Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas.

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.

In Vivo Functional Mapping of a Cortical Column at Single-Neuron Resolution.

The cerebral cortex is organized in vertical columns that contain neurons with similar functions. The cellular micro-architecture of such columns is an essential determinant of brain dynamics and cortical information processing. However, a detailed understanding of columns is incomplete, even in the best studied cortical regions, and mostly restricted to the upper cortical layers. Here, we developed a two-photon Ca2+-imaging-based method for the serial functional mapping of all pyramidal layers of the mouse primary auditory cortex at single-neuron resolution in individual animals. We demonstrate that the best frequency-responsive neurons are organized in all-layers-crossing narrow columns, with fuzzy boundaries and a bandwidth of about one octave. This micro-architecture is, in many ways, different from what has been reported before, indicating the region and stimulus specificity of functional cortical columns in vivo.

Primary Traumatic Axonopathy in Mice Subjected to Impact Acceleration: A Reappraisal of Pathology and Mechanisms with High-Resolution Anatomical Methods.

Traumatic axonal injury (TAI) is a common neuropathology in traumatic brain injury and is featured by primary injury to axons. Here, we generated TAI with impact acceleration of the head in male Thy1-eYFP-H transgenic mice in which specific populations of neurons and their axons are labeled with yellow fluorescent protein. This model results in axonal lesions in multiple axonal tracts along with blood-brain barrier disruption and neuroinflammation. The corticospinal tract, a prototypical long tract, is severely affected and is the focus of this study. Using optimized CLARITY at single-axon resolution, we visualized the entire corticospinal tract volume from the pons to the cervical spinal cord in 3D and counted the total number of axonal lesions and their progression over time. Our results divulged the presence of progressive traumatic axonopathy that was maximal at the pyramidal decussation. The perikarya of injured corticospinal neurons atrophied, but there was no evidence of neuronal cell death. We also used CLARITY at single-axon resolution to explore the role of the NMNAT2-SARM1 axonal self-destruction pathway in traumatic axonopathy. When we interfered with this pathway by genetically ablating SARM1 or by pharmacological strategies designed to increase levels of Nicotinamide (Nam), a feedback inhibitor of SARM1, we found a significant reduction in the number of axonal lesions early after injury. Our findings show that high-resolution neuroanatomical strategies reveal important features of TAI with biological implications, especially the progressive axonopathic nature of TAI and the role of the NMNAT2-SARM1 pathway in the early stages of axonopathy.SIGNIFICANCE STATEMENT In the first systematic application of novel high-resolution neuroanatomical tools in neuropathology, we combined CLARITY with 2-photon microscopy, optimized for detection of single axonal lesions, to reconstruct the injured mouse brainstem in a model of traumatic axonal injury (TAI) that is a common pathology associated with traumatic brain injury. The 3D reconstruction of the corticospinal tract at single-axon resolution allowed for a more advanced level of qualitative and quantitative understanding of TAI. Using this model, we showed that TAI is an axonopathy with a prominent role of the NMNAT2-SARM1 molecular pathway, that is also implicated in peripheral neuropathy. Our results indicate that high-resolution anatomical models of TAI afford a level of detail and sensitivity that is ideal for testing novel molecular and biomechanical hypotheses.

Intensify3D: Normalizing signal intensity in large heterogenic image stacks.

Three-dimensional structures in biological systems are routinely evaluated using large image stacks acquired from fluorescence microscopy; however, analysis of such data is muddled by variability in the signal across and between samples. Here, we present Intensify3D: a user-guided normalization algorithm tailored for overcoming common heterogeneities in large image stacks. We demonstrate the use of Intensify3D for analyzing cholinergic interneurons of adult murine brains in 2-Photon and Light-Sheet fluorescence microscopy, as well as of mammary gland and heart tissues. Beyond enhancement in 3D visualization in all samples tested, in 2-Photon in vivo images, this tool corrected errors in feature extraction of cortical interneurons; and in Light-Sheet microscopy, it enabled identification of individual cortical barrel fields and quantification of somata in cleared adult brains. Furthermore, Intensify3D enhanced the ability to separate signal from noise. Overall, the universal applicability of our method can facilitate detection and quantification of 3D structures and may add value to a wide range of imaging experiments.

Biosafety Level 3 setup for multiphoton microscopy in vivo.

Multiphoton microscopy has revealed important insights into cellular behavior in vivo. However, its application in infectious settings often encounters technical, safety and regulatory limitations that prevent its wider use with highly virulent human pathogens. Herein, we present a method that renders multiphoton microscopy in vivo compatible with biosafety level 3 regulations and present an example of its application and potential to visualize a Mycobacterium tuberculosis infection of the mouse lung.

Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-dependent way, unraveling in vitro dissociation constants K(D) of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

Assessing and benchmarking multiphoton microscopes for biologists.

Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.

Calcium imaging of living astrocytes in the mouse spinal cord following sensory stimulation.

Astrocytic Ca(2+) dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca(2+) signaling in living central nervous system. Ca(2+) waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca(2+) levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca(2+) levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca(2+) levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma.

