A large‐field polarisation‐resolved laser scanning microscope: applications to CARS imaging

Vito, Giuseppe de; Canta, A; Marmiroli, P; Piazza, Vincenzo

doi:10.1111/jmi.12282

Cited by 9 publications

(13 citation statements)

References 28 publications

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“…Based on these results, RP-CARS microscope analysis was exploited to detect the myelin-associated α values in the sciatic nerve of slow- and fast-progressing mice. α value is an indicator of the myelin molecular order, and it is tightly associated with the myelin health status [ 19 , 20 , 28 ]. Representative RP-CARS images of sciatic nerve longitudinal optical sections are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The samples were then thawed by immersion into warm (~38 °C) Dulbecco’s phosphate buffer saline (D8537; Sigma-Aldrich, Saint Luis, Missouri, U.S.A.) and immobilized with a custom-made electrophysiology-type anchor. The explanted sciatic nerves from C57SOD1 G93A , 129SvSOD1 G93A , and relative NTG mice were observed with an RP-CARS microscope [ 19 ] tuned to CH 2 molecular vibrations yielding the average in-plane orientation anisotropy ( α ) of the chosen molecular vibrations and their average orientation ( φ ) within the sub-micrometric excitation volume [ 20 ]. The acquired z-stacks were analyzed with a custom-made Python software (Python Software Foundation, Beaverton, OR, USA) to threshold the myelin walls and to extract the stack distribution of the α values (we employed the same algorithm described in [ 21 ], but computed the threshold on the α value images).…”

Section: Methodsmentioning

confidence: 99%

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Immune response in peripheral axons delays disease progression in SOD1G93A mice

Nardo

Trolese

Vito

et al. 2016

J Neuroinflammation

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BackgroundIncreasing evidence suggests that the immune system has a beneficial role in the progression of amyotrophic lateral sclerosis (ALS) although the mechanism remains unclear. Recently, we demonstrated that motor neurons (MNs) of C57SOD1G93A mice with slow disease progression activate molecules classically involved in the cross-talk with the immune system. This happens a lot less in 129SvSOD1G93A mice which, while expressing the same amount of transgene, had faster disease progression and earlier axonal damage. The present study investigated whether and how the immune response is involved in the preservation of motor axons in the mouse model of familial ALS with a more benign disease course.MethodsFirst, the extent of axonal damage, Schwann cell proliferation, and neuromuscular junction (NMJ) denervation were compared between the two ALS mouse models at the disease onset. Then, we compared the expression levels of different immune molecules, the morphology of myelin sheaths, and the presence of blood-derived immune cell infiltrates in the sciatic nerve of the two SOD1G93A mouse strains using immunohistochemical, immunoblot, quantitative reverse transcription PCR, and rotating-polarization Coherent Anti-Stokes Raman Scattering techniques.ResultsMuscle denervation, axonal dysregulation, and myelin disruption together with reduced Schwann cell proliferation are prominent in 129SvSOD1G93A compared to C57SOD1G93A mice at the disease onset, and this correlates with a faster disease progression in the first strain. On the contrary, a striking increase of immune molecules such as CCL2, MHCI, and C3 was seen in sciatic nerves of slow progressor C57SOD1G93A mice and this was accompanied by heavy infiltration of CD8+ T lymphocytes and macrophages. These phenomena were not detectable in the peripheral nervous system of fast-progressing mice.ConclusionsThese data show for the first time that damaged MNs in SOD1-related ALS actively recruit immune cells in the peripheral nervous system to delay muscle denervation and prolong the lifespan. On the contrary, the lack of this response has a negative impact on the disease course.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0732-2) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Immune response in peripheral axons delays disease progression in SOD1G93A mice

Nardo

Trolese

Vito

et al. 2016

J Neuroinflammation

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“…Axial and lateral CARS resolution are respectively about 0.70 and 0.28 μm (Wang et al, 2005 ). As CARS relies on the endogenous chemical contrast provided by lipids in the myelin sheath it abolishes the need for staining and dehydration/embedding (the critical step for myelin damage), therefore allowing for more realistic measures of g-ratio and myelin sheath thickness (Wang et al, 2005 ; Bélanger et al, 2009 ; De Vito et al, 2015 ). Additionally, this technique can image myelin in vivo , allowing researchers to follow demyelination in animal models of MS or nerve crush injury (Henry et al, 2009 ; Bélanger et al, 2011 ; Imitola et al, 2011 ).…”

