (em)=517 nm), presenting desirable compatibility with commonly used fluorescence equipment. Polysaccharide AAG-FTSC derivatives exhibited rather low levels of cytotoxicity against rat thymus cells, and the fluorescent labeling procedure had slight impact on their anti-tumor activity. Results indicated that the assay neither introduces discernible cytotoxicity against living cells nor obviously alters the functional activities of polysaccharides, and provides a convenient, highly efficient fluorescent labeling approach for imaging the transport of polysaccharides in living cells.

Cyanines
Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging. Depending on the structure, they cover the spectrum from IR to UV.

Figure 7. Generic Structure for Cyanine Dye (n = 1-3, R = alkyl groups or a specific labelling site)

16

Cyanine dye chromophore is, in fact, a sulfoindocyanine. These sulphonated cyanine dyes have good solubility in biological media at physiological pH. Another important feature of these compounds is the site for biological attachment. N-Hydroxy-succinimidyl esters are most commonly employed for labelling amino groups present in antibodies, lipids, drugs, cell membranes and oligonucleotides, and in most cases the dyes exhibit low non-specific binding. There are also numerous examples whereby other groups have been employed for more specialised labelling, such as maleimides and iodoacetamides6 for labelling cysteine residues. Cy3 and Cy5 are reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are red (~550 nm excitation, ~570 nm emission and therefore appear red), while Cy5 is fluorescent in the far red region (~650/670 nm) but absorbs in the orange region (~649 nm). They are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically linked to either nucleic acids or protein molecules. Labeling is done for visualization and quantification purposes. They are used in a wide variety of biological applications including comparative genomic hybridizationand in gene chips, which are used in transcriptomics. They are also used to label proteins and nucleic acid for various studies including proteomics and RNA localization

APPLICATIONS 1. Detection of Oligosaccharides Labeled with Cyanine Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
A Kameyama, Y Kaneda, H Yamanaka (2004). Anal. Chem. 76 (15): 45374542

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. Its described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard Nglycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5dihydroxybenzoic acid and -cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.

17

2. Cyanine dyes and their applications as luminescence quenching compounds

Zhenjun Diwu, Sunnyvale,J ianheng Zhang, Santa Clara. US Patent 7,910,753, 2011

The quenching compounds of the invention are weakly luminescent cyanines that are substituted by one or more heteroaromatic quenching moieties. The quenching compounds of the invention exhibit little or no observable luminescence and efficiently quench a broad spectrum of luminescent compounds. The chemically reactive quenching compounds possess utility for labeling a wide variety of substances, including biomolecules. These labeled substances are highly useful for a variety of energy-transfer assays and applications. 3. Cyanine dyes as Labelling Reagents for Detection of Biological and other Materials by Luminescence Methods.
Waggoner Alan. US Patent No. 5627027

Cyanine and related dyes, are strongly light absorbing and highly luminescent. These dyes are covalently attached to proteins and other biological and nonbiological materials to make these materials fluorescent so that they can be detected. The labeled materials can then be used in assays employing excitation light sources and luminescence detectors. Avidin labeled with cyanine type dyes can be used to quantify biotinylated materials and antibodies conjugated with cyanine-type dyes can be used to detect and measure antigens and haptens. In addition, cyanine-conjugated lectins can be used to detect specific carbohydrate groups. Also, cyanine-conjugated fragments of DNA or RNA can be used to identify the presence of complementary nucleotide sequences in DNA or RNA. The cyanine dyes have the advantage that by synthesizing structural modifications of the chromophore portion the molecule, different fluorescent labeling reagents can be made that will absorb and fluoresce light at many different wavelengths in the visible and near infrared region of the spectrum. Also, the cyanine and related dyes have an advantage in their structural versatility. That is, they can be synthesized in many structural forms and with a variety of functional groups attached. This versatility permits control over such factors as the solubility of the dye and labeled product and helps reduce nonspecific binding of the labeled material to irrelevant components in the assay mixture. This versatility also allows for selection of labeling reagents that minimally perturb the function of the labeled product. 4. Application of near-infrared absorbing heptamethine cyanine dyes as sensitizers for zinc oxide solar cell.
Masaki Matsui, Yoshimi Hashimoto, Kazumasa Funabiki, Ji-Ye Jin, Tsukasa Yoshida, Hideki Minoura (2005). Synthetic Metals 148 (2): 147-153

Novel near-infrared absorbing heptamethine cyanine dyes derived from indole, benzoxazole, benzothiazole, and quinoline were synthesized and examined as sensitizers for a zinc oxide solar cell. A 2-carboxyphenylthio-substituted indolium heptamethine cyanine dye showed incident photon-to-current efficiency (IPCE) of 4.17% at 804 nm.

