Dual-color Fluorescence Research Articles

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

The availability of cost-effective, highly portable and easy to use high resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the tracking of virus particles across virological synapses that form between infected and uninfected T cells. The dynamics of HIV-1 proteins studied at the cellular level and the formation of live virological synapses reveals mechanisms by which cell-cell interactions facilitate infection between cells. Dual-color 3D fluorescence deconvolution microscopy of HIV-1 particles at frames rates of 100 frames per second allows us to follow the transfer of HIV-1 particles across the T cell virological synapse between living T cells. We confirm the successful transfer of virus by imaging T cell samples fixed at specific time points during cell-cell virus transfer by super-resolution structured illumination microscopy.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery.Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen.Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing.Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

A versatile synthetic approach to tunable dual-emissive Pdots with very small-size based on amphiphilic block copolymers for cell imaging

An effective strategy was constructed for the small-scale preparation of AIE-active Pdots, which displayed reversibly distinct dual-color fluorescence in cancer cells.

A unique self-reporting photosensitizer enabling simultaneous photodynamic therapy and real-time monitoring of phototheranostic process in a dynamic dual-color mode.

Phototheranostics has attracted great interest in cancer therapy. Small-molecule self-reporting photosensitizers, one kind of idea agent in phototheranostics, enables simultaneous photodynamic therapy (PDT) and feedback of therapeutic efficacy. However, previous such photosensitizers exclusively employed the change of single emission to monitor cell death, which can be disturbed by variations in photosensitizer concentration and the excitation intensity. Herein, we report a unique self-reporting photosensitizer TPA-3PyA+ constructed from a twisted triphenylamine unit (TPA), three benzene ring units and three cyanovinyl-pyridinium units (PyA) for PDT and its real-time monitoring in a dynamic dual-color mode. TPA-3PyA+ possesses a rotatable electron donor-π bridge-electron acceptor framework and exhibits high singlet oxygen quantum yield (124%) and a twisted intramolecular charge transfer (TICT) effect. TPA-3PyA+ not only enables effective staining of cancer cells with dual-color fluorescence due to the TICT effect but also shows excellent PDT performance. The simultaneous change in emission color, intensity and intracellular location of TPA-3PyA+ during cell death allows it to self-report cell death. Moreover, the change of dual-emission color allows distinguishing living and dead cells and effectively avoids interference in previous single-emission self-reporting photosensitizers. This work highlights the great potential of a self-reporting photosensitizer with dual-color emissions for efficient feedback of theranostics.

Ratiometric detection of p-nitrophenol and its derivatives using a dual-emissive neuron cell-like carbonized probe based on a ππ stacking quenching mechanism.

p-Nitrophenol and its derivatives can cause serious harm to the health of mankind and the earth's ecosystem. Therefore, it is necessary to develop a novel and rapid detection technology for p-nitrophenol and its derivative. Herein, excellent water-soluble, large-size and dual-emissive neuron cell-analogous carbon-based probes (NCNPs) have been prepared via a solvothermal approach, using o-phenylenediamine as the only precursor, which exhibit two distinctive fluorescence (FL) peaks at 420 and 555 nm under 345 nm excitation. The NCNPs show a neuron cell-like branched structure, are cross-connected, and are in the range of 10-20 nm in skeleton diameter. Interestingly, their blue-green dual-colour fluorescence is quenched by p-nitrophenol or its derivative due to the specific mechanism of the ππ stacking interactions or internal filtration effect. Accordingly, a simple, rapid, direct and free-label ratiometric FL detection of p-nitrophenol is proposed. An excellent linear relationship shows linear regions over the range of 0.1-50 μM between the ratio of the FL intensity (FL555 nm/FL420 nm) and the concentrations of p-nitrophenol. The detection limit is as low as 43 nM (3σ). Importantly, the NCNP-based probe also shows acceptable repeatability and reproducibility for the detection of p-nitrophenol and its derivatives, and the recovery results for p-nitrophenol in real wastewater samples are favourable.

A Novel Platform to Test In Vivo Single Gene Dependencies in t(8,21) and t(15,17) AML Confirms Zeb2 as Leukemia Target.

