Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1 , based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe— P2 , based on a SNAP-tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.     

Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies

Visualizing and manipulating the behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labeling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labeling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small-molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, current technical advances in the HaloTag and SNAP-tag systems means that they are becoming powerful tools to enable the visualization and manipulation of biological processes, providing invaluable scientific insights that are difficult to obtain by traditional methodologies. As the multiplex of self-labeling protein tag systems continues to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.     

Synergistically integrated nanoparticles as multimodal probes for nanobiotechnology

Current biomedical imaging techniques including magnetic resonance imaging (MRI), positron emission tomography (PET), and computed X-ray tomography (CT) are vital in the diagnosis of various diseases. Each imaging modality has its own merits and disadvantages, and a single technique does not possess all the required capabilities for comprehensive imaging. Therefore, multimodal imaging methods are quickly becoming important tools for state-of-the-art biomedical research and clinical diagnostics and therapeutics. In this Account, we will discuss synergistically integrated nanoparticle probes, which will be an essential tool in multimodal imaging technology. When inorganic nanoparticles are introduced into biological systems, their extremely small size and their exceptional physical and chemical properties make them useful probes for biological diagnostics. Nanoparticle probes can endow imaging techniques with enhanced signal sensitivity, better spatial resolution, and the ability to relay information about biological systems at the molecular and cellular levels. Simple magnetic nanoparticles function as MRI contrast enhancement probes. These magnetic nanoparticles can then serve as a core platform for the addition of other functional moieties including fluorescence tags, radionuclides, and other biomolecules for multimodal imaging, gene delivery, and cellular trafficking. For example, MRI-optical dual-modal probes composed of a fluorescent dye-doped silica (DySiO(2)) core surrounded by magnetic nanoparticles can macroscopically detect neuroblastoma cancer cells via MRI along with subcellular information via fluorescence imaging. Magnetic nanoparticles can also be coupled to radionuclides ((124)I) to construct MRI-PET dual-modal probes. Such probes can accurately detect lymph nodes (LNs), which are critical for assessing cancer metastasis. In vivo MRI/PET images can clearly identify small (approximately 3 mm) LNs along with precise anatomical information. Systems using multicomponent nanoparticles modified with biomolecules can also monitor gene expression and other markers in cell therapeutics studies. We have used hybrid stem cell-magnetic nanoparticle probes with MRI to monitor in vivo stem cell trafficking. MRI with hybrid probes of magnetic nanoparticles and adenovirus can detect target cells and can monitor gene delivery and the expression of green fluorescent proteins optically. Each component of such multimodal probes complements the other modalities, and their synergistic materials properties ultimately provide more accurate information in in vitro and in vivo biological systems.     

Bioconjugation of luminescent silicon quantum dots to gadolinium ions for bioimaging applications

Luminescent imaging agents and MRI contrast agents are desirable components in the rational design of multifunctional nanoconstructs for biological imaging applications. Luminescent biocompatible silicon quantum dots (SiQDs) and gadolinium chelates can be applied for fluorescence microscopy and MRI, respectively. Here, we report the first synthesis of a nanocomplex incorporating SiQDs and gadolinium ions (Gd(3+)) for biological applications. The nanoconstruct is composed of a PEGylated micelle, with hydrophobic SiQDs in its core, covalently bound to DOTA-chelated Gd(3+). Dynamic light scattering reveals a radius of 85 nm for these nanoconstructs, which is consistent with the electron microscopy results depicting radii ranging from 25 to 60 nm. Cellular uptake of the probes verified that they maintain their optical properties within the intracellular environment. The magnetic resonance relaxivity of the nanoconstruct was 2.4 mM(-1) s(-1) (in terms of Gd(3+) concentration), calculated to be around 6000 mM(-1) s(-1) per nanoconstruct. These desirable optical and relaxivity properties of the newly developed probe open the door for use of SiQDs in future multimodal applications such as tumour imaging.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.     

Importance of DOTA derivatives in bimodal imaging

The clinical applications of multimodal probes are numerous since a few decades. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has played an important role in diagnostic and therapeutic areas. The vast applications of DOTA as chelator have been explored in magnetic resonance imaging (MRI) and in radioisotope chemistry. Moreover, the possibility to functionalize the macrocycle with pendant arms has allowed to explore new functionalities as bimodal imaging agents. Different combinations are possible between the different possible imaging techniques like Magnetic Resonance Imaging, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Optical imaging (OI). The main use of DOTA and its derivatives was for MRI as gadolinium complexes. It was then further extended to the complexation with europium or terbium for optical imaging. Although other chelates are available such as DTPA or NOTA, derivatives of DOTA were often the primary choice due to their versatility. DOTA derivatives can indeed also be complexed with radioisotopes and conjugated to peptides which leads to targeted contrast agents for PET or SPECT. Depending on the chosen imaging modality, a variety of radiometals can be complexed with DOTA, e.i. ⁶⁴Cu and ⁶⁸Ga for PET, or ¹¹¹In and ⁹⁰Y for SPECT. Conjugation of chromophores to gadolinium complexes of DOTA derivatives can also lead to bimodal agents for MRI and OI. In this review, we will provide the applications of DOTA and its derivatives in different imaging modalities and their clinical applications.     

Small BODIPY Probes for Combined Dual 19F MRI and Fluorescence Imaging

The combination of the two complementary imaging modalities ¹⁹F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual ¹⁹F MRI–fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T₁ and the transverse relaxation times T₂, both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual ¹⁹F MRI–fluorescence agent was also successfully imaged in a mouse post‐mortem within a 9.4 T small‐animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing.     

