Membrane fouling is the bottleneck that restricts the sustainability of membrane technology for environmental applications. Therefore, the development of novel analytical tools for characterizing membrane fouling processes is essential. In this work, we demonstrate a capability of probing the chemical structure of foulants and detecting their 3-dimentional spatial distribution on membranes based on stimulated Raman scattering (SRS) microscopy as a vibrational spectroscopic imaging approach. The adsorption process of foulants onto membrane surfaces and their aggregation process within membrane pores during the microfiltration of protein and polysaccharide solutions were clearly monitored. Pore constriction and cake layer formation were found to be the coupled membrane fouling mechanisms. This work establishes an ultrafast, highly sensitive, nondestructive and label-free imaging platform for the characterization of membrane fouling evolution. Furthermore, this work provides new insights into membrane fouling and offers a powerful tool for membrane-based process exploration.

Dysregulation of lipid metabolism is associated with many diseases including cancer. Lipid droplet (LD), a ubiquitous organelle in mammalian cells, serves as a hub for lipid metabolism. Conventional assays on the measurement of lipid metabolism rely on the quantification of the lipid composition or amount. Such methods cannot distinguish LDs having different biofunctionalities in living cells, and thus could be inaccurate in measuring the instantaneous lipogenesis of the living cells. We applied label-free stimulated Raman scattering microscopy to quantify the LDs’ spatial-temporal dynamics, which showed direct links to cellular lipid metabolisms and can separate LDs involved in different metabolic events. In human cancer cells, we found that changes in the maximum displacement of LDs reflected variations in cellular lipogenic activity, and changes in the average speed of LDs revealed alterations in LD size. The LD dynamics analysis allowed for more accurate measurement in the lipogenesis and LD dimensions, and can break the optical diffraction limit to detect small variation in lipid metabolism that was conventionally undetectable. By this method, we revealed changes in the lipogenic activity and LD sizes during glucose starvation of HeLa cells and transforming growth factor beta-induced epithelial-to-mesenchymal transition of SKOV-3 cells. This method opens a way to quantify lipid metabolism in living cells during cellular development and transition.

Recent advances in atherosclerotic plaque detection have shown that not only does lipid core size and depth play important roles in plaque rupture and thrombi formation, but lipid composition, especially cholesterol deposition, is equally important in determining lesion vulnerability. Here, we demonstrate a spectral analysis assisted photoacoustic imaging approach to differentiate and map lipid compositions within an artery wall. The approach is based on the classification of spectral curves obtained from the sliding windows along time-of-flight photoacoustic signals via a numerical k-means clustering method. The evaluation result on a vessel-mimicking phantom containing cholesterol and olive oil shows accuracy and efficiency of this method, suggesting the potential to apply this approach in assessment of atherosclerotic plaques.

AbstractThienoisoindigo-based semiconducting polymer with a strong near-infrared absorbance is synthesized and its water-dispersed nanoparticles (TSPNs) are investigated as a contrast agent for photoacoustic (PA) imaging in the second near-infrared (NIR-II) window (1000–1350 nm). The TSPNs generate a strong PA signal in the NIR-II optical window, where background signals from endogenous contrast agents, including blood and lipid, are at the local minima. By embedding a TSPN-containing tube in chicken-breast tissue, an imaging depth of more than 5 cm at 1064 nm excitation is achieved with a contrast-agent concentration as low as 40 µg mL−1. The TSPNs under the skin or in the tumor are clearly visualized at 1100 and 1300 nm, with negligible interference from the tissue background. TSPN as a PA contrast in the NIR-II window opens new opportunities for biomedical imaging of deep tissues with improved contrast.

•Ovarian cancer stem cells have high levels of unsaturated lipids•Blocking lipid desaturation impairs cancer stemness and tumor initiation capacity•The NF-κB pathway directly regulates the expression of lipid desaturases•Lipid desaturase inhibitors inactivate the NF-κB pathwayLack of sensitive single-cell analysis tools has limited the characterization of metabolic activity in cancer stem cells. By hyperspectral-stimulated Raman scattering imaging of single living cells and mass spectrometry analysis of extracted lipids, we report here significantly increased levels of unsaturated lipids in ovarian cancer stem cells (CSCs) as compared to non-CSCs. Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells. Inhibition of lipid desaturases effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo. Mechanistically, we demonstrate that nuclear factor κB (NF-κB) directly regulates the expression levels of lipid desaturases, and inhibition of desaturases blocks NF-κB signaling. Collectively, our findings reveal that increased lipid unsaturation is a metabolic marker for ovarian CSCs and a target for CSC-specific therapy.Download high-res image (250KB)Download full-size image