Simple SummaryMouse models are extensively used to study human diseases, including cancer. They are particularly useful to evaluate the role of specific genes in the tumorigenic process. The platform we present allows to effectively induce in vivo silencing of any potential candidate gene in two acute myeloid leukemia mouse models, with the scope of furthering the understanding of this gene’s role in the biology of leukemia.The increased usage of high-throughput technologies in cancer research, including genetic and drug screens, generates large sets of candidate targets that need to be functionally validated for their roles in tumor development. Thus, reliable and robust in vivo model systems are needed to perform reverse genetic experiments. Ideally, these models should allow for a conditional silencing of the target and an unambiguous identification of engineered cancer cells. Here, we present a platform consisting of: (i) t(8;21) and t(15;17) driven acute myeloid leukemia (AML) transgenic mice with constitutive expression of green fluorescent protein (GFP) and inducible expression of Cre recombinase, and (ii) REX, a modified pSico lentiviral vector for inducible shRNA expression and red fluorescent protein (RFP) as a selection marker. In this system, leukemic cells from transgenic mice are transduced with REX, flow sorted, and transplanted into syngeneic hosts. Gene interference is induced in established tumors by tamoxifen treatment. Dual-color cell fluorescence guides the in vivo identification of shRNA interfered AML cells, monitoring engraftment and disease progression. We tested the platform by inducing knockdown of Zeb2, a gene upregulated by AML1-ETO and PML-RARα oncogenes in pre-leukemic hematopoietic stem cell compartment, and observed a significant delay in leukemia onset. This proves the power and utility of the platform and confirms Zeb2 contribution to the pathogenesis of AML.

Chromosome Mapping of 5S Ribosomal Genes in Indo-Pacific and Atlantic Muraenidae: Comparative Analysis by Dual Colour Fluorescence In Situ Hybridisation.

The Muraenidae is one of the largest and most complex anguilliform families. Despite their abundance and important ecological roles, morays are little studied, especially cytogenetically, and both their phylogenetic relationships and the taxonomy of their genera are controversial. With the aim of extending the karyology of this fish group, the chromosomal mapping of the 5S ribosomal gene family was performed on seven species belonging to the genera Muraena and Gymnothorax from both the Atlantic and Pacific oceans. Fluorescence in situ hybridisation (FISH) experiments were realized using species-specific 5S rDNA probes; in addition, two-colour FISH was performed to investigate the possible association with the 45S ribosomal gene family. Multiple 5S rDNA clusters, located either in species-specific or in possibly homoeologous chromosomes, were found. Either a syntenic or different chromosomal location of the two ribosomal genes was detected. Our results revealed variability in the number and location of 5S rDNA clusters and confirmed a substantial conservation of the number and location of the 45S rDNA.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

Phase separation at the nanoscale quantified by dcFCCS

Liquid-liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

Chromosomal mapping of 5S and 18S-5.8S-25S rRNA genes in Saccharina japonica (Phaeophyceae) as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta. In regard to the usual two positions of linked 5S rDNA to the 3' end of 25S rDNA, two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica, one of species in Ochrophyta. This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp. In order to simultaneously visualize this unlinkage of two genes, dual-color fluorescence in situ hybridization (FISH) technique was applied to the cytogenetics of S. japonica. Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte, respectively, metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA. Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23, whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27. These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene, and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp. Therefore, dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S. japonica.

The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat

ABSTRACTEukaryotic flagella undertake different beat types as necessary for different functions; for example, the Leishmania parasite flagellum undergoes a symmetric tip-to-base beat for forward swimming and an asymmetric base-to-tip beat to rotate the cell. In multi-ciliated tissues or organisms, the asymmetric beats are coordinated, leading to movement of the cell, organism or surrounding fluid. This coordination involves a polarisation of power stroke direction. Here, we asked whether the asymmetric beat of the single Leishmania flagellum also has a fixed polarisation. We developed high frame rate dual-colour fluorescence microscopy to visualise flagellar-associated structures in live swimming cells. This showed that the asymmetric Leishmania beat is polarised, with power strokes only occurring in one direction relative to the asymmetric flagellar machinery. Polarisation of bending was retained in deletion mutants whose flagella cannot beat but have a static bend. Furthermore, deletion mutants for proteins required for asymmetric extra-axonemal and rootlet-like flagellum-associated structures also retained normal polarisation. Leishmania beat polarisation therefore likely arises from either the nine-fold rotational symmetry of the axoneme structure or is due to differences between the outer doublet decorations.

Single-Color Fluorescence Lifetime Cross-Correlation Spectroscopy In Vivo

The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.