Facile synthesis of gadolinium (III) chelates functionalized carbon quantum dots for fluorescence and magnetic resonance dual-modal bioimaging

Multi-modal imaging probes through synergistically combining two or more imaging modalities into single nanoparticle, offer possibilities to address multiple issues such as resolution, sensitivity, and tissue penetration. In this study, we report a facile strategy to synthesize a dual-modal fluorescence/magnetic resonance (MR) imaging nanoprobe with bright fluorescence and significantly improved relaxivity, where Gd³⁺ ions are simply chelated onto the outer surface of the cyclic DTPA dianhydride (cDTPAA) functionalized carbon quantum dots (CQDs). Transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and zeta potential measurements validated the successful preparation of CQD–DTPA–Gd. The surface conjugation of CQDs with cDTPAA and further Gd³⁺ chelation does not perturb core optical properties of CQDs, and the thus prepared CQD–DTPA–Gd contain minimum Gd³⁺ content (8.06%, w/w), while exhibiting significantly improved longitudinal relaxivity (56.72 mM⁻¹ s⁻¹). Excellent water solubility, good cell-membrane permeability and negligible cytotoxicity make CQD–DTPA–Gd an ideal dual-modal fluorescence/MR imaging probe, suggesting its potential and significance in practical bioimaging applications and medical translation in the future.     

Tagging Live Cells that Express Specific Peptidase Activity with Solid‐State Fluorescence

A three‐component probe harnesses the extraordinary properties of a solid‐state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off–on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal‐to‐background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long‐term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live‐cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high‐fidelity flow cytometry and sensitive colony assays.     

Fast-Response Fluorogenic Probe for Visualizing Hypochlorite in Living Cells and in Zebrafish

A fast-response fluorogenic probe—compound D1 —for monitoring hypochlorite (ClO⁻), based on specific ClO⁻ cleavage of a C=N bond and producing results observable to the naked eye, has been developed. The response of the probe to ClO⁻ increases linearly, and the fluorescence intensity was heightened by a factor of about 25. D1 responses to ClO⁻, with high selectivity and sensitivity, were observable by naked eye within 10 s. D1 can not only detect levels of hypochlorite in vitro, such as in urine, but is also capable of monitoring hypochlorite content under extremely cold conditions, as low as −78 °C. Meanwhile, its good biocompatibility permitted the use of D1 to detect intracellular ClO⁻ by confocal microscopy. Moreover, D1 was successfully applied to monitor exogenous and endogenous ClO⁻ in zebrafish through fluorescence imaging.     

Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications

Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation, and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted through these mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell-specific activatable probes have considerable flexibility and can be adapted to specific diagnostic needs. A multitude of cell surface molecules, such as overexpressed growth factor receptors, are directly related to carcinogenesis and thus provide numerous targets highly specific for cancer. This discussion of the chemical, pharmacological, and biological basis of target-cell-specific activatable imaging probes, and methods for successfully designing them, underscores the systematic, rational basis for further developing in vivo cancer imaging.     

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.     

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.     

Amplified Expansion Stimulated Emission Depletion Microscopy

Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of ≈4 in one dimension and a factor of ≈64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. However, photobleaching is a critical issue in ExSTED because the sample expansion lowers the fluorophore density whereas high-resolution STED requires high depletion intensity. To overcome these issues, we developed extremely bright expansion nanoscopy by using biotin–avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GW cm⁻². We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.     

Design,Synthesis and In Vivo Fluorescence Imaging Study of a Cytochrome P450 1B1 Targeted NIR Probe Containing a Chelator Moiety

Cytochrome P450 (CYP) 1B1 has been found to be overexpressed specifically in tumor tissues at an early stage, which makes it a potential cancer biomarker for molecular imaging. Multimodal imaging combines different imaging modalities and offers more comprehensive information. Thus, imaging probes bearing more than one kind of signal fragment have been extensively explored and display great promise. Herein, we developed a near infrared (NIR) probe with a chelator moiety targeting CYP1B1 by conjugating α-naphthoflavone (ANF) derivatives with both an NIR dye and a chelator for potential application in bimodal imaging. Enzymatic inhibitory studies demonstrated inhibitory activity against CYP1B1 and selectivity among CYP1 were successfully retained after chemical modification. Cell-based saturation studies indicated nanomolar range binding affinity between the probe and CYP1B1 overexpressed cancer cells. In vitro competitive binding assays monitored by confocal microscopy revealed that the probe could specifically accumulate in tumor cells. In vivo and ex vivo imaging studies demonstrated that the probe could effectively light-up the tumor tissues as early as 2 hours post-injection. In addition, the fluorescence was significantly blocked by co-injection of CYP1B1 inhibitor, which indicated the probe accumulation in tumor sites was due to specific binding to CYP1B1.     

Chloro‐Functionalized Photo‐crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2‐Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH‐labeled probe (pcBD2–GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S‐transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH–GST complex in live cells. Interactions between probe and GST were confirmed by means of photo‐crosslinking under intact live‐cell conditions. Interestingly, isomers of chloro‐functionalized 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co‐staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide‐mediated reactive oxygen species induction experiments showed that pcBD2–GSH accumulated in mitochondria. This is the first example of a live‐cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.     

HER2‐ and EGFR‐Specific Affiprobes: Novel Recombinant Optical Probes for Cell Imaging

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell‐surface receptors/tyrosine kinases. EGFR‐ and HER2‐positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR‐ or HER2‐negative cancers. The details of HER2 proto‐oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009 , 315, 683–696). We have created photostable and relatively simple‐to‐produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2‐ or EGFR‐specific Affibody® molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell‐surface antigens/ receptors.     

A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore ( 1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.     

COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15–30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)—synthetic DNA analogues with a neutral backbone—which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488^® showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10–20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.