18

Rhodamine
Rhodamine dyes are used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA. The laser dye rhodamine 123 is also used in biochemistry to inhibit mitochondrion function, especially the electron transport chain, thus slowing down inner respiration. It is a substrate of Pglycoprotein (Pgp), which is usually overexpressed in cancer cells. Rhodamine B has its maximum excitation wavelength at 540 nm and emission maxima occurs at 625 nm. There are many rhodamine derivatives used for imaging purposes, for example Carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC) and, sulforhodamine 101 (and its sulfonyl chloride form Texas Red) and Rhodamine Red. Other derivatives of rhodamine include newer fluorophores such as Alexa 546, Alexa 555, Alexa 633, DyLight 549 and DyLight 633, have been tailored for various chemical and biological applications where higher photostability, increased brightness, different spectral characteristics, or different attachment groups are needed. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with instruments called fluorometers.

Figure 8.Rhodamine

Figure 9.Rhodamine B molecule

APPLICATIONS 1. Localization of mitochondria in living cells with rhodamine 123

L V Johnson, M L Walsh, and L B Chen (1980). PNAS 77 ( 2): 990-994

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of

19

rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. 2. Rhodamine immunohistofluorescence applied to plant tissue
S J Hapner and K D Hapner (1978) J Histochem Cytochem 26 (6): 478-482

Rhodamine is used as a coupled immunohistochemical reagent for studying plant tissues by the localization of plant lectin in root tissue. Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence. 3. A RhodamineHydroxamic Acid-Based Fluorescent Probe for Hypochlorous Acid and Its Applications to Biological Imagings
Young-Keun Yang, Hyungseoph Jason Cho, Jihyun Lee, Injae Shin and Jinsung Tae (2009). Org. Lett. 11 (4): 859861

A new rhodaminehydroxamic acid-based fluorescent chemosensor for the rapid detection of HOCl in aqueous media was developed. The system, which utilizes an irreversible HOClpromoted oxidation reaction, responds instantaneously at room temperature with linear proportionality to the amount of HOCl. This system is highly selective for HOCl over other reactive oxygen species (ROS) and highly sensitive in aqueous solutions. Biological imaging studies using living cells and organisms (A549 cells and zebrafish) to detect HOCl are successfully demonstrated.

20

Novel Application of Fluorophore

Comparative analysis of antioxidant content in various aromatic, medicinal and spice plants and measuring the effect of antioxidant therapies using cell-permeable fluorogenic probe 2, 7-Dichlorodihydrofluorescin diacetate (DCFH-DA). The plants are susceptible to damage caused by active oxygen and thus develop numerous antioxidant defence systems resulting in formation of numerous potent antioxidants. Many aromatic, medicinal and spice plants contain compounds that possess confirmed strong anti oxidative components. The recent researches have also proved that these can also be used as antioxidants in order to protect our body from various disastrous and chronic diseases, like cancer, arthritis, common cold cough, cataracts etc. which weaken the immune system of the body.

Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. In humans, oxidative stress is involved in many diseases. Examples include Sickle Cell Disease, atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, Schizophrenia, Bipolar disorder, fragile X syndrome and chronic fatigue syndrome.

21

1. Various aromatic and medicinal plants like Ashwagandha (Withania somnifera), Black Pepper (Piper nigrum), Garlic (Allium sativum), Ginger (Zingiber officinalis), Green Tea (Camalia bensgalensis), Turmeric (Curcuma longa) etc. is evaluated for the content of antioxidants by various tests like: GSH Assay (Kelly BM et al. 1963, Owens CWI et al. 1965), CATALASE assay (Aebi H et al. 1984), GST Assay (Habig WH et al. 1974), SOD Assay etc. 2. A cell-based assay for measuring reactive oxygen species activity within a cell is performed. The assay (DCF assay) employs the cell-permeable fluorogenic probe 2, 7-Dichlorodihydrofluorescin diacetate (DCFH-DA). In brief, DCFH-DA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent 2, 7Dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2, 7-Dichlorodihydrofluorescein (DCF) by ROS. The fluorescence intensity is proportional to the ROS levels within the cell cytosol. The effect of antioxidant or free radical compounds on DCFDA can be measured against the fluorescence of the provided DCF standard. 3. The ROS levels can be checked again using DCF assay after the cells are exposed to test products or extracts from various medicinal and aromatic plants mentioned above after making them bio-available. The cells are allowed time to absorb compounds from the test product. 4. In this way, the difference in fluorescence in absence and presence of antioxidants measures the effect of antioxidant therapies and ROS activity. This is crucial for suppressing or treating oxidative stress inducers.