Section: Novel Techniques and Perspectivesmentioning

confidence: 99%

Axon and Myelin Morphology in Animal and Human Spinal Cord

et al. 2017

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Characterizing precisely the microstructure of axons, their density, size and myelination is of interest for the neuroscientific community, for example to help maximize the outcome of studies on white matter (WM) pathologies of the spinal cord (SC). The existence of a comprehensive and structured database of axonal measurements in healthy and disease models could help the validation of results obtained by different researchers. The purpose of this article is to provide such a database of healthy SC WM, to discuss the potential sources of variability and to suggest avenues for robust and accurate quantification of axon morphometry based on novel acquisition and processing techniques. The article is organized in three sections. The first section reviews morphometric results across species according to range of densities and counts of myelinated axons, axon diameter and myelin thickness, and characteristics of unmyelinated axons in different regions. The second section discusses the sources of variability across studies, such as age, sex, spinal pathways, spinal levels, statistical power and terminology in regard to tracts and protocols. The third section presents new techniques and perspectives that could benefit histology studies. For example, coherent anti-stokes Raman spectroscopy (CARS) imaging can provide sub-micrometric resolution without the need for fixation and staining, while slide scanners and stitching algorithms can provide full cross-sectional area of SC. In combination with these acquisition techniques, automatic segmentation algorithms for delineating axons and myelin sheath can help provide large-scale statistics on axon morphometry.

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“…The two beams are synchronized by means of a delay line ("DL") present along the Stokes path and spatially recombined using a 900-nm long-pass dichroic mirror ("DM"). The beams are finally routed to a high-numerical-aperture (NA) objective ("Obj"; C-Achroplan W; 32X, NA=0.85, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) of an inverted microscope (Axio Observer Z1; Carl Zeiss MicroImaging GmbH, Göttingen, Germany) through a pair of galvo-scanning mirrors ("XY"; GVS002; Thorlabs , Newton, New Jersey, U.S.A.), a scan lens, a compensation lens [29] and the tube lens of the microscope. The CARS signal generated from the sample ("S") is collected both in the trans direction, using a condenser lens ("Cond", NA=0.55), and in the epi direction, by means of a dichroic mirror ("DM'"), and then detected with red-sensitive photomultiplier tubes ("PMT"; R3896; Hamamatsu, Hamamatsu City, Japan) after being spectrally filtered in order to remove the pump photons and to select the Raman band of the CH2 bonds (2850 cm -1 , "BP", band-pass filter centred at 650 nm and with a full width at half maximum of 50 nm).…”

Section: Description Of the Rp-cars Microscopementioning

confidence: 99%

RP‐CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease

Vito

Cappello

Tonazzini

et al. 2016

Journal of Biophotonics

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Krabbe disease (KD) is a rare demyelinating sphingolipidosis, often fatal in the first years of life. It is caused by the inactivation of the galactocerebrosidase (GALC) enzyme that causes an increase in the cellular levels of psychosine considered to be at the origin of the tissue-level effects. GALC is inactivated also in the Twitcher (TWI) mouse: a genetic model of KD that is providing important insights into the understating of the pathogenetic process and the development of possible treatments. In this article an innovative optical technique, RP-CARS, is proposed as a tool to study the degree of order of the CH bonds inside the myelin sheaths of TWI-mice sciatic-nerve fibres. RP-CARS, a recently developed variation of CARS microscopy, is able to combine the intrinsic chemical selectivity of CARS microscopy with molecular-bond-spatial-orientation sensibility. This is the first time RP-CARS is applied to the study of a genetic model of a pathology, leading to the demonstration of a post-onset progressive spatial disorganization of the myelin CH bonds. The presented result could be of great interest for a deeper understanding of the pathogenic mechanisms underlying the human KD and, moreover, it is an additional proof of the experimental validity of this microscopy technique. RP-CARS image (2850 cm , CH bonds) of a sciatic-nerve optical longitudinal section from a Twitcher P23 (symptomatic) mouse. Scale bar: 10 microns. The image was constructed by colour-mapping the degree of molecular order of the CH bonds inside the myelin walls, as displayed in the colour bar on the right.

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A large‐field polarisation‐resolved laser scanning microscope: applications to CARS imaging

Cited by 9 publications

References 28 publications

Immune response in peripheral axons delays disease progression in SOD1G93A mice

Immune response in peripheral axons delays disease progression in SOD1G93A mice

Axon and Myelin Morphology in Animal and Human Spinal Cord

RP‐CARS reveals molecular spatial order anomalies in myelin of an animal model of Krabbe disease

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