Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.We acknowledge support from the National Institute of Health 5-P41-EB015871-28, 2R01EY017656-06, R21-NS091982-01, 1-U01-NS090438-01, 1U01CA202177-01, and 1R01HL121386-01A1, Singapore-MIT Alliance for Science and Technology, Connecticut Children’s Medical Center, Hamamatsu Corp., and Samsung Advanced Institute of Technology. C.J.R. further acknowledges support from the Wellcome Trust 093831/Z/10/Z

Tumor cell nuclei soften during transendothelial migration

During cancer metastasis, tumor cells undergo significant deformation in order to traverse through endothelial cell junctions in the walls of blood vessels. As cells pass through narrow gaps, smaller than the nuclear diameter, the spatial configuration of chromatin must change along with the distribution of nuclear enzymes. Nuclear stiffness is an important determinant of the ability of cells to undergo transendothelial migration, yet no studies have been conducted to assess whether tumor cell cytoskeletal or nuclear stiffness changes during this critical process in order to facilitate passage. To address this question, we employed two non-contact methods, Brillouin confocal microscopy (BCM) and confocal reflectance quantitative phase microscopy (QPM), to track the changes in mechanical properties of live, transmigrating tumor cells in an in vitro collagen gel platform. Using these two imaging modalities to study transmigrating MDA-MB-231, A549, and A375 cells, we found that both the cells and their nuclei soften upon extravasation and that the nuclear membranes remain soft for at least 24 h. These new data suggest that tumor cells adjust their mechanical properties in order to facilitate extravasation

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.1152sciescopu

Luminescent surfaces with tailored angular emission for compact dark-field imaging devices

Dark-field microscopy is a standard imaging technique widely employed in biology that provides high image contrast for a broad range of unstained specimens1. Unlike bright-field microscopy, it accentuates high spatial frequencies and can therefore be used to emphasize and resolve small features. However, the use of dark-field microscopy for reliable analysis of blood cells, bacteria, algae and other marine organisms often requires specialized, bulky microscope systems, as well as expensive additional components, such as dark-field-compatible objectives or condensers2,3. Here, we propose to simplify and downsize dark-field microscopy equipment by generating the high-angle illumination cone required for dark-field microscopy directly within the sample substrate. We introduce a luminescent photonic substrate with a controlled angular emission profile and demonstrate its ability to generate high-contrast dark-field images of micrometre-sized living organisms using standard optical microscopy equipment. This new type of substrate forms the basis for miniaturized lab-on-chip dark-field imaging devices that are compatible with simple and compact light microscopes

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids

Sucrose Monoester Micelles Size Determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, Rh. As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured

Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging

Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy, two-photon microscopy and optical coherence tomography, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than ~1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO_2) of single vessels in animals and total hemoglobin concentration in humans

Imaging of Zebrafish In Vivo with Second-Harmonic Generation Reveals Shortened Sarcomeres Associated with Myopathy Induced by Statin

We employed second-harmonic generation (SHG) imaging and the zebrafish model to investigate the myopathy caused by statin in vivo with emphasis on the altered microstructures of the muscle sarcomere, the fundamental contractile element of muscles. This approach derives an advantage of SHG imaging to observe the striated skeletal muscle of living zebrafish based on signals produced mainly from the thick myosin filament of sarcomeres without employing exogenous labels, and eliminates concern about the distortion of muscle structures caused by sample preparation in conventional histological examination. The treatment with statin caused a significantly shortened sarcomere relative to an untreated control (1.73±0.09 µm vs 1.91±0.08 µm, P<0.05) while the morphological integrity of the muscle fibers remained largely intact. Mechanistic tests indicated that this microstructural disorder was associated with the biosynthetic pathway of cholesterol, or, specifically, with the impaired production of mevalonate by statins. This microstructural disorder exhibited a strong dependence on both the dosage and the duration of treatment, indicating a possibility to assess the severity of muscle injury according to the altered length of the sarcomeres. In contrast to a conventional assessment of muscle injury using clinical biomarkers in blood, such as creatine kinase that is released from only disrupted myocytes, the ability to determine microstructural modification of sarcomeres allows diagnosis of muscle injury before an onset of conventional clinical symptoms. In light of the increasing prevalence of the incidence of muscle injuries caused by new therapies, our work consolidates the combined use of the zebrafish and SHG imaging as an effective and sensitive means to evaluate the safety profile of new therapeutic targets in vivo

Collagen reorganization at the tumor-stromal interface facilitates local invasion

BACKGROUND: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. METHODS: Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen. RESULTS: We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers. CONCLUSION: The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues