Interoperability of Imaging Data File Formats: The MPScope Case Quoc-Thang NGUYEN Physics Department, UCSD

Multiphoton laser fluorescence scanning microscopy. Multiphoton Laser Fluorescence Scanning Microscope (MPLFSM): Focus high energy, femtosecond laser pulse to excite fluorescent dye: nm ~ nm MPLFSM versus other Fluorescence Microscopes: No out of focusz-sectioning, sharp contrast excitation Longer Better penetration in scattering tissue Smaller excitation Less dye bleaching volumeBetter resolution More photons collected

Brightfield vs. Multiphoton Trigeminal Ganglion Neurons loaded with Calcium Green Zeiss Axioplan 2 Microscope Cooled CCD Camera Fuzzy images, severe bleaching Multiphoton Laser Scanning Fluorescence Microscope z-sectioning 500  m 40 x Water Immersion Objective

Custom-built multiphoton microscopes Used in Neuroscience, Immunology, Developmental Biology...  Commercial two-photon scopes: $750K  Custom-made 2P scope: <$250K Better optics, detectors and layout for in vivo/in vitro experiments. Extensive customization (bringing other laser lines etc...)

The MPScope Software Suite Software choice for custom multiphoton microscope was limited: - ScanImage (K. Svoboda's lab - Matlab) - LabView based software MPScope: - Comprehensive suite of programs to control custom multiphoton microscopes and analyze results - Downloaded by ~ 50 laboratories worldwide (executable or source code) - Extensive community, now on NITRC - Commercial scopes using MPScope Nguyen et al. (2006) MPScope: a versatile software suite for multiphoton microscopy. J. Neurosci. Methods Nguyen, Q.-T. and Kleinfeld, D. (2008) Book chapter in "In vivo optical imaging of brain function"

MPScope: Description MPScope = MPScan + MPView + MPFile Microsoft Windows Platform

Important features of MPScope High performance suite of software: - Very fast, compact (native compiled code) - Symmetrical line scans for fast frame rate - Easy deployment: no need for run-time libraries - Professional looking GUI - Supports wide variety of microscope hardware - Multithreaded - Intel Integrated Performance Primitive Library COM (Component Object Model) -based technologies: - ActiveX Automation client and server - ActiveX Scripting - Structured Storage

File Format Considerations Goal of File Format : Uncompromizing performance and seamless integration with Windows to allow different types of data (imaging, electrophys) to be stored Native (i.e., binary with Intel byte ordering) - Fast - Opaque header Text/XML-based data file: - Bulky (storing binary data is convoluted) - Slower

The MPScope File Format (I) Structured Storage: "A File System Inside a File" - COM: Component Object Model (Windows) - Compound Files: Implementation of Structured Storage Structured Storage - Hierarchical organizations of folders and data streams inside a data file - Previously used as the Microsoft Office file format mechanism, CAD programs, etc...

The MPScope File Format (II) "A File System Inside a File" - Extensible file structure (displacement deltas) - Allow e-phys, imaging + other info to be stored independently - Queriable, extensible, programmable Property Information Stream Solves the opaque, non-extensible header problem! Data mining

Interoperability with other applications (I) Data Interchange Via Disk File MPScan (acquisition): - Save asBMP file MPView: - Save as:TIFF AVI BMP Convenient, but ackward and slow!

Interoperability with other applications (II) Data Interchange from adressing space to adressing space - Faster, more accurate memory to memory transfer - ActiveX Automation + ActiveX Scripting Possibilities: - MPView -> Matlab (images to matrices automatically) One mouse click - MPFile.OCX -> Matlab, Visual Basic, C++ clients A few lines of code (high-level component) - MPScan -> Header additions in near real-time - MPScan -> MySQL database for logging files - MPScan -> whole frames to clients in near real-time

Interoperability with other applications (III) The MPFILE.OCX component - ActiveX Automation standard component - Opens MPScope files from any ActiveX Automation- enabled application (Matlab, C/C++, Visual Basic, Delphi) - High-level interface, no need to think about headers, file offsets - Backward and forward compatible -Easy to use (Matlab example): f = figure( [300, 300, 500, 500]) mpfile = actxcontrol('MPFile.Data', [0, 0, 500, 500], f) openResult = invoke(mpfile, 'OpenMPFile', 'c:\MCC\050105_006.MPD') openResult = 0 frameCount = invoke(mpfile, 'ReadParameter', 'Frame Count') frameCount = 150

Conclusion What we learned: It's OK to have a proprietary file format as long as there are software translators for major image formats and software bridges to other applications. Future Directions: - Enhanced Windows Integration. Thumbnail images in Explorer, Windows 64-bit - Cross-Platform Tools for Mac OSX and Linux. Java libraries for reading Compound Files (Apache POIFS). Compound File specs opened by Microsoft as part of the Microsoft Open Specification Promise (2007)