Optical Neural System Optical Neural System Imaging SurveyImaging Survey
November 15, 1999November 15, 1999Andreas G. NowatzykAndreas G. Nowatzyk
OutlineOutline
• Background and Motivation
• System Overview
• Backscatter Imager
• Fluorescent Imager
• Microtome
• Staining Unit
• Computational Aspects
• Summary
Context• Enabling technologies
– Moore’s Law: Billions of Billions of cycles– Interconnect networks: Multi-Gbytes/s I/O– Optics: confocal microscopy, lasers– Biochemistry: selective staining– Computer science: Image processing– Mechanics: Chip fabrication technologies
• Interdisciplinary research: Combining all these new tools to open up new research areas
Ultimate Goal: Scan a Mouse in Ultimate Goal: Scan a Mouse in 3D3D• Eventually, Eventually,
reverse reverse engineer a engineer a mouse brain, mouse brain, including the including the entire rodent entire rodent nervous nervous systemsystem
Computational ChallengeComputational Challenge
• 2 x 2 x 4 cm2 x 2 x 4 cm3 3 specimen volume specimen volume
• 0.2 0.2 m resolutionm resolution
• = 100,000 x 100,000 x 200,000 = 100,000 x 100,000 x 200,000 voxelsvoxels
• 6 x 12 bit per voxel6 x 12 bit per voxel
• 16,763,806 Gbytes of raw data16,763,806 Gbytes of raw data
• 200:1 compression (lossy)200:1 compression (lossy)
• => 1700 tapes (8mm, 50Gbytes/tape)=> 1700 tapes (8mm, 50Gbytes/tape)
Computational ChallengeComputational Challenge
• 20 Msamples/s per channel (12 bits 20 Msamples/s per channel (12 bits each)each)
• 6 x 64 channels6 x 64 channels
• ~50% scan duty cycle~50% scan duty cycle
• 2x for complex phase sensing2x for complex phase sensing
• = 11,520 Mbytes/s average data = 11,520 Mbytes/s average data raterate
System OverviewSystem Overview
Basic approach, instrument Basic approach, instrument architecture, and computing architecture, and computing
infrastructureinfrastructure
Functional ImagingFunctional Imaging
• Selective labeling with Fluorescent Selective labeling with Fluorescent DyesDyes
• Genetically engineered Labels (GFP)Genetically engineered Labels (GFP)
VS.
Basic ApproachBasic Approach
• Use of light microscopy plus selective Use of light microscopy plus selective functional imagingfunctional imaging
• Five step processFive step process– Bulk imaging into freshly cut sampleBulk imaging into freshly cut sample
– Mechanical sectioning via integrated Mechanical sectioning via integrated microtomemicrotome
– Automated, continuous stainingAutomated, continuous staining
– Functional, fluorescent imagingFunctional, fluorescent imaging
– Data fusion, compression and archival Data fusion, compression and archival storagestorage
Confocal Light MicroscopyConfocal Light Microscopy
• Using one objective lens twiceUsing one objective lens twice
• Point-spread function squaredPoint-spread function squared
Instrument OverviewInstrument Overview
BackscatterImager
Staining Unit
SpecimenHolder
Transfer Unit
Carrier Tape
Microtom
FluorescentImager
Backscatter ImagerBackscatter Imager
Optical system, mechanical Optical system, mechanical components, scan pathcomponents, scan path
Design ObjectivesDesign Objectives
• Need to maximize throughputNeed to maximize throughput
• Maximize practical resolutionMaximize practical resolution
• Optical sectioning into the exposed Optical sectioning into the exposed sample surfacesample surface
XZ Detection FunctionXZ Detection Function
High NA (0.9) objective with pinhole High NA (0.9) objective with pinhole detectordetector
PMT
Acousto-Optical ModulatorAcousto-Optical Modulator
• Exploit Doppler shift to split laser Exploit Doppler shift to split laser into two components with differing into two components with differing frequencyfrequency
RF
Heterodyne DetectionHeterodyne Detection
• Elimination of black-level drift through AC-Elimination of black-level drift through AC-coupled amplifierscoupled amplifiers
• Contrast independent of reference beam Contrast independent of reference beam intensity (within dynamic range of detector)intensity (within dynamic range of detector)
• Optical phase transferred to electrical Optical phase transferred to electrical domaindomain
