Descriptions for Grant Applications*
Multiphoton Intravital and Tissue Imaging (MITI) Core Facility (MITI Facility Description)
The MITI Core Facility, part of the Preclinical Imaging Center housed within the Fred & Pamela Buffett Cancer Center and Cognitive Neuroscience of Development & Aging Center, is equipped with an Olympus FVMPE-RS Multiphoton Laser Scanning Microscope. This system is specially configured for deep tissue imaging in both living and fixed samples. Dual beam, multi photon excitation is accomplished using a Spectra Physics Dual line InSight X3 near infrared laser. The main output from the InSight laser is a tunable line ranging from 680 to 1300 nm with a second multiphoton excitation line at 1045 nm. This extended excitation range supports both traditional fluorophores (blue to long range red) and fluorescent proteins, as well as second and third harmonic imaging. Potential signal crosstalk during simultaneous imaging is reduced by transitioning to sequential scanning (line or frame). Likewise, this system utilizes a Quadralign 4 axis alignment module to autocorrect potential laser beam misalignment and pixel shift during multi-color excitation.
Equipped with 5, 10 and 25x objectives, Z-axis and xy stage motors, this system can image both intravital and fixed samples. Deep Tissue imaging is facilitated using a highly specialized light path containing silver coated, high reflectance scanner mirrors equipped to increase detection of scattered and low intensity fluorescence. This system uses both a traditional Galvanometer scanner and a high-speed Resonant scanner capable of imaging 512 x 512 pixels at 30 frames per section or up to 438 frames per section when imaging 512 x 32 pixels. Two multi-alkaline and two gallium arsenide phosphide (GaAsP) are available for multichannel detection, the latter delivering optimum quantum efficiency and improved signal to noise ratios.
The Olympus FVMP-RS system is additionally equipped with 458 and 588 nm stimulation laser lines exclusively positioned for photobleaching, light stimulation and/or photoactivation of various fluorescent or caged constructs. In addition to automating a number of imaging procedures, the Olympus software is available for collecting and interacting with various types of data including optical Z-sectioning, 3D imaging, multi-location imaging at individual and multiple time points and fluorescence quantification. A Neurotar Mobile Home Cage allows researchers to integrate real-time imaging (brain, spinal cord) with behavioral/locomotion tracking. Locomotion tracking software facilitating behavioral analyses is available to download/use by researchers.
Multiphoton Intravital and Tissue Imaging (MITI) Reproducibility and Rigor (MITI Rigor Description)
To ensure consistent equipment performance, the MITI Research Core performs weekly maintenance on all instruments and maintains comprehensive preventative maintenance and service contracts with Olympus and Newport/Spectra Physics to maintain the research core’s microscope and laser systems in optimal condition. To further ensure consistent reproducibility and rigor during ongoing/long term imaging experiments (individual studies which may vary greatly in system configuration), both IR laser output (power at a given wavelength) and detector responses (intensity under identical collection parameters) are measured prior to each imaging session and are not allowed to vary by more than 10% throughout the complete imaging process. In addition to maintaining equivalent system functioning, all imaging configurations for a given experimental series are kept identical throughout the imaging series.
* Please notify multiphotonintravital@unmc.edu to discuss research proposal development, inquiries regarding logistics of desired studies, and/or updates in proposed or active funding utilizing the imaging core in a timely fashion.
Details for Developing Data Management and Sharing Plans (MITI_Core Data Managment_2023)
Researchers are responsible for their data once it is collected (i.e. data should be immediately transferred to another location). Everything that happens to your data after collection (i.e., imaging, analyses) is the researcher’s responsibility.
Research data is stored in the Olympus, or IMARIS file formats but can be exported to OME tif (*.oir, *.ome tif, *.ims).
The Olympus FVMPE-RS acquisition workstation is connected to the internet. Data may be securely transferred to individual researchers/research groups and/or temporarily stored (≤ 6 months) using the your personal SharePoint location or BOX account. Data may reside on the acquisition workstation 3 months and BOX location for up to 6 months. After this time, data will be removed from the acquisition workstation without prior notification. Once a month, data will be backed up and/or fully transferred to a network location.
Long-Term Data Transfer and Storage Plans (underway, NOT currently implemented): The AMCF (an affiliated imaging core) is working the RITO to establish additional temporary and long-term biomedical image storage, management, and sharing options using the OMERO platform. OMERO is a critical organizational infrastructure for managing imaging data, is universally/internationally accepted for publication-ready datasets, recognized by existing NIH data repositories, will allow us to eventually offer our own shareable data repository, directly reads/interfaces/integrates all imaging data collected on our imaging core acquisition workstations, and is capable of organizing curated datasets with meta data meeting community standards for biomedical imagery. The OMERO server will automatically read and organize meta data contained within biomedical images from hundreds of different systems (both inside and outside the imaging cores). As soon as the OMERO server is active, researchers will be notified regarding specific features and storage options.
Additional Data Management Considerations
- BEFORE IMAGING, make sure you have an appropriate (funding specific) data sharing and management plan in place. DISCUSS your plan with the MITI core. Each repository has its own requirements with significant variability across data repositories. Researchers must let core staff know prior to imaging if additional meta data is required.
- Per NIH, "Regardless of the mechanism used to share data, each dataset will require documentation. Documentation provides information about the methodology and procedures used to collect the data, details about codes, definitions of variables, variable field locations, frequencies, and the like. The precise content of documentation will vary by scientific area, study design, the type of data collected, and characteristics of the dataset." This is your meta data. When not otherwise stipulated by funding agency, researchers are strongly encouraged to use community metadata standards (reference manuscripts below describe evolving community standards). Community standards generally exceed repository reporting requirements. Individual data repositories often provide guidance regarding appropriate metadata standards.
- Montero Llopis, P., et al. Best practices and tools for reporting reproducible fluorescence microscopy methods. Nat Methods 18, 1463–1476 (2021).
https://rebecca-senft.shinyapps.io/MicCheck/
- Hammer, M., et al. Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model. Nat Methods 18, 1427–1440 (2021).
3) Researcher (funding source)-specific data management and sharing plans may require differing types of meta data/information. Basic system information (instrument type, date, time, excitation type/power, detector and scan settings) are automatically contained in the image meta data. Additional information regarding objective specification (i.e., NA), filter parameters (i.e. center wavelength and bandwidth) can be found on the MITI website under equipment. The MITI will assist researchers in obtaining additional information regarding global system configurations, as needed.
Equipment | VCR | University of Nebraska Medical Center (unmc.edu)
4) Olympus files (*.oir) can be opened outside the MITI using open-source software including ImageJ with bioformats plugin or FIJI, and QuPath. Individual repositories have specific requirements for acceptable file formats. If required by a repository, conversion of image files to opensource file formats such as OME tiffs (Open Microscopy Exchange) can be used to preserve meta data in a universal file format. This option is available in most programs readily interfacing with *.oir files.
https://imagej.net/formats/bio-formats
https://imagej.net/software/fiji/downloads
5) Raw/unaltered image data is maintained in the oir file, if anything other than the initial raw data is analyzed for the final dataset, researchers should verify and record all downstream analysis parameters.
6) Volumetric mosaic images from the UltraMicroscope II/Light Sheet Microscope (TIBA affiliated instrumentation) can be stitched together using ImageJ/FIJI BigStitcher (ome tiff) or using the IMARIS stitch program (requires initial storage as, or conversion to *.ims file type). IMS files can be viewed using free viewer software.
https://imaris.oxinst.com/imaris-viewer
https://imagej.net/plugins/bigstitcher/advanced-stitching
7) Conversion/storage as *.ome tiff can significantly increase files sizes (2-3x). Please ensure adequate storage space and upload times. The AMCF has installed 10 Gb ethernet connections between the new instrumentation (Axioscan whole slide imager, Light Sheet Microscope) and the Data analysis room. Additional high-speed connections will be established as quickly as possible. Many locations on campus are 10 Gb 'ready,' not actively installed/configured. Researchers should verify individual transfer capabilities in their location/building and plan accordingly.