Alan S. Waggoner

Fluorogen-activating single-chain antibodies for imaging cell surface proteins

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Microenvironments engineered by inkjet bioprinting spatially direct adult stem cells toward muscle- and bone-like subpopulations

In vivo, growth factors exist both as soluble and as solid‐phase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle‐derived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP‐2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time‐lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP‐2 pattern. This work provides proof‐of‐concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.

Fluorophores for Confocal Microscopy: Photophysics and Photochemistry

Fluorescence is probably the most important optical readout mode in biological confocal microscopy, because it can be so much more sensitive and specific than absorbance or reflectance, and because it works so well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on the availability of suitable fluorophores that can either be tagged onto biological macro-molecules to show their location, or whose optical properties are sensitive to the local environment. Despite the pivotal importance of good fluorophores, little is known about how to rationally design good ones. Whereas the concept of confocal microscopy is only a few decades old and nearly all the optical, electronic and computer components to support it have been developed or redesigned in the last few years, the most popular fluorophores were developed more than a century ago (in the case of fluoresceins or rhodamines) or several billion years ago (in the case of phycobiliproteins). References

Fluorescence Imaging of Tumors In Vivo

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, κ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG2a, κ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Fluorophores for Confocal Microscopy

Fluorescence is probably the most important optical readout mode in biological confocal microscopy, because it can be so much more sensitive and specific than absorbance or reflectance, and because it works so well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on the availability of suitable fluorophores that can either be tagged onto biological macromolecules to show their location, or whose optical properties are sensitive to the local environment. Despite the pivotal importance of good fluorophores, little is known about how to rationally design good ones. Whereas the concept of confocal microscopy is only a few decades old and nearly all the optical, electronic, and computer components to support it have been developed or redesigned in the last few years, the most popular fluorophores were developed more than a century ago (in the case of fluoresceins or rhodamines) or several billion years ago (in the case of phycobiliproteins). Moreover, whereas competition between commercial makers of confocal microscopes stimulates ardent efforts to refine the instrumentation, relatively few companies or academic scientists are interested in improving fluorophores.

Long Term Persistence and Spectral Blue Shifting of Quantum Dots in vivo

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo. We have observed fluorescence of quantum dots injected into Balb/c and nude mice for up to two-years post injection using both whole-body and microscopic fluorescence techniques. Two-photon spectral microscopy was used to verify the existence of quantum dots within two-year tissues, but also revealed a range of significantly blue-shifted emission peaks with increased bandwidths. Systemically administered quantum dots persist and retain fluorescence for up to two-years in vivo, but with significantly blue-shifted emission.

Molecular Mechanism Controlling the Incorporation of Fluorescent Nucleotides Into DNA by PCR

The efficiency and yield of incorporation of fluorescent nucleotides into DNA by polymerase chain reaction (PCR) have been investigated with linear amplification (PCR with single-stranded template and single primer). In the present study, we prepared single-stranded templates with defined sequences and used dUTP attached to the fluorescent label with linkers of different lengths. Incorporation and yield of the modified dUTP were reduced when the sequence demanded that multiple dyes be inserted at adjacent sites. The interactions between the polymerase and cyanine-labeled sites on the extending strand probably terminated the chain extension. Thus, because labeling density was increased, the yield of PCR was reduced. We also found that the interactions between the primer and dye-labeled sites on template disturb primer annealing and lead to a decrease in PCR yield.

Leishmania donovani: Surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown).