Search

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near-infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold-ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low-toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a break-through in PA flow cytometry and near-infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-μm microspheres as cell phantoms on slides and in 90-μm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Circulating cells, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo multicolor photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits.

In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique.

Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single-channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 x 10(-9) mol/L (80 attomols in the signal-generation zone); that is ca. 10³ lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 x 10(-21) mol) in the ultrasmall irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted.

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic-PT switching of conventional and gold-coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple-modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near-infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super-resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed.

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic-PT switching of conventional and gold-coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple-modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near-infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super-resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed.