Fluorescence imaging developed as a unique visualization technique for the non-invasive study of the distribution and kinetics of labelled molecules, e.g. drug candidates, as well as for the expression, localization, and activity of drug targets, e.g. specific enzymes. As further benefit, fluorometric assays avoid the typical problems of radiolabeling and are easy to handle.

Typically, fluorogenic dyes show a low emission, but a change in the surrounding conditions or enzymatic activity inside the target cell leads to an increase in brightness. This can either occur after binding of the fluorophore to the target or after intracellular modification of a chemical moiety. For the latter one, typically, the fluorophore is covalently bound to a distinct enzyme substrate through an amide or ester bond. Upon enzymatic cleavage, the free amino or hydroxyl compound is liberated as active fluorophore, which leads to an increase in fluorescence and a red shift of the absorbance maximum.

In the following, we are summarizing information on different fluorophores available at Iris Biotech:

DAPI (LS-3590) is a popular nuclear counterstain for use in multicolor fluorescent techniques, as it binds strongly to A-T rich regions in DNA. It can be used to stain both living and fixed cells without side effects. Its blue fluorescence stands out in vivid contrast to green, yellow, or red fluorescent probes of other structures. λ_Exλ_Em (DNA-bound) = 358/461 nm.

Indocyanine Green (ICG) dye, a material approved by the FDA for various applications, is a powerful tool for imaging in live cells and tissues. ICG exhibits an absorption maximum in the near infrared region (NIR) at ca. 800 nm with a slight absorption in the visible range, which results in low auto-fluorescence. The emission maximum is at 810 nm. This absorption/emission profile allows for tissue-penetrating excitation without causing tissue damage. Consequently, ICG has found use in fields as diverse as angiography, detection of solid tumors and fluorescence image-guided surgery. Iris Biotech offers variously functionalized derivatives (see related products).

One fluorophore combining high photostability and fluorescence yield with a large Stokes shift is 7-amino-4-methylcoumarin (AMC), which can be used for the preparation of fluorogenic 7-amido-4-methylcoumarin based substrates for the detection of proteolytic enzyme activity. When bound to a peptide, the AMC-amide fluoresces very weakly and excitation/emission wavelengths are shorter (ca. 330/390 nm). When the free AMC-amine is released by proteolytic cleavage, the fluorescence increases by a factor of approx. 700 and excitation and emission wavelengths are red-shifted. Thus, the low fluorescence of the AMC-amide substrate does not interfere with the fluorometric assay.

Its acetic acid derivative 7-amino-4-methyl coumarin-3-acetic acid (AMCA) emits in the blue region 440-460 nm upon activation with UV light of 350 nm.

Additionally, Iris Biotech offers fluorescence dyes with the chromophore system known from BODIPY. Those derivatives absorb light at 500 nm and emit at 510 nm.

Chemical structures of fluorophores available at Iris Biotech.

References: 

Clinical applications of indocyanine green (ICG) enhanced fluorescence in laparoscopic surgery; L. Boni, G. David, A. Mangano, G. Dionigi, S. Rausei, S. Spampatti, E. Cassinotti, A. Fingerhut; Surg. Endosc. 2015; 29: 2046-2055. https://doi.org/10.1007/s00464-014-3895-x

A Review of Indocyanine Green Fluorescent Imaging in Surgery; J. T. Alander, I. Kaartinen, A. Laakso, T. Pätilä, T. Spillmann, V. V. Tuchin, M. Venermo, P. Välisuo; Int. J. Biomed. Imaging 2012; 2012: 940585. https://doi.org/10.1155/2012/940585

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update; M. V. Marshall, J. C. Rasmussen, I. C. Tan, M. B. Aldrich, K. E. Adams, X. Wang, C. E. Fife, E. A. Maus, L. A. Smith, E. M. Sevick-Muraca; Open Surg. Oncol. J. 2010; 2: 12-25. https://doi.org/10.2174/1876504101002010012

Application of a biodegradable macromolecular contrast agent in dynamic contrast enhanced MRI for assessing the efficacy of indocyanine green enhanced photothermal cancer therapy; Y. Feng, L. Emerson, E.-K. Jeong, D. L. Parker, Z.-R. Lu; JMRI 2009; 30: 401-406. https://doi.org/10.1002/jmri.21838

DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites, F. A. Tanious, J. M. Veal, H. Buczak, L. S. Ratmeyer, W. D. Wilson; Biochemistry 1992; 31(12): 3103-3112. https://doi.org/10.1021/bi00127a010

Synthesis of a New Fluorogenic Substrate for Cystine Aminopeptidase; Y. Kanaoka, T. Takahashi, H. Nakayama, T. Ueno, T. Sekine; Chem. Pharm. Bull. 1982; 30(40): 1485-1487. https://doi.org/10.1248/cpb.30.1485

Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates; M. Zimmerman, B. Ashe, E. C. Yurewicz, G. Patel; Anal. Biochem. 1977; 78(1): 47-51. https://doi.org/10.1016/0003-2697(77)90006-9

Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins; H. Khalfan, R. Abuknesha, M. Rand-Weaver, R. G. Price, D. Robinson; Histochem J. 1986; 18(9): 497-499. https://doi.org/10.1007/BF01675617

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries; J. L. Harris, B. J. Backes, F. Leonetti, S. Mahrus, J. A. Ellman, C. S Craik; Proc. Natl. Acad. Sci. USA. 2000; 97(14): 7754-7759. https://doi.org/10.1073/pnas.140132697

A Simple Fluorescent Labeling Method for Studies of Protein Oxidation, Protein Modification, and Proteolysis; A. M. Pickering, K. J. A. Davies; Free Radic Biol Med. 2012; 52(2): 239-246. https://doi.org/10.1016/j.freeradbiomed.2011.08.018

Fluorescence imaging of drug target proteins using chemical probes; H. Zhu, I. Hamachi; J. Pharm. Anal. 2020; 10: 426-433. https://doi.org/10.1016/j.jpha.2020.05.013

A New Fluorogenic Substrate for Chymotrypsin; M. Zimmerman, E. Yurewicz, G. Patel; Anal. Biochem. 1976; 70: 258-262. https://doi.org/10.1016/S0003-2697(76)80066-8

Determination of Caspase Specificities Using a Peptide Combinatorial Library; N. A. Thornberry, K. T. Chapman, D. W. Nicholson; Method Enzymol 2000; 322: 100-110. https://doi.org/10.1016/S0076-6879(00)22011-9

BODIPY: A Highly Versatile Platform for the Design of Bimodal Imaging Probes; D. Lhenry, M. Larrouy, C. Bernhard, V. Goncalves, O. Raguin, P. Provent, M. Moreau, B. Collin, A. Oudot, J.-M. Vrigneaud, F. Brunotte, C. Goze, F. Denat; Chemistry 2015; 21(37): 13091-9. https://doi.org/10.1002/chem.201501676