A facile method for the preparation of antibody–quantum dot (QD) conjugates using the immunoglobulin binding (B1) domain of protein G is presented. The utility of antibody–QD conjugates using the B1 domain is demonstrated for fluorescence imaging of breast tumor cells in vitro and in vivo.

Indocyanine green (ICG) is the only near-infrared (NIR) fluorescent dye which is approved for medical applications. However, ICG has several drawbacks such as aqueous instability, photodegradation, and low fluorescence quantum yield (2.5% in water), which lead to the limitation on the use of ICG for in vitro and in vivo NIR fluorescence imaging. Free ICG rapidly aggregates in physiological buffer solutions, and its fluorescence diminishes within several days. The objective of this work is to provide an easy method for the enhancement of the stability and fluorescence brightness of ICG in aqueous solutions for NIR fluorescence imaging. Herein, we report that the incorporation of ICG into small calix[4]arene (S4-6) micelles (<5 nm in diameter) significantly improves the aqueous stability and fluorescence brightness of ICG. The fluorescence quantum yields of ICG-calix[4]arene micelles are increased up to ∼6% in aqueous solutions. Using the ICG-calix[4]arene micelles, we achieved non-invasive NIR fluorescence imaging of the liver and lymph system in mice. Furthermore, we achieved NIR fluorescence imaging of nude mice bearing human breast tumors using an ICG conjugated antibody which is incorporated into the calix[4]arene micelles. Preparation of the calix[4]arene micelles including ICG is very easy and the micelle system does not show significant cytotoxicity. The ICG-calix[4]arene micelle system acts as a highly stable and bright probe for in vitro and in vivo NIR fluorescence imaging.

We report a one-step synthetic strategy for the preparation of recombinant protein (EGFP-Protein G)-coated PbS quantum dots for dual (visible and second-NIR) fluorescence imaging of breast tumors at the cellular and whole-body level.

Compact SNAP ligand-conjugated quantum dots (<10 nm) with high colloidal stability over a wide range of pH (5–9) have been synthesized as fluorescent probe for the single-molecule imaging of dynein motor protein.

For the non-invasive visualization of cell migration in deep tissues, we synthesized a short-wavelength infrared (SWIR) emitting multimodal probe that contains PbS/CdS quantum dots, rhodamine 6G and iron oxide nanoparticles. This probe enables multimodal (SWIR fluorescence/magnetic resonance) imaging of phagocyte cell migration in living mice.

Near-infrared (NIR) fluorescence imaging at wavelengths from 1000 to 1500 nm (2nd-NIR window) is a promising modality for in vivo fluorescence imaging because of the deeper tissue penetration with lower tissue scattering of the 2nd-NIR light. For such in vivo fluorescence imaging, highly fluorescent probes in the 2nd-NIR wavelength region are needed. Although single-walled carbon nanotubes and Ag2S quantum dots (QDs) have recently appeared as 2nd-NIR fluorescent probes, their fluorescence brightness is relatively low (quantum yields <6%). In this study, we developed a synthetic method for preparing highly fluorescent PbS/CdS core–shell QDs (quantum yields, 17% in water) with narrow band widths (<200 nm) that emit in the 2nd-NIR region. By overcoating of a CdS shell onto a PbS QD core, we could easily control the emission wavelengths of the PbS/CdS QDs at 1000 to 1500 nm. To use the QDs for in vivo imaging, we investigated the optical properties of QDs (penetration depth and blurring of fluorescence images in slices of skin, brain, and heart in mice) in the 2nd-NIR region. We found that the 2nd-NIR fluorescence imaging at ca.1300 nm using the PbS/CdS QDs results in the highest signal to background ratio with a low blurring for in vivo imaging. To confirm the capabilities of the PbS/CdS QDs for in vivo imaging, we conducted fluorescence angiography imaging of a mouse head.

Glutathione-coated PbS quantum dots with tunable emission were synthesized in the aqueous phase and used for non-invasive tissue imaging in the second near-infrared biological window.

Bioluminescence resonance energy transfer coupled near-infrared quantum dots using glutathione-s-transferase (GST) tagged luciferase were synthesized as luminescent probes for in vivo imaging.

We report that bovine serum albumin (BSA)-coated quantum dots can be used as a fluorescent probe for measuring the cytoplasmic viscosity of a single living cell: fluorescence correlation spectroscopy measurements for a HeLa cell incorporating BSA-quantum dots show that the viscosity of cytoplasm is 4.7 times greater than that of water.

•In vivo cryotechnique revealed early events in a model of lung metastasis.•Fibrinogen was detected beside cancer cells and edge of platelet mass.•Platelets start gathering at 1 min, and their Src was activated at 5 min.•Fibrin was mainly formed in areas of impairment of blood circulation.•We successfully detected fibrin formation on tissue sections.Microscopic bioimaging of blood flow and distribution of cancer cells in lungs is essential to analyze mechanism of lung metastasis. Such cancer metastasis has been well known to induce hypercoagulable states and thrombosis. In histopathological tissue sections, however, it has been difficult to capture rapid phenomenon of thrombus formation due to technical problems associated with much less retention of soluble serum components as well as dynamic histological features reflecting their living states. In this study, to achieve bioimaging of both hypercoagulable states and thrombosis induced by early metastasis of mouse B16-BL6 melanoma, “in vivo cryotechnique” (IVCT) was used, which retained soluble components at their original sites. Glutathione-coated quantum dots (QDs) were subsequently injected after melanoma cells via right ventricles to examine plasma flow with fluorescence emission. At 5 s after the melanoma injection, melanoma cells were mostly stacked and intruded in alveolar capillaries with changing their shapes. Assembly of platelets initially appeared at 1 min, and they aggregated around the stacked melanoma cells at 5 min. Such aggregated platelets were immunopositive for both phospho-tyrosine 418 and 527 of Src, indicating their partial signal activation. Fibrin monomers were also immunolocalized around both melanoma cells and platelet aggregates, and massive immunoreaction deposits of fibrinogen were also detected near the same areas, but more strongly detected around the melanoma cells, indicating initial thrombus formation. In those areas, QDs were rarely detected, probably because of the lack of blood supply. Thus, IVCT revealed histopathological features of initial thrombosis under their circulatory conditions.Download full-size image

Bioluminescence resonance energy transfer coupled near-infrared quantum dots using glutathione-s-transferase (GST) tagged luciferase were synthesized as luminescent probes for in vivo imaging.

Compact SNAP ligand-conjugated quantum dots (<10 nm) with high colloidal stability over a wide range of pH (5–9) have been synthesized as fluorescent probe for the single-molecule imaging of dynein motor protein.

We report that bovine serum albumin (BSA)-coated quantum dots can be used as a fluorescent probe for measuring the cytoplasmic viscosity of a single living cell: fluorescence correlation spectroscopy measurements for a HeLa cell incorporating BSA-quantum dots show that the viscosity of cytoplasm is 4.7 times greater than that of water.

For the non-invasive visualization of cell migration in deep tissues, we synthesized a short-wavelength infrared (SWIR) emitting multimodal probe that contains PbS/CdS quantum dots, rhodamine 6G and iron oxide nanoparticles. This probe enables multimodal (SWIR fluorescence/magnetic resonance) imaging of phagocyte cell migration in living mice.

Glutathione-coated PbS quantum dots with tunable emission were synthesized in the aqueous phase and used for non-invasive tissue imaging in the second near-infrared biological window.