•Metabolism is highly dynamic and intrinsically heterogeneous at the cellular level.•CRS microscopy visualizes metabolic activities of biomolecules in single live cells.•CRS study of single cell metabolism is facilitated by incorporation of Raman tags.•CRS study offers new insights into the role of cell metabolism in pathogenesis.Metabolism is highly dynamic and intrinsically heterogeneous at the cellular level. Although fluorescence microscopy has been commonly used for single cell analysis, bulky fluorescent probes often perturb the biological activities of small biomolecules such as metabolites. Such challenge can be overcome by a vibrational imaging technique known as coherent Raman scattering microscopy, which is capable of chemically selective, highly sensitive, and high-speed imaging of biomolecules with submicron resolution. Such capability has enabled quantitative assessments of metabolic activities of biomolecules (e.g. lipids, proteins, nucleic acids) in single live cells in vitro and in vivo. These investigations provide new insights into the role of cell metabolism in maintenance of homeostasis and pathogenesis of diseases.

The quantized vibration of chemical bonds provides a way of detecting specific molecules in a complex tissue environment. Unlike pure optical methods, for which imaging depth is limited to a few hundred micrometers by significant optical scattering, photoacoustic detection of vibrational absorption breaks through the optical diffusion limit by taking advantage of diffused photons and weak acoustic scattering. Key features of this method include both high scalability of imaging depth from a few millimeters to a few centimeters and chemical bond selectivity as a novel contrast mechanism for photoacoustic imaging. Its biomedical applications spans detection of white matter loss and regeneration, assessment of breast tumor margins, and diagnosis of vulnerable atherosclerotic plaques. This review provides an overview of the recent advances made in vibration-based photoacoustic imaging and various biomedical applications enabled by this new technology.

Highly lignified vascular plant cell walls represent the majority of cellulosic biomass. Complete release of the biomass to deliver renewable energy by physical, chemical, and biological pretreatments is challenging due to the “protection” provided by polymerized lignin, and as such, additional tools to monitor lignin deposition and removal during plant growth and biomass deconstruction would be of great value. We developed a hyperspectral stimulated Raman scattering microscope with 9 cm–1 spectral resolution and submicrometer spatial resolution. Using this platform, we mapped the aromatic ring of lignin, aldehyde, and alcohol groups in lignified plant cell walls. By multivariate curve resolution of the hyperspectral images, we uncovered a spatially distinct distribution of aldehyde and alcohol groups in the thickened secondary cell wall. These results collectively contribute to a deeper understanding of lignin chemical composition in the plant cell wall.

High-speed coherent Raman scattering imaging is opening a new avenue to unveil cellular machinery by visualizing the spatiotemporal dynamics of target molecules or intracellular organelles. By extracting signals from the laser at megahertz modulation frequency, current stimulated Raman scattering (SRS) microscopy has reached shot-noise-limited detection sensitivity. The laser-based local oscillator in SRS microscopy not only generates high levels of signal but also delivers a large shot noise that degrades image quality and spectral fidelity. Here, we demonstrate a denoising algorithm that removes the noise in both spatial and spectral domains by total variation minimization. The signal-to-noise ratio of SRS spectroscopic images was improved by up to 57 times for diluted dimethyl sulfoxide solutions and by 15 times for biological tissues. Weak Raman peaks of target molecules originally buried in the noise were unraveled. Coupling the denoising algorithm with multivariate curve resolution allowed discrimination of fat stores from protein-rich organelles in Caenorhabditis elegans. Together, our method significantly improved detection sensitivity without frame averaging, which can be useful for in vivo spectroscopic imaging.

Real-time vibrational spectroscopic imaging is desired for monitoring cellular states and cellular processes in a label-free manner. Raman spectroscopic imaging of highly dynamic systems is inhibited by relatively slow spectral acquisition on millisecond to second scale. Here, we report microsecond scale vibrational spectroscopic imaging by lock-in free parallel detection of spectrally dispersed stimulated Raman scattering signal. Using a homebuilt tuned amplifier array, our method enables Raman spectral acquisition, within the window defined by the broadband pulse, at the speed of 32 µs and with close to shot-noise limited detection sensitivity. Incorporated with multivariate curve resolution analysis, our platform allows compositional mapping of lipid droplets in single live cells, observation of intracellular retinoid metabolism, discrimination of fat droplets from protein-rich organelles in Caenorhabditis elegans, spectral detection of fast flowing tumor cells and monitoring drug diffusion through skin tissue in vivo. The reported technique opens new opportunities for compositional analysis of cellular compartment in a microscope setting and high-throughput spectral profiling of single cells in a flow cytometer setting.

