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

Multiphoton laser fluorescence scanning microscopy. Multiphoton Laser Fluorescence Scanning Microscope (MPLFSM):

Focus high energy, femtosecond laser pulse to excite fluorescent dye:

2 photons @ 800 nm ~ 1 photon @ 400 nm

MPLFSM versus other Fluorescence Microscopes:

No out of focusz-sectioning, sharp contrast excitation

Longer l Better penetration in scattering tissue

Smaller excitation Less dye bleaching volumeBetter resolutionMore photons collected

Brightfield vs. MultiphotonTrigeminal Ganglion Neurons loaded with Calcium GreenZeiss Axioplan 2MicroscopeCooled CCD CameraFuzzy images, severe bleachingMultiphoton LaserScanning FluorescenceMicroscopez-sectioning500 mm40 xWaterImmersionObjective

Custom-built multiphoton microscopesUsed in Neuroscience, Immunology, Developmental Biology... Commercial two-photon scopes: $750K Custom-made 2P scope:

The MPScope Software SuiteSoftware choice for custom multiphoton microscope was limited:

ScanImage (K. Svoboda's lab - Matlab)- LabView based software

MPScope:

Comprehensive suite of programs to control custom multiphotonmicroscopes 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. MethodsNguyen, Q.-T. and Kleinfeld, D. (2008) Book chapter in "In vivo optical imaging of brain function"

MPScope: DescriptionMPScope =

MPScan

+

MPView

+

MPFileMicrosoft Windows Platform

Important features of MPScopeHigh 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 ConsiderationsGoal of File Format :

Uncompromizing performance and seamless integration withWindows 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 insidea 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, programmableProperty Information StreamSolves the opaque, non-extensible header problem!Data mining

Interoperability with other applications (I)Data Interchange Via Disk File

MPScan (acquisition):- Save asBMP fileMPView:- Save as:TIFFAVIBMP

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++ clientsA 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- enabledapplication (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 =0frameCount = invoke(mpfile, 'ReadParameter', 'Frame Count')frameCount =150

ConclusionWhat we learned:

It's OK to have a proprietary file format as long as there aresoftware translators for major image formats and softwarebridges 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 ofthe Microsoft Open Specification Promise (2007)