A split aptamer sensing platform for highly sensitive detection of theophylline based on dual-color fluorescence colocalization and single molecule photobleaching

A new split aptamer sensing platform is developed for highly sensitive and selective detection of theophylline based on single molecule photobleaching (SMPB) technique. The sensing system contains two probes. One is formed by one streptavidin and four biotinylated RNA fragments labelled with fluorescein isothiocyanate (FITC). Each biotinylated RNA fragment contains two repeating aptamer fragments. The other probe is the complementary aptamer fragment labelled with Cy5 dye. The existence of theophylline can trigger the first probe to bind as many as eight Cy5-labelled probes. The average combined number depends on the theophylline concentration and can be measured by SMPB technique. In the sensing system, the dual-color fluorescence colocalization is performed by the red fluorophore (Cy5) and green fluorophore (FITC), in which the red fluorophore is utilized for quantitative counting of photobleaching steps, while the green fluorophore serves as a counting reference to increase detection efficiency. On basis of the principle, an ultra-sensitive sensing platform of theophylline is created with a low limit of detection (LOD) of 0.092 nM. This work provides not only a highly sensitive method for theophylline detection but also a novel perspective for the applications of SMPB technology to construct biosensors.

A framework for quantitative analysis of spectral data in two channels

Simultaneous acquisition of emission intensity from two channels in dual-color fluorescence measurements can be used to determine the spectral properties of an emitter. We detail a method to extract quantitative information from dual-color measurements by considering two models: a single spectral component model for peak wavelength determination and a dual spectral component model for relative strength determination. The former model is useful for spectral characterization and establishing heterogeneity within an emitter species, while the latter model is useful for analyzing energy transfer efficiency. Both models are formalized to yield probability distributions that reflect uncertainties due to shot noise. We applied these analysis tools to single-particle measurements of CdSe/CdS quantum dots. Isolated emitters had narrow spectral-intensity distributions that reflected their static emission profiles and fluorescence stability. Small assemblies of mixed donor/acceptor particles exhibited dynamic behavior where emission configuration changes reveal different spectral characteristics due to energy transfer.

MiR-146b-5p suppresses the malignancy of GSC/MSC fusion cells by targeting SMARCA5.

Recent studies have confirmed that both cancer-associated bone marrow mesenchymal stem cells (BM-MSCs, MSCs) and glioma stem-like cells (GSCs) contribute to malignant progression of gliomas through their mutual interactions within the tumor microenvironment. However, the exact ways and relevant mechanisms involved in the actions of GSCs and MSCs within the glioma microenvironment are not fully understood. Using a dual-color fluorescence tracing model, our studies revealed that GSCs are able to spontaneously fuse with MSCs, yielding GSC/MSC fusion cells, which exhibited markedly enhanced proliferation and invasiveness. MiR-146b-5p was downregulated in the GSC/MSC fusion cells, and its overexpression suppressed proliferation, migration and invasion by the fusion cells. SMARCA5, which is highly expressed in high-grade gliomas, was a direct downstream target of miR-146b-5p in the GSC/MSC fusion cells. miR-146b-5p inhibited SMARCA5 expression and inactivated a TGF-β pathway, thereby decreasing GSC/MSC fusion cell proliferation, migration and invasion. Collectively, these findings demonstrate that miR-146b-5p suppresses the malignant phenotype of GSC/MSC fusion cells in the glioma microenvironment by targeting a SMARCA5-regulated TGF-β pathway.

Molecular cloning and expression analysis of hybrid hazelnut (Corylus heterophylla×Corylus avellana) ChaSRK1/2 genes and their homologs from other cultivars and species.