Undesirable side effects remain a significant challenge in cancer chemotherapy. Here we report a strategy for cancer-selective chemotherapy by blocking acyl-CoA cholesterol acyltransferase-1 (ACAT-1)-mediated cholesterol esterification. To efficiently block cholesterol esterification in cancer in vivo, we developed a systemically injectable nanoformulation of avasimibe (a potent ACAT-1 inhibitor), called avasimin. In cell lines of human prostate, pancreatic, lung, and colon cancer, avasimin significantly reduced cholesteryl ester storage in lipid droplets and elevated intracellular free cholesterol levels, which led to apoptosis and suppression of proliferation. In xenograft models of prostate cancer and colon cancer, intravenous administration of avasimin caused the concentration of avasimibe in tumors to be 4-fold higher than the IC50 value. Systemic treatment of avasimin notably suppressed tumor growth in mice and extended the length of survival time. No adverse effects of avasimin to normal cells and organs were observed. Together, this study provides an effective approach for selective cancer chemotherapy by targeting altered cholesterol metabolism of cancer cells.Keywords: ACAT-1 inhibitor; avasimibe; cancer; cholesterol; cholesteryl ester; human serum albumin;

In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10 000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.

Spinal cord injury results in significant mortality and morbidity, lifestyle changes, and difficult rehabilitation. Treatment of spinal cord injury is challenging because the spinal cord is both complex to treat acutely and difficult to regenerate. Nanomaterials can be used to provide effective treatments; their unique properties can facilitate drug delivery to the injury site, enact as neuroprotective agents, or provide platforms to stimulate regrowth of damaged tissues. We review recent uses of nanomaterials including nanowires, micelles, nanoparticles, liposomes, and carbon-based nanomaterials for neuroprotection in the acute phase. We also review the design and neural regenerative application of electrospun scaffolds, conduits, and self-assembling peptide scaffolds.

Spectroscopic imaging has been an increasingly critical approach for unveiling specific molecules in biological environments. Toward this goal, we demonstrate hyperspectral stimulated Raman loss (SRL) imaging by intrapulse spectral scanning through a femtosecond pulse shaper. The hyperspectral stack of SRL images is further analyzed by a multivariate curve resolution (MCR) method to reconstruct quantitative concentration images for each individual component and retrieve the corresponding vibrational Raman spectra. Using these methods, we demonstrate quantitative mapping of dimethyl sulfoxide concentration in aqueous solutions and in fat tissue. Moreover, MCR is performed on SRL images of breast cancer cells to generate maps of principal chemical components along with their respective vibrational spectra. These results show the great capability and potential of hyperspectral SRL microscopy for quantitative imaging of complicated biomolecule mixtures through resolving overlapped Raman bands.

Although nanocarriers hold promise for cancer chemotherapy, their intracellular drug delivery pathways are not fully understood. In particular, the influence of nanocarrier stability on cellular uptake is still uncertain. By physically loading hydrophobic FRET probes, we revealed different intracellular drug delivery routes of self-assembled and disulfide bonded micelles. The self-assembled micelles were structurally dissociated by micelle–membrane interactions, and the hydrophobic probes were distributed on the plasma membrane. Alternatively, intact disulfide bonded micelles carrying hydrophobic probes were internalized into cancer cells via multiple endocytic pathways. Following internalization, disulfide bonded micelles were decomposed in early endosomes by glutathione-mediated disulfide bond reduction, exposing the probes to intracellular organelles.Keywords: cancer; disulfide bonded micelles; fluorescence resonance energy transfer (FRET); glutathione; intracellular drug delivery; nanomedicine;

The quantized vibration of chemical bonds provides a way of imaging target molecules in a complex tissue environment. Photoacoustic detection of harmonic vibrational transitions provides an approach to visualize tissue content beyond the ballistic photon regime. This method involves pulsed laser excitation of overtone transitions in target molecules inside of tissue. Fast relaxation of the vibrational energy into heat results in a local temperature rise on the order of milliKelvins and a subsequent generation of acoustic waves detectable with an ultrasonic transducer. In this Perspective, we review recent advances that demonstrate the advantages of vibration-based photoacoustic imaging and illustrate its potential in diagnosing cardiovascular plaques. An outlook into future development of vibrational photoacoustic endoscopy and tomography is provided.

