Many current biomedical applications require the development of advanced non-linear optical microscopy methods to provide functional and non-invasive imaging of live tissues with sub-cellular resolution. In the first part of the talk I will present recent advances in label-free metabolic imaging of living tissues by two-photon fluorescence lifetime microscopy (2P-FLIM) of endogenous biomarkers. We implement simultaneous two-photon excitation of NAD(P)H and FAD by wavelength mixing to acquire 2P-FLIM data of the two biomarkers at the same time and perform efficient multiparametric metabolic imaging in dynamic biological system. We demonstrate how 2P-FLIM of the two metabolic coenzymes reveals the richness and complexity of several metabolic processes in intact tissues with minimal phototoxicity and can be implemented non-invasively for longitudinal studies in vitro and in vivo such as stem cell differentiation, T cell activation and embryo development. In the second part of the talk I will present recent advances in polarization resolved second harmonic generation (pSHG) and modelling from protein molecular structure. We establish a general multi-scale numerical framework relating the micrometer-scale SHG measurements at the optical wavelength to the atomic-scale and molecular structure of the proteins under study and their supramolecular arrangement.
KEYWORDS: Red blood cells, Oxygenation, Microscopy, Tissues, Absorption, Third harmonic generation, Sum frequency generation, Spatial resolution, Resonance enhancement, Multiphoton microscopy
We present color third-order sum-frequency generation (color TSFG) microscopy, a multiphoton imaging strategy based on the simultaneous detection of several third-order coherent signals produced by two synchronized femtosecond pulse trains. We demonstrate that it can be used to obtain red blood cell (RBC)-specific label-free contrast in live zebrafish and is a promising tool for probing RBC oxygenation.
Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess overtime the UVA1- induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of the two endogenous biomarkers nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios We measure NAD(P)H and FAD fluorescence lifetime and fraction of bound coenzymes both in keratinocytes in the epidermis basal layer and in fibroblasts in the dermis superficial layer. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to OXPHOS or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamics and sensitivities to skin cell types and UVA1 dose. While FAD biomarker is UVA1 dose-dependent in keratinocytes, NAD(P)H biomarker shows earlier time points modulation in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter MCE prevents UVA1 metabolic stress response from occurring.
The application range of P-THG microscopy has been so far restricted to studies on molecular order and anisotropy of static specimen removed from their biological environment. Slow polarization commutation limits the investigation of highly dynamic systems because of motion artifacts. Here we have developed a new fast-P-THG microscope enabling efficient in vivo studies in dynamic biological samples. Our P-THG scheme benefits from a built-in EOM that switch polarization states at kilohertz between image lines to provide artefact-free P-THG images with micrometric resolution. Furthermore, we have developed a fast Fourier analysis enabling rapid P-THG processing to quantify lipid order and angular maps. We demonstrated that fast-P-THG is suitable in two major applications. Using first a linear polarization configuration, fast P-THG imaging revealed molecular order changes in MLVs undergoing phase-transition upon heating despite sample distortions. Anisotropy properties of small endogenous microparticles swimming in the otolith cavity embryos were also reported in early zebrafish embryos. A second configuration with linear-circular polarization commutation enabled efficient detection of birefringent media such as anisotropic vesicles in C-elegans gut cells.
Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.
We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.
We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.
Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon
microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in
vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly
presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have
developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the
disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have
been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as
deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue
imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more
shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in
overlapping tissue regions) between the different technologies.
We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers in
conjunction with image segmentation and the concept of cell phasor for deriving metabolic maps of cells and living
tissues in vivo. In issues we identify and separate intrinsic fluorophores such as collagen, retinol, retinoic acid,
porphyrin, flavins, free and bound nicotinamide adenine dinucleotide (NADH). Metabolic signatures of tissues are
obtained by calculating the phasor fingerprint of single cells and by mapping the relative concentration of
metabolites. This method detects small changes in metabolic signatures and redox states of cells. Phasor fingerprints
of stem cells cluster according to their differentiation state in a living tissue such as the C. elegans germ line and the
crypt base of small intestine and colon. Phasor FLIM provides a label-free and fit-free sensitive method to identify
metabolic states of cells and to classify stem cells, normal differentiated cells and cancer cells both in vitro and in a
live tissue. Our method could identify symmetric and asymmetric divisions, predict cell fate and identify pre-cancer
stages in vivo. This method is a promising non-invasive optical tool for monitoring metabolic pathways during
differentiation and carcinogenesis, for cell sorting and high throughput screening.
Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored
with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of
proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries
and provides selective information on the structural order and orientation of the emitting proteins and the
dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We
characterise the polarization-dependence of SHG intensity in three different physiological states: resting,
rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The
orientation of motor domains of the myosin molecules is dependent on their physiological states and
modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different
molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor.
We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from
its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the
fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We
estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the
dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an
isometric contraction.
The structural modifications of the collagen lattice induced in corneal stroma by laser welding were investigated with second-harmonic-generation (SHG) imaging. Corneal laser welding lies in the staining of the wound with a saturated solution of ICG followed by irradiation with a near-infrared-laser operated in continuous (CWLW) or pulsed (PWL) mode. After CWLW the lamellar arrangement was lost although a densely packing of collagen bundles increasingly disordered remained still clearly visible pointing out the lack of complete collagen denaturation. PLW produced welding spots which were characterized by a severe loss of SHG signal suggesting the occurrence of a complete collagen denaturation.
The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic
Generation (SHG). As previously found (Vanzi et al., J. Muscle Cell Res. Motil. 2006) by fractional
extraction of proteins, myosin is the source of SHG signal. A full characterization of the polarization-dependence
of the SHG signal can provide very selective information on the orientation of the emitting
proteins and their dynamics during contraction. We developed a line scan polarization method, allowing
measurements of a full polarization curve in intact muscle fibers from skeletal muscle of the frog to
characterize the SHG polarization dependence on different physiological states (resting, rigor and isometric
tetanic contraction). The polarization data have been interpreted by means of a model in terms of the
average orientation of SHG emitters.The different physiological states are characterized by distinct patterns
of SHG polarization. The variation of the orientation of emitting molecules in relation to the physiological
state of the muscle demonstrates that one part of SHG signal arises from the globular head of the myosin
molecule that cross-links actin and myosin filaments. The dependence of the SHG modulation on the
degree of overlap between actin and myosin filaments during an isometric contraction, provides the
constraints to estimate the fraction of myosin heads generating the isometric force in the active muscle
fiber.
V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, C. Tesi, N. Piroddi, C. Poggesi, C. Castiglioni, A. Milani, M. Linari, G. Piazzesi, V. Lombardi, F. Pavone
The intrinsically ordered arrays of proteins in skeletal muscle allows imaging of this tissue by Second Harmonic
Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution
of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very
powerful tool in the investigation of structural dynamics occurring in muscle during active production of force. A full
characterization of the polarization-dependence of the SHG signal represents a very selective information on the
orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of
muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are
obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in
terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber
axis. Optimizing the setup for accurate polarization measurements with SHG, we developed a line scan imaging method
allowing measurement of SHG polarization curves in different physiological states. We demonstrate that muscle fiber
displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.
Valentina Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, C. Tesi, N. Pirrodi, C. Poggesi, C. Castiglioni, A. Milani, M. Linari, G. Piazzesi, V. Lombardi, F. Pavone
The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation
(SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the
molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very
powerful tool in the investigation of structural dynamics occurring in muscle during active production of force and/or
shortening. A full characterization of the polarization-dependence of the SHG signal represents a very selective
information on the orientation of the emitting proteins and their dynamics during contraction, provided that different
physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here
polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by
means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical
symmetry about the fiber axis. The setup is optimized for accurate polarization measurements with SHG, combined with
a line scan imaging method allowing acquisition of SHG polarization curves in different physiological states. We
demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from
rest to isometric contraction.
The intrinsically ordered arrays of proteins (mainly actin and myosin) constituting the myofibrils within muscle cells are at the basis of a strong Second Harmonic Generation (SHG) from muscle fibers and isolated myofibrils. We have characterized the SHG signal with regard to its polarization and potential source within the muscle cell. The lateral resolution that can be achieved through SHG imaging of muscle strongly depends on sample depth. In fact, a comparison between intact muscle fibers and single myofibrils demonstrates that, whereas in both cases the alternation of dark I bands and bright A bands is visible, the contours of these bands are much better resolved in myofibrils than in fibers. Further, imaging of myofibrils revealed the presence of a darker zone in the centre of the A band. These effects of scattering by tissue on the image resolution were also studied with regard to the polarization of the SHG signal. The polarization-dependence of SHG intensity represents a powerful tool for the investigation of the structural dynamics occurring in the emitting proteins during the active cycle of muscle contraction. The prospective to perform functional studies requires a complete characterization of the effects of scattering and possibly multiple emitting populations on the measured SHG signal. Also, SHG is extremely sensitive to the degree of order present in the filament array, offering an interesting potential in the development of non-invasive tools for the diagnosis of degenerative diseases affecting skeletal muscles.
In eukaryotic cells, proper position of the mitotic spindle and the division plane is necessary for successful cell division and development. In this work the nature of forces governing the positioning and elongation of the mitotic spindle and the spatio-temporal regulation of the division plane positioning in fission yeast was studied. By using a mechanical perturbations induced by laser dissection of the spindle and astral microtubules, we found that astral microtubules push on the spindle poles. Further, laser dissection of the spindle midzone induced spindle collapse inward. This suggests that the spindle is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Exploiting a combination of non-linear microscopy and optical trapping, we performed an optical manipulation procedure designed to displace the cell nucleus away from its normal position in the center of the cell. After the laser-induced displacement, the nucleus typically returned towards the cell center, in a manner correlated with the extension of a microtubule from the nucleus to the closer tip of the cell. This observation suggests that the centering of the nucleus is provided by microtubule pushing force. Moreover the cells in which the nucleus was displaced during interphase displayed asymmetric division, whereas when the nucleus was displaced during late prophase or metaphase, the division plane formed at the cell center as in non-manipulated cells. This result suggests that in fission yeast the division plane is selected before pro-metaphase and that the signal is not provided by the mitotic spindle.
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