In vivo imaging of white matter is important for the mechanistic understanding of demyelination and evaluation of remyelination therapies. Although white matter can be visualized by a strong coherent anti-Stokes Raman scattering (CARS) signal from axonal myelin, in vivo repetitive CARS imaging of the spinal cord remains a challenge due to complexities induced by the laminectomy surgery. We present a careful experimental design that enabled longitudinal CARS imaging of de- and remyelination at single axon level in live rats. In vivo CARS imaging of secretory phospholipase A2 induced myelin vesiculation, macrophage uptake of myelin debris, and spontaneous remyelination by Schwann cells are sequentially monitored over a 3 week period. Longitudinal visualization of de- and remyelination at a single axon level provides a novel platform for rational design of therapies aimed at promoting myelin plasticity and repair.

High-frequency electrical stimulation is becoming a promising therapy for neurological disorders, however the response of the central nervous system to stimulation remains poorly understood. The current work investigates the response of myelin to electrical stimulation by laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging of myelin in live spinal tissues in real time. Paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation. Retraction was seen to begin minutes after the onset of stimulation and continue for up to 10 min after stimulation was ceased, but was found to reverse after a 2 h recovery period. The myelin retraction resulted in exposure of Kv 1.2 potassium channels visualized by immunofluorescence. Accordingly, treating the stimulated tissue with a potassium channel blocker, 4-aminopyridine, led to the appearance of a shoulder peak in the compound action potential curve. Label-free CARS imaging of myelin coupled with multiphoton fluorescence imaging of immuno-labeled proteins at the nodes of Ranvier revealed that high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down.

The integration of near IR picosecond pulse excitation, collinear beam geometry, epi-detection, and laser-scanning has produced a coherent anti-Stokes Raman scattering (CARS) microscope with a detection sensitivity of 105 vibrational oscillators, sub-micron 3D resolution, and video-rate acquisition speed. The incorporation of spectral detection and other imaging modalities has added versatility to the CARS microscope. These advances allowed sensitive interrogation of biological samples, particularly lipids that have a high density of CH2 groups. With initial applications to membrane domains, lipid bodies, demyelinating diseases, obesity, and cardiovascular diseases, CARS microscopy is poised to become a powerful bio-imaging tool with the availability of a multifunctional, affordable, easy-to-operate CARS microscope, and the development of CARS endoscopy for in vivo diagnosis.

Background Lipid-rich tumours have been associated with increased cancer metastasis and aggressive clinical behaviours. Nonetheless, pathologists cannot classify lipid-rich tumours as a clinically distinctive form of carcinoma due to a lack of mechanistic understanding on the roles of lipids in cancer development. Methods Coherent anti-Stokes Raman scattering (CARS) microscopy is employed to study cancer cell behaviours in excess lipid environments in vivo and in vitro . The impacts of a high fat diet on cancer development are evaluated in a Balb/c mice cancer model. Intravital flow cytometry and histology are employed to enumerate cancer cell escape to the bloodstream and metastasis to lung tissues, respectively. Cancer cell motility and tissue invasion capability are also evaluated in excess lipid environments. Results CARS imaging reveals intracellular lipid accumulation is induced by excess free fatty acids (FFAs). Excess FFAs incorporation onto cancer cell membrane induces membrane phase separation, reduces cell-cell contact, increases surface adhesion, and promotes tissue invasion. Increased plasma FFAs level and visceral adiposity are associated with early rise in circulating tumour cells and increased lung metastasis. Furthermore, CARS imaging reveals FFAs-induced lipid accumulation in primary, circulating, and metastasized cancer cells. Conclusion Lipid-rich tumours are linked to cancer metastasis through FFAs-induced physical perturbations on cancer cell membrane. Most importantly, the revelation of lipid-rich circulating tumour cells suggests possible development of CARS intravital flow cytometry for label-free detection of early-stage cancer metastasis.

Spinal cord injury results in immediate disruption of neuronal membranes, followed by extensive secondary neurodegenerative processes. A key approach for repairing injured spinal cord is to seal the damaged membranes at an early stage. Here, we show that axonal membranes injured by compression can be effectively repaired using self-assembled monomethoxy poly(ethylene glycol)-poly(d,l-lactic acid) di-block copolymer micelles. Injured spinal tissue incubated with micelles (60 nm diameter) showed rapid restoration of compound action potential and reduced calcium influx into axons for micelle concentrations much lower than the concentrations of polyethylene glycol, a known sealing agent for early-stage spinal cord injury. Intravenously injected micelles effectively recovered locomotor function and reduced the volume and inflammatory response of the lesion in injured rats, without any adverse effects. Our results show that copolymer micelles can interrupt the spread of primary spinal cord injury damage with minimal toxicity.

Multimodal nonlinear optical (NLO) imaging is poised to become a powerful tool in bioimaging given its ability to capitalize on the unique advantages possessed by different NLO imaging modalities. The integration of different imaging modalities such as two-photon-excited fluorescence, sum frequency generation, and coherent anti-Stokes Raman scattering on the same platform can facilitate simultaneous imaging of different biological structures. Parameters to be considered in constructing a multimodal NLO microscope are discussed with emphasis on achieving a compromise in these parameters for efficient signal generation with each imaging modality. As an example of biomedical applications, multimodal NLO imaging is utilized to investigate the central nervous system in healthy and diseased states.

Obesity is an established risk factor for breast cancer incidence and mortality. However, the mechanism that links obesity to tumorigenesis is not well understood. Here we combined nonlinear optical imaging technologies with an early-onset diet-induced obesity breast cancer animal model to evaluate the impact of obesity on the composition of mammary gland and tumor stroma. Using coherent anti-Stokes Raman scattering and second harmonic generation on the same platform, we simultaneously imaged mammary adipocytes, blood capillaries, collagen fibrils, and tumor cells without any labeling. We observed that obesity increases the size of lipid droplets of adipocytes in mammary gland and collagen content in mammary tumor stroma, respectively. Such impacts of obesity on mammary gland and tumor stroma could not be analyzed using standard two-dimensional histologic evaluation. Given the importance of mammary stroma to the growth and migration of tumor cells, our observation provides the first imaging evidence that supports the relationship between obesity and breast cancer risk.

Gold nanorods coated with cetyltrimethylammonium bromide (CTAB), a cationic micellar surfactant used in nanorod synthesis, were rapidly and irreversibly internalized by KB cells via a nonspecific uptake mechanism. Internalized nanorods near the cell surface were monitored by two-photon luminescence (TPL) microscopy and observed to migrate toward the nucleus with a quadratic rate of diffusion. The internalized nanorods were not excreted but formed permanent aggregates within the cells, which remained healthy and grew to confluence over a 5-day period. Nonspecific nanorod uptake could be greatly reduced by displacing the CTAB surfactant layer with chemisorptive surfactants, particularly by the conjugation of poly(ethylene glycol) chains onto nanorods using in situ dithiocarbamate formation.

Coherent anti‐Stokes Raman scattering (CARS) microscopy, which allows vibrational imaging of myelin sheath in its natural state, was applied to characterize lysophosphatidylcholine (lyso‐PtdCho)‐induced myelin degradation in tissues and in vivo. After the injection of lyso‐PtdCho into ex vivo spinal tissues or in vivo mouse sciatic nerves, myelin swelling characterized by the decrease of CARS intensity and loss of excitation polarization dependence was extensively observed. The swelling corresponds to myelin vesiculation and splitting observed by electron microscopy. The demyelination dynamics were quantified by the increase of g ratio measured from the CARS images. Treating spinal tissues with Ca2+ ionophore A23187 resulted in the same kind of myelin degradation as lyso‐PtdCho. Moreover, the demyelination lesion size was significantly reduced upon preincubation of the spinal tissue with Ca2+ free Krebs' solution or a cytosolic phospholipase A2 (cPLA2) inhibitor or a calpain inhibitor. In accordance with the imaging results, removal of Ca2+ or addition of cPLA2 inhibitor or calpain inhibitor in the Krebs' solution remarkably increased the mean compound action potential amplitude in lyso‐PtdCho treated spinal tissues. Our results suggest that lyso‐PtdCho induces myelin degradation via Ca2+ influx into myelin and subsequent activation of cPLA2 and calpain, which break down the myelin lipids and proteins. The current work also shows that CARS microscopy is a potentially powerful tool for the study of demyelination. © 2007 Wiley‐Liss, Inc.

Plasmon-resonant gold nanorods have outstanding potential as multifunctional agents for image-guided therapies. Nanorods have large absorption cross sections at near-infrared (NIR) frequencies, and produce two-photon luminescence (TPL) when excited by fs-pulsed laser irradiation. The TPL signals can be detected with single-particle sensitivity, enabling nanorods to be imaged in vivo while passing through blood vessels at subpicomolar concentrations. Furthermore, cells labeled with nanorods become highly susceptible to photothermal damage when irradiated at plasmon resonance, often resulting in a dramatic blebbing of the cell membrane. However, the straightforward application of gold nanorods for cell-specific labeling is obstructed by the presence of CTAB, a cationic surfactant carried over from nanorod synthesis which also promotes their nonspecific uptake into cells. Careful exchange and replacement of CTAB can be achieved by introducing oligoethyleneglycol (OEG) units capable of chemisorption onto nanorod surfaces by in situ dithiocarbamate formation, a novel method of surface functionalization. Nanorods with a dense coating of methyl-terminated OEG chains are shielded from nonspecific cell uptake, whereas nanorods functionalized with folate-terminated OEG chains accumulate on the surface of tumor cells overexpressing their cognate receptor, with subsequent delivery of photoinduced cell damage at low laser fluence.

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.

We report in vivo molecular imaging of mouse sciatic nerve by epi-detected coherent anti-Stokes Raman Scattering (E-CARS) microscopy with vibrational selectivity, high signal-to-background ratio, 3D spatial resolution, and real-time imaging capability. The large CARS signal from the CH2 stretch vibration allows highly sensitive and selective imaging of the myelin membrane which possesses a high lipid to protein ratio. The underlying contrast mechanism of in vivo CARS is explored by 3D imaging of fat cells that surround the nerve as well as dermal adipocytes in the mouse ear. Simultaneous E-CARS imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibers were performed in vivo without any labeling. Finally, we show that CARS microscopy is able to distinguish between healthy myelin and disintegrated myelin induced by lysolecithin based on decrease in E-CARS intensity as well as loss of dependence on excitation polarization. Our system provides a multimodality in vivo imaging tool for studying neurodegenerative disorders.

Light-activated therapies can be used to eradicate diseased cells and tissues in a noninvasive manner. Much attention has been focused on the emerging potential of photothermolysis (also referred to as optical hyperthermia), which involves the conversion of absorbed light into heat via nonradiative mechanisms. Photoactivated effects can be localized and intensified by employing exogenous agents with large absorption cross sections, confining damage to areas of interest with minimal collateral effects.[1] In particular, targeted photothermolysis may be most effective when mediated by photothermal agents that absorb strongly at near infrared (NIR) frequencies, to enable deeper penetration into biological tissues.[2]

Plasmon-resonant gold nanorods, which have large absorption cross sections at near-infrared frequencies, are excellent candidates as multifunctional agents for image-guided therapies based on localized hyperthermia. The controlled modification of the surface chemistry of the nanorods is of critical importance, as issues of cell-specific targeting and nonspecific uptake must be addressed prior to clinical evaluation. Nanorods coated with cetyltrimethylammonium bromide (a cationic surfactant used in nanorod synthesis) are internalized within hours into KB cells by a nonspecific uptake pathway, whereas the careful removal of cetyltrimethylammonium bromide from nanorods functionalized with folate results in their accumulation on the cell surface over the same time interval. In either case, the nanorods render the tumor cells highly susceptible to photothermal damage when irradiated at the nanorods' longitudinal plasmon resonance, generating extensive blebbing of the cell membrane at laser fluences as low as 30 J/cm2.

We demonstrate laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging with two excitation laser beams delivered by a large-mode-area photonic crystal fiber. The group-velocity dispersion and self-phase modulation effects are largely suppressed due to the large mode area of the fiber and the use of picosecond pulses. The fiber delivery preserves the signal level and image spatial resolution well. High-quality images of live spinal cord tissues are acquired using the fiber-delivered laser source. Our method provides a basic platform for developing a flexible and compact CARS imaging system.

Haifeng Wang et al. use the bright two-photon luminescence of gold nanorods to turn these rods into probes for in vitro and in vivo imaging. Wang et al. determined that one gold nanorod has a two-photon luminescence signal 58 times brighter than that of a two-photon fluorescence signal from a single rhodamine molecule. The brightness is due to a plasmon-enhanced two-photon absorption cross section. Wang et al. excited and imaged prepared nanorods with an 830-nm laser-scanning microscope, using this setup to image the blood vessels of a live mouse after nanorod injection into the tail vein. Broadband emissions were detected from individual nanorods found to be flowing in mouse ear blood vessels. The rods were visible 5 min after the injection and dissipated fully by 30 min. The nanorods’ brightness depended on the incident polarization, providing a way to calculate the rods’ orientation in a given sample. The authors suggest that the rods’ brightness and the ability to measure the nanorods’ orientation may advance biological imaging. — P.D.

Gold nanorods excited at 830 nm on a far-field laser-scanning microscope produced strong two-photon luminescence (TPL) intensities, with a cos(4) 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.