Proof-of-concept of vibrational photoacoustic tomography is demonstrated with a home-built Raman laser generating greater than 100 mJ of energy per pulse at a 1197 nm wavelength. We employed this system for excitation of the second overtone transition of C–H bonds. The vibrational photoacoustic signal from a C–H-rich polyethylene tube phantom placed under 3 cm thick chicken breast tissue was obtained with a signal-to-noise ratio of 2.5. Further, we recorded a photoacoustic image of a polyethylene ring placed under 5 mm chicken tissue with excellent contrast. This development opens new opportunities of performing label-free vibrational imaging in the deep tissue regime.Keywords: lipid; molecular vibration; photoacoustic tomography; Raman laser;

Desorption electrospray ionization (DESI) mass spectrometry (MS) is used in an imaging mode to interrogate the lipid profiles of 15 μm thin tissue cross sections of injured rat spinal cord and normal healthy tissue. Increased relative intensities of fatty acids, diacylglycerols, and lysolipids (between +120% and +240%) as well as a small decrease in intensities of lipids (−30%) were visualized in the lesion epicenter and adjacent areas after spinal cord injury. This indicates the hydrolysis of lipids during the demyelination process due to activation of phospholipase A2 enzyme. In addition, signals corresponding to oxidative degradation products, such as prostaglandin and hydroxyeicosatetraenoic acid, exhibited increased signal intensity by a factor of 2 in the negative ion mode in lesions relative to the normal healthy tissue. Analysis of malondialdehyde, a product of lipid peroxidation and marker of oxidative stress, was accomplished in the ambient environment using reactive DESI mass spectrometry imaging. This was achieved by electrospraying reagent solution containing dinitrophenylhydrazine as high-velocity charged droplets onto the tissue section. The hydrazine reacts selectively and rapidly with the carbonyl groups of malondialdehyde, and signal intensity of twice the intensity was detected in the lesions compared to healthy spinal cord. With a small amount of tissue sample, DESI-MS imaging provides information on the composition and distribution of specific compounds (limited by the occurrence of isomeric lipids with very similar fragmentation patterns) in lesions after spinal cord injury in comparison with normal healthy tissue allowing identification of the extent of the lesion and its repair.

Si−Au core−shell nanowires were synthesized with a two-step strategy combining chemical vapor deposition and wet chemistry methods. Si core diameter, Au shell thickness, and wire length were controlled during synthesis. Under a far-field two-photon excitation scanning microscope, spatially modulated two-photon luminescence (TPL) was observed from the core−shell nanowires prepared with several micrometers in length. The mode spacing scales linearly with the length of nanowires for samples with 15 and 40 nm Au shells. The spatial modulation pattern was found to be independent of the excitation wavelength. The TPL intensity displayed a cos2 dependence on the excitation field polarization, indicating a sequential two-photon excitation process enhanced by the longitudinal plasmon field. The spatially modulated TPL is interpreted by a standing wave model of surface plasmon polaritons along the core−shell nanowire which acts as a Fabry−Pérot resonator. These results suggest TPL imaging of Si−Au core−shell nanowires as a new system providing useful insights regarding a plasmonic wave in a 1-D nanostructure.

Nonlinear vibrational imaging of live cells and organisms is demonstrated by detecting femtosecond pulse stimulated Raman loss. Femtosecond pulse excitation produced a 12 times larger stimulated Raman loss signal than picosecond pulse excitation. The large signal allowed real-time imaging of the conversion of deuterated palmitic acid into lipid droplets inside of live cells and three-dimensional sectioning of fat storage in live C. elegans. With the majority of the excitation power contributed by the Stokes beam in the 1.0–1.2 μm wavelength range, photodamage of biological samples was not observed.Keywords: 3D imaging; coherent Raman scattering; femtosecond laser; lipid storage; nonlinear microscopy; quantitative analysis; stimulated Raman scattering (SRS);

We demonstrate that P11LRR, a recently developed amphiphilic polyproline, cell penetrating agent, is able to locate inside the mitochondria of various cell lines when administrated at high concentrations. Mitochondrial targeting was verified by confocal fluorescence co-localization of P11LRR-fluorescein with Mitotracker Red. Elimination of mitochondrial membrane potential dramatically inhibits the localization of P11LRR to mitochondria. Concentration-dependency experiments suggest that cellular internalization of P11LRR occurs via two different pathways: endocytosis and direct transport. Results indicate that the latter pathway predominates at high concentrations of P11LRR, resulting in localization of the agent to the mitochondria. The membrane translocation pathway was further confirmed by two endocytosis inhibitors, cytochalasin D and phenylarsine oxide, and by modulation of plasma membrane potential. The potential of using P11LRR as a mitochondrial drug delivery vector was demonstrated through the delivery of a covalently linked small antioxidant, dimethyltyrosine (Dmt), which allowed for the reduction of chemically induced reactive oxygen species within the mitochondria.

Proper chemical imaging tools are critical to the pharmaceutical industry due to growing regulatory demand for intermediate and end-product content uniformity testing. Herein we demonstrate stimulated Raman scattering (SRS) imaging of active pharmaceutical ingredient (API) and four excipients within tablets. Tablets from six manufactures were imaged with a speed of 53 s per frame of 512 × 512 pixels (i.e., 200 μs per pixel) and a lateral spatial resolution as high as 0.62 μm. The SRS chemical imaging was compared to confocal Raman mapping and coherent anti-Stokes Raman scattering (CARS) chemical imaging in terms of speed and chemical selectivity. The acquisition speed of SRS imaging is ca. 104 times faster than confocal Raman mapping and SRS technique showed superior to CARS chemical selectivity for studied samples. Our data demonstrate the potential of SRS microscopy in high-speed screening of pharmaceutical solid dosage forms.

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

The current work investigates the four-wave mixing (FWM) signal from gold nanorods (NRs) using two synchronized lasers and its potential applications in bioimaging. Using the lightning rod model, we show that the strongest FWM occurs when the pump laser wavelength is tuned to be resonant with the longitudinal plasmon resonance wavelength of NR. The calculation is experimentally demonstrated by comparing the intensities of FWM from NRs with different plasmon resonance wavelengths. The FWM signal is further found to be enhanced by aggregation of NRs and is strongly dependent on pulse width. The FWM intensity from NRs is ∼39 times stronger than the coherent anti-Stokes Raman scattering intensity from melamine beads. This plasmon-resonance-enhanced FWM signal enables NRs to be used as a nonlinear optical (NLO) imaging probe.

Characterization of systemic performance of gold nanostructures is critical to the advancement of biomedical applications of these nanomaterials as imaging or therapeutic agents. The accuracy of current in vitro methods, however, is limited by interanimal variability. We present a novel method capable of monitoring the pharmacokinetics of PEGylated gold nanorods (GNRs) in the same animal by using intravital two-photon luminescence (TPL) imaging of GNRs flowing through a surface blood vessel. The TPL imaging with high speed and submicrometer resolution allowed for studying the clearance of GNRs as a function of surface coating. PEGylated-GNRs (PEG-NRs) were found to exhibit a biphasic clearance mode, with a significantly prolonged blood residence time for branched poly(ethylene glycol) (PEG) as compared to the linear PEG. With spectral detection to distinguish GNR TPL from tissue autofluorescence, we also mapped the cellular distribution of GNRs in the explanted organs, and found most GNRs resided in the macrophages in liver and spleen.

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin’s autofluorescence and SFG signals arising from collagen fibrils’ non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using dual-labeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Förster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC₁₈₍₃₎ and DiOC₁₈₍₃₎, was loaded into monomethoxy poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed.

Understanding the in vivo behavior of nanoparticles is critical for the translation of nanomedicine from laboratory research to clinical trials. In this work, in vivo Förster resonance energy transfer (FRET) imaging was employed to monitor the release of hydrophobic molecules from circulating poly(ethylene glycol)-poly(d,l-lactic acid) (PEG-PDLLA) micelles. A lipophilic FRET pair (DiIC18 and DiOC18) was physically entrapped into micelle cores by mimicking the loading of hydrophobic drugs. The FRET efficiency was found significantly reduced within 15 min after intravenous injection, implying that DiIC18 and DiOC18 quickly escaped from the circulating micelles. FRET spectroscopy studies further demonstrated that α- and β-globulins were major factors for the observed fast release, while γ-globulins, albumin, and red blood cells played minor roles. These results provide useful information for developing blood-stable micelles to deliver hydrophobic drugs to the target site via prolonged circulation and extravasation from the vascular system.

Mechanisms underlying the release of paclitaxel (PTX) from poly(ethylene glycol)/poly(lactic-co-glycolic acid) (PEG/PLGA) blends were investigated by coherent anti-Stokes Raman scattering (CARS) microscopy. PLGA, PEG, and PTX were selectively imaged by using the resonant CARS signal from the CH3, CH2, and aromatic CH stretch vibrations, respectively. Phase segregation was observed in PLGA films containing 10 to 40 wt.% PEG in the absence of PTX loading. The PEG phase existed in the form of crystalline fibers in the (8:2, weight ratio) and (7:3) PLGA/PEG films. CARS observation indicated that PTX preferentially partitioned into the PEG domains in the (9:1) and (8:2) PLGA/PTX films, but exhibited a uniform mixing with both PLGA and PEG in the (7:3) PLGA/PEG film. The solid dispersion of PTX into PEG domains was attributed to a strong interaction between PTX and PEG, supported by the disappearance of PEG crystallization in the PTX-loaded PLGA/PEG film evidenced through X-ray diffraction analysis. PTX release was induced by exposing the film to an aqueous solution and monitored in real time by CARS and two-photon fluorescence microscopy. Fast dissolution of both PEG and PTX was observed at the film surface. Upon infiltration of water into the film, the PEG domains were rearranged into ring structures enriched by both PTX and PEG. The CARS data provided visual evidence explaining the accelerated burst release followed by more sustained release of PTX from the PLGA/PEG films as measured by HPLC.

Gold nanorods excited at 830 nm on a far-field laser-scanning microscope produced strong two-photon luminescence (TPL) intensities, with a cos⁴ dependence on the incident polarization. The TPL excitation spectrum can be superimposed onto the longitudinal plasmon band, indicating a plasmon-enhanced two-photon absorption cross section. The TPL signal from a single nanorod is 58 times that of the two-photon fluorescence signal from a single rhodamine molecule. The application of gold nanorods as TPL imaging agents is demonstrated by in vivo imaging of single nanorods flowing in mouse ear blood vessels.

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger the breast epithelium. A Raman scattering-based platform was used for label-free determination of apical polarity in live breast glandular structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces of an acinus. Raman microspectroscopy subsequently revealed that polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca2+-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore, the disruption of apical polarity by a dietary breast cancer risk factor, ω6 fatty acid, could be observed with this method, even when the effect was too moderate to permit a conclusive assessment by the traditional immunostaining method.

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed “femtosecond-stimulated Raman loss” microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.

The role of cholesterol in the regulation of endosome motility was investigated by monitoring the intracellular trafficking of endocytosed folate receptors (FRs) labeled with fluorescent folate conjugates. Real-time fluorescence imaging of HeLa cells transfected with green fluorescent protein-tubulin revealed that FR-containing endosomes migrate along microtubules. Moreover, microinjection with antibodies that inhibit microtubule-associated motor proteins demonstrated that dynein and kinesin I participate in the delivery of FR-containing endosomes to the perinuclear area and plasma membrane, respectively. Further, single-particle tracking analysis revealed bidirectional motions of FR endosomes, mediated by dynein and kinesin motors associated with the same endosome. These experimental tools allowed us to use FR-containing endosomes to evaluate the impact of cholesterol on intracellular membrane trafficking. Lowering plasma membrane cholesterol by metabolic depletion or methyl-β-cyclodextrin extraction was found to both increase FR-containing endosome motility and change endosome distribution from colocalization with Rab7 to colocalization with Rab4. These data provide evidence that cholesterol regulates intracellular membrane trafficking via modulation of the distribution of low molecular weight G-proteins that are adaptors for microtubule motors.

Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the spinal cord, between adjacent axons, and along the blood vessels. Using 20-μm-thick tissue slices, the ratio of forward to backward SHG signal from large bundles was found to be much larger than that from small single fibrils, indicating a phase-matching effect in coherent microscopy. Based on the intensity profiles across fibrils and the size dependence of forward and backward signal from the same fibril, we concluded that the main SHG signal directly originates from the fibrils, but not from surface SHG effects. Further polarization analysis of the SHG signal showed that the symmetry property of the fibril could be well described with a cylindrical model. Colocalization of the SHG signal with two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrated that SHG arises from astroglial filaments. This assignment was further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by a Ca2+ indicator and sulforhodamine 101. This work shows that a combination of three nonlinear optical imaging techniques—coherent anti-Stokes Raman scattering, TPEF, and SHG (SFG) microscopy—allows simultaneous visualization of different structures in a complex biological system.