Mechanism of DCF Assay

22

References
1. Roger Y. Tsien (1998). The Green Fluorescent Protein. Annu. Rev. Biochem. 67:50944. 2. The green fluorescent protein: discovery, expression and development. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/chemadv08.pdf 3. George H. Patterson and Jennifer Lippincott-Schwartz (2002). A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 297(5588): 1873-1877 4. Takashi Aoki, Shirou Tsuchida, Taemi Yahara, Naoya Hamaue (2008). Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk. Analytical Biochemistry 378 (2): 132-137 5. Wan-ling Chiu, Yasuo Niwa, Weike Zeng, Takanori Hirano, Hirokazu Kobayashi, Jen Sheen (1996). Engineered GFP as a vital reporter in plants. Current Biology 6 (3): 325-330. 6. Hiroyuki Kishimoto, Ming Zhao, Katsuhiro Hayashi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara, Sheldon Penman and Robert M. Hoffman (2009). In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation. PNAS 106 (34): 14514-14517. 7. Katrin I Willig, Robert R Kellner, Rebecca Medda, Birka Hein, Stefan Jakobs & Stefan W Hell (2006). Nanoscale resolution in GFP-based microscopy. Nature Methods 3(9): 721-723. 8. Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel, and Douglas R. Green (2006). Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. Cold Spring Harb Protoc 9. J. Keiser, J. Utzinger, Z. Premji, Y. Yamagata and B. H. Singer (2002). Acridine Orange for malaria diagnosis: its diagnostic performance, its promotion and implementation in Tanzania, and the implications for malaria control. Annals of Tropical Medicine & Parasitology 96 (7): 643654. 10. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S(1984). A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steri. 42(1):87-91. 11. Tolga ava (2008). In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells. Food and Chemical Toxicology 46(1): 352-358. 12. T Matsubara, K Kusuzaki, A Matsumine (2010). Photodynamic therapy with acridine orange in musculoskeletal sarcomas. Journal of Bone and Joint Surgery 92-B(6): 760-762. 13. Kapuscinski J (1995). "DAPI: a DNA-specific fluorescent probe". Biotech Histochem 70 (5): 22033. 14. Sanna PP, Jirikowski GF, Lewandowski GA, Bloom FE (1992). Applications of DAPI cytochemistry to neurobiology. Biotech Histochem. 67(6):346-50. 15. TN Siegel, DR Hekstra (2008). Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging. Molecular and Biochemical Parasitology 160(2): 171-174. 16. Anna N. Kulakova, Darragh Hobbs (2011). Direct Quantification of Inorganic Polyphosphate in Microbial Cells Using DAPI. Environ. Sci. Technol. 45 (18): 77997803. 17. Konrad Ocalewicz, Jose C. Mota-Velasco, Rafael Campos-Ramos and David J. Penman (2009). FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene. Chromosome Research 17(6): 773-782.

23

18. Takahiro Isaka, Andrea L. Nestor, Tadahiro Takada and David C. Allison (2003). Chromosomal Variations Within Aneuploid Cancer Lines. J Histochem Cytochem 51(10): 1343-1353. 19. Noga E. J., Udomkusonsri, P. (2002). "Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish". Vet Pathol 39 (6): 726731 20. Tianrong Li, Zhengyin Yang, Yong Li, Zengchen Liu, Gaofei Qi, Baodui Wang (2011). A novel fluorescein derivative as a colorimetric chemosensor for detecting copper(II) ion. Dyes and Pigments 88 (1): 103-108 21. S. Madhwani, R. Ameta, J. Vardia, P. B. Punjabi & V. K. Sharma (2007). Use of FluorosceinEDTA System in Photogalvanic Cell for Solar Energy Conversion. Energy Sources 29(8): 721729. 22. Mark J. Waner , David P. Mascotti (2008). A simple spectrophotometric streptavidinbiotin binding assay utilizing biotin-4-fluorescein. Journal of Biochemical and Biophysical Methods70(6): 873-877. 23. Ying Zhang, Zhongfu Wanga, Xiaorui Zhang, Wenxia Zhou, Linjuan Huang (2011). One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells. Carbohydrate Research 346(14):2156-64. 24. Michael E. Cooper. Fluorescent Probes for Biological Applications: Cyanine Dyes Revisited. Amersham Biosciences Ltd, R & D, Cardiff, UK. 25. A Kameyama, Y Kaneda, H Yamanaka (2004). Detection of Oligosaccharides Labeled with Cyanine Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Chem. 76 (15): 45374542. 26. Zhenjun Diwu, Sunnyvale,J ianheng Zhang, Santa Clara (2011). Cyanine dyes and their applications as luminescence quenching compounds. US Patent 7,910,753 27. Waggoner Alan. Cyanine dyes as Labelling Reagents for Detection of Biological and other Materials by Luminescence Methods. US Patent No. 5627027 28. Masaki Matsui, Yoshimi Hashimoto, Kazumasa Funabiki, Ji-Ye Jin, Tsukasa Yoshida, Hideki Minoura (2005). Application of near-infrared absorbing heptamethine cyanine dyes as sensitizers for zinc oxide solar cell. Synthetic Metals 148 (2): 147-153 29. L V Johnson, M L Walsh, and L B Chen (1980). Localization of mitochondria in living cells with rhodamine 123. PNAS 77 ( 2): 990-994. 30. S J Hapner and K D Hapner (1978). Rhodamine immunohistofluorescence applied to plant tissue. J Histochem Cytochem 26 (6): 478-482. 31. Young-Keun Yang, Hyungseoph Jason Cho, Jihyun Lee, Injae Shin and Jinsung Tae (2009). A RhodamineHydroxamic Acid-Based Fluorescent Probe for Hypochlorous Acid and Its Applications to Biological Imagings. Org. Lett. 11 (4): 859861.