The incidence of malignant melanoma has shown a dramatic increase over the past three decades. Patient outcome and curability depend on early diagnosis. In vivo multiphoton laser tomography represents a recently developed diagnostic tool that allows non-invasive tissue imaging. We aim to demonstrate the application of multiphoton laser tomography for the in vivo differentiation and diagnosis of melanoma. Laser radiation in the near infrared spectrum was used to image endogenous fluorophores by multiphoton excitation. Eighty-three melanocytic skin lesions have been investigated. The results showed distinct morphological differences in melanoma compared with melanocytic nevi. In particular, six characteristic features of malignant melanoma were specified and statistically evaluated. Sensitivity values up to 95% (range: 71-95%) and specificity values up to 97% (range: 69-97%) were achieved for diagnostic classification. Logistic regression analysis was performed to identify the most significant diagnostic criteria. We found that architectural disarray of the epidermis, poorly defined keratinocyte cell borders as well as the presence of pleomorphic or dendritic cells were of prime importance. By means of this procedure accuracy values up to 97% were reached. These findings underline the potential applicability of multiphoton laser tomography in melanoma diagnosis of melanocytic skin lesions.

Exploring the nature of photo-damage in two-photon excitation by fluorescence intensity modulation.

We investigate the relative photo-damage effects during one- and two-photon excitations and demonstrate that there exist fundamental differences in the damage induced by a high repetition rate laser as compared to that of a CW laser. This difference is evident from the degree of enhanced fluorescence intensity achieved by blanking the excitation with an optical chopper. Such an enhancement in fluorescence intensity provides better signal-to-noise ratio that could have immediate applications in multiphoton imaging of live specimens.

Two-photon absorption standards in the 550-1600 nm excitation wavelength range.

We present absolute two-photon absorption (2PA) spectra of 15 commercial organic dyes covering an extended range of excitation wavelengths, 550-1600 nm. The 2PA is measured with an estimated accuracy +/-10% using a femtosecond fluorescence excitation method. The data are corrected for the variations of the pulse duration and the beam profile with the excitation wavelength, and are applicable as reference standards for 2PA measurements.

Extended analysis of benchmark datasets for Agilent two-color microarrays.

BACKGROUND: As part of its broad and ambitious mission, the MicroArray Quality Control (MAQC) project reported the results of experiments using External RNA Controls (ERCs) on five microarray platforms. For most platforms, several different methods of data processing were considered. However, there was no similar consideration of different methods for processing the data from the Agilent two-color platform. While this omission is understandable given the scale of the project, it can create the false impression that there is consensus about the best way to process Agilent two-color data. It is also important to consider whether ERCs are representative of all the probes on a microarray. RESULTS: A comparison of different methods of processing Agilent two-color data shows substantial differences among methods for low-intensity genes. The sensitivity and specificity for detecting differentially expressed genes varies substantially for different methods. Analysis also reveals that the ERCs in the MAQC data only span the upper half of the intensity range, and therefore cannot be representative of all genes on the microarray. CONCLUSION: Although ERCs demonstrate good agreement between observed and expected log-ratios on the Agilent two-color platform, such an analysis is incomplete. Simple loess normalization outperformed data processing with Agilent's Feature Extraction software for accurate identification of differentially expressed genes. Results from studies using ERCs should not be over-generalized when ERCs are not representative of all probes on a microarray.

CALIB: a Bioconductor package for estimating absolute expression levels from two-color microarray data.

UNLABELLED: In this article we describe a new Bioconductor package 'CALIB' for normalization of two-color microarray data. This approach is based on the measurements of external controls and estimates an absolute target level for each gene and condition pair, as opposed to working with log-ratios as a relative measure of expression. Moreover, this method makes no assumptions regarding the distribution of gene expression divergence. AVAILABILITY: http://bioconductor.org/packages/2.0/bioc Open Source.

Goulphar: rapid access and expertise for standard two-color microarray normalization methods.

BACKGROUND: Raw data normalization is a critical step in microarray data analysis because it directly affects data interpretation. Most of the normalization methods currently used are included in the R/BioConductor packages but it is often difficult to identify the most appropriate method. Furthermore, the use of R commands for functions and graphics can introduce mistakes that are difficult to trace. We present here a script written in R that provides a flexible means of access to and monitoring of data normalization for two-color microarrays. This script combines the power of BioConductor and R analysis functions and reduces the amount of R programming required. RESULTS: Goulphar was developed in and runs using the R language and environment. It combines and extends functions found in BioConductor packages (limma and marray) to correct for dye biases and spatial artifacts. Goulphar provides a wide range of optional and customizable filters for excluding incorrect signals during the pre-processing step. It displays informative output plots, enabling the user to monitor the normalization process, and helps adapt the normalization method appropriately to the data. All these analyses and graphical outputs are presented in a single PDF report. CONCLUSION: Goulphar provides simple, rapid access to the power of the R/BioConductor statistical analysis packages, with precise control and visualization of the results obtained. Complete documentation, examples and online forms for setting script parameters are available from http://transcriptome.ens.fr/goulphar/.

Clinically validated benchmarking of normalisation techniques for two-colour oligonucleotide spotted microarray slides.

Acquisition of microarray data is prone to systematic errors. A correction, called normalisation, must be applied to the data before further analysis is performed. With many normalisation techniques published and in use, the best way of executing this correction remains an open question. In this study, a variety of single-slide normalisation techniques, and different parameter settings for these techniques, were compared over many replicated microarray experiments. Different normalisation techniques were assessed through the distribution of the standard deviation of replicates from one biological sample across different slides. It is shown that local normalisation outperformed global normalisation, and intensity-based 'LOWESS' outperformed trimmed mean and median normalisation techniques. Overall, the top performing normalisation technique was a print-tip-based LOWESS with zero robust iterations. Lastly, we validated this evaluation methodology by examining the ability to predict oestrogen receptor-positive and -negative breast cancer samples with data that had been normalised using different techniques.