micron-scale-resolution-optical-tomography-entire-mouse-brains-with

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Laser Nanosurgery of Cerebellar Axons <em>In Vivo</em>

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

European Laboratory for Non-linear Spectroscopy (LENS)

National Institute of Optics (CNR-INO)

University of Florence

ICON Foundation, Sesto Fiorentino, Italy

national research council

International Center for Computational Neurophotonics (ICON Foundation)

Laser beams have been demonstrated to be capable of exerting torque as well as forces on microparticles. Using a custom magneto-optic manipulator, we directly measured the torque exerted by laser light on absorbing microspheres as a result of the transfer of spin angular momentum. A general method for measuring torque has been developed, and the experimental apparatus has shown a sensitivity of approximately 1 pN/nm.

High-precision measurements of light-induced torque on absorbing microspheres.

The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 + 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disruption and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation.

Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion.

Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.

Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping.

FÃ¶rster (or Fluorescence) resonance energy transfer (FRET) is a physical process in which energy is transferred nonradiatively from an excited fluorophore, serving as a donor, to another chromophore (acceptor). Among the techniques related to fluorescence microscopy, FRET is unique in providing signals sensitive to intra- and intermolecular distances in the 1-10 nm range. Because of its potency, FRET is increasingly used to visualize and quantify the dynamics of protein-protein interaction in living cells, with high spatio-temporal resolution. Here we describe the physical bases of FRET, detailing the principal methods applied: (1) measurement of signal intensity and (2) analysis of fluorescence lifetime (FLIM). Although several technical complications must be carefully considered, both methods can be applied fruitfully to specific fields. For example, FRET based on intensity detection is more suitable to follow biological phenomena at a finely tuned spatial and temporal scale. Furthermore, a specific fluorescence signal occurring close to the plasma membrane (< or = 100 nm) can be obtained using a total internal reflection fluorescence (TIRF) microscopy system. When performing FRET experiments, care must be also taken to the method chosen for labeling interacting proteins. Two principal tools can be applied: (1) fluorophore tagged antibodies; (2) recombinant fluorescent fusion proteins. The latter method essentially takes advantage of the discovery and use of spontaneously fluorescent proteins, like the green fluorescent protein (GFP). Until now, FRET has been widely used to analyze the structural characteristics of several proteins, including integrins and ion channels. More recently, this method has been applied to clarify the interaction dynamics of these classes of membrane proteins with cytosolic signaling proteins. We report two examples in which the interaction dynamics between integrins and ion channels have been studied with FRET methods. Using fluorescent antibodies and applying FRET-FLIM, the direct interaction of beta1 integrin with the receptor for Epidermal Growth Factor (EGF-R) has been proved in living endothelial cells. A different approach, based on TIRFM measurement of the FRET intensity of fluorescently labeled recombinant proteins, suggests that a direct interaction also occurs between integrins and the ether-a-go-go-related-gene 1 (hERG1) K+ channel.

Optical methods in the study of protein-protein interactions.

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues.

Non-linear fluorescence lifetime imaging of biological tissues.

Second-harmonic generation (SHG) microscopy is a fantastic tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. In fact, SHG combines the advantages of a non-linear microscopy approach with a coherent modality able to probe molecular organization. In this manuscript we review the physical concepts describing SHG from collagen, highlighting how this optical process allows to probe structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward - backward SHG detection, we then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods, highlighting advantages and limitations of the methods applied to tissue imaging and to potential clinical applications.

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy.

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only âˆ¼10 Î¼s, allows detection of interactions as brief as âˆ¼100 Î¼s and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.

Two optical fibre-based probes for spectroscopic measurements on human tissues were designed and developed. The two probes combine fluorescence and Raman spectroscopy in a multimodal approach. The fluorescence excitation was provided by two laser diodes emitting in the UV (378 nm) and in the visible (445 nm) range, while a third source in the NIR (785 nm) was used for Raman. The device was tested on freshly excised human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin. Discrimination of lesions based on their fluorescence and Raman spectra showed good correlation with the subsequent histological examination. (Â© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Combined fluorescence-Raman spectroscopic setup for the diagnosis of melanocytic lesions.

Information processing inside the central nervous system takes place on multiple scales in both space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.

Correlative two-photon and light sheet microscopy.

Atherosclerosis is characterized by the accumulation of lipids within the arterial wall and is commonly diagnosed using standard histology. Non-linear microscopy represents a possible label-free alternative to standard diagnostic methods for imaging various tissue components. Here we employ SHG and CARS microscopy for imaging thin cross-sections of atherosclerotic arterial tissue, demonstrating that both cholesterol deposition in the lumen and collagen in the normal arterial wall can be imaged and discriminated using SHG and CARS microscopy. A simultaneous detection of both forward and backward scattered SHG signals allows distinguishing collagen fibres from cholesterol. Further analysis, based on image pattern evaluation algorithms, is used to characterize collagen organization in the healthy arterial wall against collagen found within plaques. Different values of fibre mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue. (Â© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Francesco Saverio Pavone

European Laboratory for Non-linear Spectroscopy (LENS)

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Laser Nanosurgery of Cerebellar Axons In Vivo