The self-incompatibility system of Corylus is a sporophytic type that is phenotypically similar to that of Brassica. While the molecular mechanism of sporophytic self-incompatibility (SSI) has been investigated extensively in Brassica (Brassicaceae), little is known about the corresponding mechanism in Corylus (Betulaceae). Here, we discuss the SSI mechanism with respect to S-locus receptor kinase (SRK) gene homologs. To obtain two SRK candidate unigenes, we compared all of the unigenes in a transcriptional protein database from our previous study with BnSRK-1 (AB270767) using BLASTX with a cutoff e-value of 10-5. We then cloned the full-length cDNA of ChaSRK1 and ChaSRK2 genes from Ping'ou hybrid hazelnut (Corylus heterophylla×Corylus avellana) using RACE techniques. Bioinformatics approaches were used to analyze the cDNA sequences, protein sequences, and domains of the encoded proteins. The full-length ChaSRK1 cDNA was 2883 base pairs (bp) with a coding sequence (CDS) of 2,547bp encoding 849 amino acid residues. The full-length ChaSRK2 cDNA was 2,693bp, with a CDS of 2,433bp encoding 811 amino acids. The ChaSRK1/2 proteins contained an S-domain (extracellular domain), a transmembrane domain, a Ser/Thr protein kinase active site (kinase domain), and DUF3660 and/or DUF3403 domains. The lengths of 18 partial SRK homologs ranged from 1347 to 1451bp, and they contained the same structural domains as ChaSRK1 and ChaSRK2. Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica. The expression patterns of ChaSRK1 and ChaSRK2 differed: ChaSRK2 was predominantly expressed in mature stigmatic styles, while ChaSRK1 was expressed in other tissues with the highest in the root tips of Corylus. Using dual-color fluorescence in situ hybridization, ChaSRK1/2 expression was found to be localized in papillar cells. Collectively, these results revealed that SRKs from Corylus had similar characteristics to SRKs from Brassica. We therefore speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.

Fusion Pore Formation Observed during SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve elementary steps of the neuronal fusion process. However, in previous studies, we monitored only lipid mixing between fusing large unilamellar vesicles and PSMs and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into large unilamellar vesicles containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on gold-coated porous silicon substrates. By dual-color spinning disk fluorescence microscopy with a time resolution of ∼20 ms, we could unambiguously distinguish between bursting vesicles, which was only rarely observed (<0.01%), and fusion pore formation. From the time-resolved dual-color fluorescence time traces, we were able to identify different fusion pathways, including remaining three-dimensional postfusion structures with released content and transient openings and closings of the fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation after the merger of the two lipid membranes occurs almost simultaneously.

Downregulation of miRNA-146a-5p promotes malignant transformation of mesenchymal stromal/stem cells by glioma stem-like cells.

Mesenchymal stromal/stem cells (MSCs) are promising carriers in cell-based therapies against central nervous system diseases, and have been evaluated in various clinical trials in recent years. However, bone marrow-derived MSCs (BMSCs) are reportedly involved in tumorigenesis initiated by glioma stem-like cells (GSCs). We therefore established three different orthotopic models of GSC-MSC interactions in vivo using dual-color fluorescence tracing. Cell sorting and micropipetting techniques were used to obtain highly proliferative MSC monoclones from each model, and these cells were identified as transformed MSC lines 1, 2 and 3. Nineteen miRNAs were upregulated and 24 miRNAs were downregulated in all three transformed MSC lines compared to normal BMSCs. Reduced miR-146a-5p expression in the transformed MSCs was associated with their proliferation, malignant transformation and overexpression of heterogeneous nuclear ribonucleoprotein D. These findings suggest that downregulation of miR-146a-5p leads to overexpression of its target gene, heterogeneous nuclear ribonucleoprotein D, thereby promoting malignant transformation of MSCs during interactions with GSCs. Given the risk that MSCs will undergo malignant transformation in the glioma microenvironment, targeted glioma therapies employing MSCs as therapeutic carriers should be considered cautiously.

Aptamer-based ratiometric fluorescent nanoprobe for specific and visual detection of zearalenone

We firstly proposed a ratiometric fluorescent nanoprobe with aptamer-labeled CdTe quantum dots (QDs) for the determination of zearalenone (ZEN). The nanoprobe was designed with dual-color emission fluorescence by single-wavelength excitation. Red-emitting silica-coated QDs (r-QDs@SiO2) with the maximum excitation/emission at 400/608 nm were used as a reference. Green-emitting QDs linked with aptamer and covalently bonded to r-QDs@SiO2 with the maximum excitation/emission at 400/518 nm were used as the responsive fluorophore for specific recognition of ZEN. The green fluorescence was quenched in the presence of ZEN. The ratio of fluorescence at 518 nm/608 nm decreased linearly against the increase of ZEN concentrations in the range of 31.4-628 nmol/L. The color of the solution changed from green to red as the concentration of ZEN increased. This method offered a ratiometric, specific and visual determination of ZEN. The limit of detection was 7.5 nM. The aptamer-labeled ratiometric strategy showed acceptable stability and selectivity. Therefore, the approach was successfully used to measure ZEN in food and can provide guidance for the design of point-of-care, multiplexed biosensors.

An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3–5.0 μm for excitation wavelengths of 730–1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)–poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.