Laser-assisted adsorption by photobleaching.

This chapter presents a simple method to produce substrate-bound protein patterns of micrometer resolution. Our approach uses only low power visible lasers and commercially available reagents to obtain arbitrary patterns of wide concentration range. We provide useful and detailed information on how to assemble the experimental setup to create engineered cell culture substrates using laser scanning or widefield illumination modalities. A protocol that includes the biochemistry, the optics, and the computer programming needed to fabricate functional micropatterns of single and multiple components is explained for readers without experience in optical engineering. Finally, we introduce a novel widefield illumination scheme for fabricating large surface patterns as well as how to make simple patterns using a standard commercial confocal microscope.

Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach.

We present an automated two-dimensional Fourier transform (2D-FT) approach to analyze the local organization of myelinated axons in the spinal cord. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to observe lesions in a commonly used animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). A 2D-FT was applied on the CARS images to find the average orientation and directional anisotropy of the fibers within contiguous image domains. We introduce the corrected correlation parameter (CCP), a measure of the correlation between orientations of adjacent domains. We show that in the EAE animal model of MS, the CCP can be used to quantify the degree of organization/disorganization in the myelin structure. This analysis was applied to a large image dataset from animals at different clinical scores and we show that some descriptors of the CCP probability density function are strongly correlated with the clinical scores. This procedure, compatible with live animal imaging, has been developed to perform local in situ evaluation of myelinated axons afflicted by EAE.

Retrograde and Wallerian axonal degeneration occur synchronously after retinal ganglion cell axotomy.

Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration.

High-content neurite development study using optically patterned substrates.

The study of neurite guidance in vitro relies on the ability to reproduce the distribution of attractive and repulsive guidance molecules normally expressed in vivo. The identification of subtle variations in the neurite response to changes in the spatial distribution of extracellular molecules can be achieved by monitoring the behavior of cells on protein gradients. To do this, automated high-content screening assays are needed to quantify the morphological changes resulting from growth on gradients of guidance molecules. Here, we present the use of laser-assisted protein adsorption by photobleaching (LAPAP) to allow the fabrication of large-scale substrate-bound laminin-1 gradients to study neurite extension. We produced thousands of gradients of different slopes and analyzed the variations in neurite attraction of neuron-like cells (RGC-5). An image analysis algorithm processed bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the longest neurite.

Requirement for functional DNA polymerase eta in genome-wide repair of UV-induced DNA damage during S phase.

The autosomal recessive disorder Xeroderma pigmentosum-variant (XPV) is characterized (i) at the cellular level by dramatic hypermutability and defective recovery of DNA synthesis following UV exposure, and (ii) clinically by abnormal sunlight sensitivity and remarkable predisposition to skin cancer. These phenotypes are clearly attributable to germline mutations in POLH, encoding DNA polymerase eta (poleta) normally required for accurate translesion DNA synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers. Here we demonstrate that patient-derived XPV-skin fibroblasts exposed to 15J/m(2) of UV also exhibit (in addition to abnormal TLS) a significant defect in global-genomic nucleotide excision repair (GG-NER) exclusively during S phase. This cell cycle-specific GG-NER defect can be complemented by ectopic expression of wild-type poleta, but not of poleta variants deficient in either nuclear relocalization or PCNA interaction. We highlight a previous study from our laboratory demonstrating that UV-exposed, ATR-deficient Seckel syndrome fibroblasts, like XPV fibroblasts, manifest strong attenuation of GG-NER uniquely in S phase populations. We now present further evidence suggesting that deficient S phase repair can be rescued in both XPV- and Seckel syndrome-cells if the formation of blocked replication forks post-UV is either prevented or substantially reduced, i.e., following, respectively, pharmacological inhibition of DNA synthesis prior to UV irradiation, or exposure to a relatively low UV dose (5J/m(2)). Our findings in cultured cells permit speculation that abrogation of GG-NER during S phase might partially contribute (in a synergistic manner with defective, atypically error-prone TLS) to the extreme state of UV-hypermutability leading to accelerated skin cancer development in XPV patients. Moreover, based on the overall data, we postulate that loss of either functional poleta or -ATR engenders abnormal persistence of stalled replication forks at UV-adducted sites in DNA which, in turn, can actively and/or passively trigger GG-NER inhibition.

Second harmonic generation microscopy to investigate collagen configuration: a pericarditis case study.

We have used second-harmonic-generation (SHG) to image collagen fibers in pericardial tissue removed from a patient with constrictive pericarditis and compared this to healthy pericardium. SHG imaging allowed for the visualization of collagen fibers without the need for staining or pretreatment. Images were compared to stained histology slides. Collagen fibers in SHG and histology images displayed the same structure and morphology. The mature collagen of the parietal pericardium was easily distinguishable from the new collagen accumulation due to the pericarditis. SHG imaging can provide a convenient and valuable architectural profile of collagen organization.

Rapid multicomponent optical protein patterning.

Cells sense spatial distributions of molecules which trigger signal transduction pathways that induce the cell to migrate or extend by remodelling the cytoskeleton. However, the influence of local and small variations of extracellular protein concentration on chemotaxis is not fully understood, due in part to the lack of simple and precise methods to pattern proteins in vitro. We recently developed a new technology to fabricate such patterns which relies on photobleaching fluorophores to adsorb proteins on a cell culture substrate: laser-assisted protein adsorption by photobleaching (LAPAP). Here we report several key improvements to LAPAP: we created arbitrary patterns made of several different proteins simultaneously, we reduced the fabrication time more than one order of magnitude and we used secondary antibodies to significantly enlarge the spectrum of proteins that can be employed. As a result, multicomponent protein gradients can be produced using reagents that are typically available in life science research laboratories on a standard inverted microscope equipped with a camera port.

Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP.

The study of cellular responses to changes in the spatial distribution of molecules in development, immunology and cancer, requires reliable methods to reproduce in vitro the precise distributions of proteins found in vivo. Here we present a straightforward method for generating substrate-bound protein patterns which has the simplicity required to be implemented in typical life science laboratories. The method exploits photobleaching of fluorescently tagged molecules to generate patterns and concentration gradients of protein with sub-micron spatial resolution. We provide an extensive characterization of the technique and demonstrate, as proof of principle, axon guidance by gradients of substrate-bound laminin peptide generated in vitro using LAPAP.

Sensitive detection of malaria infection by third harmonic generation imaging.

Malaria remains a major health concern worldwide, with 350-500 million cases reported annually in endemic countries. In this study, we report a novel and highly sensitive optical-based detection of malaria-infected blood cells by third harmonic generation (THG) imaging of hemozoin pigment that is naturally deposited by the parasite during its lifecycle. The THG signal from the hemozoin was greater than we have observed in any cell type with signal/noise ratios that reach 1000:1. This method allows a rapid and robust detection of early stage infections of blood cells. The immense nonlinear response of the intrinsic parasitic by-product pigments suggests that automated optical detection by THG could be used for sensitive and rapid screening of parasite infection in blood samples.