• Simultaneous capture of phase and Simultaneous capture of phase and magnitudemagnitude
• Enables wave-front reconstruction / Enables wave-front reconstruction / holographyholography
Solid State DetectorSolid State Detector
HP SS-detectory = 2.0328x + 24.826y = 2.0469x + 7.2512
y = 2.036x - 13.267
-130
-120
-110
-100
-90
-80
-70
-60
-50
-40
-30
-20
-70 -60 -50 -40 -30 -20
Optical Input Power [dbm]
Sig
nal
Ou
t [d
bm
]
Sig 0db [dbm] Sig -20db [dbm] Sig -40db [dbm]
Noise floor [dbm/Hz] PMT(700V),0db PMT(700V),-20db
PMT(700V), -40db PMT(700V) noise floor Linear (Sig 0db [dbm])
Linear (Sig -20db [dbm]) Linear (Sig -40db [dbm])
• Better quantum efficiency (~ 85 vs Better quantum efficiency (~ 85 vs 30 %)30 %)
• Better dynamic range (> 120 db)Better dynamic range (> 120 db)
Backscatter Imager SummaryBackscatter Imager Summary
• Optimized for single purposeOptimized for single purpose– less relay optic, no eye piece, no turret, etc.less relay optic, no eye piece, no turret, etc.
• Scan system optimized for bulk Scan system optimized for bulk operationoperation
• Heterodyne detection for improved Z-Heterodyne detection for improved Z-axis resolution (full sampling of the axis resolution (full sampling of the optical phase)optical phase)
• Multiple wavelengthsMultiple wavelengths
• Requires computer controlled alignmentRequires computer controlled alignment
Fluorescent ImagerFluorescent Imager
Optical system, mechanical Optical system, mechanical components, coherent detectioncomponents, coherent detection
Design ObjectivesDesign Objectives
• Need to match throughput of Need to match throughput of backscatter imagerbackscatter imager
• Film based input media, accessible Film based input media, accessible from both sides with symmetric, from both sides with symmetric, optical propertiesoptical properties
• Support for optical sectioningSupport for optical sectioning
ProblemsProblems
• Dye saturationDye saturation
• Signal to noise ratioSignal to noise ratio
• Incoherent signalIncoherent signal
Need to maximize light gathering Need to maximize light gathering ability and quantum efficiency of ability and quantum efficiency of detectordetector
Scan SystemScan System
• Sorry, the few slides were removed Sorry, the few slides were removed due to pending (but incomplete) due to pending (but incomplete) patent applicationspatent applications
Design ObjectivesDesign Objectives
• Integrated with optical systemIntegrated with optical system
• Fully automaticFully automatic
• Precise control of cutting plane and Precise control of cutting plane and related parametersrelated parameters
Transferring fragile Objects to Transferring fragile Objects to FilmFilm• Microtom with stationary knifeMicrotom with stationary knife• Floating pick-upFloating pick-up• Match surface velocity to cutting Match surface velocity to cutting
speedspeed• Minimize surface tensionMinimize surface tension• Electrostatic transferElectrostatic transfer
Knife AssemblyKnife Assembly
6.292
4.694
3.017
far end of
cutting area
1.300
5.700
5.200
2.700
1.82 in.2.051
2.103
1.300
1.719
Microscope EnvironmentMicroscope Environment
• -20 to -30 degree C, controlled -20 to -30 degree C, controlled operating temperatureoperating temperature
• dust free, controlled flow, dry dust free, controlled flow, dry nitrogen atmospherenitrogen atmosphere
• vibration isolationvibration isolation
• issue: sublimation (prevent by index issue: sublimation (prevent by index matching fluid)matching fluid)
Mechanical componentsMechanical components
• Linear air bearingsLinear air bearings
• Voice-coil motor direct driveVoice-coil motor direct drive
• Laser interferometer position Laser interferometer position sensing with 10nm resolutionsensing with 10nm resolution
• Piezo-actuators for knife positioningPiezo-actuators for knife positioning
Film as Sample Slice CarrierFilm as Sample Slice Carrier
• Candidate: DuPont Cronar 410 Candidate: DuPont Cronar 410 polyester flim, gelatin coated, polyester flim, gelatin coated, 100100mm
• Need to maintain sample adhesion Need to maintain sample adhesion during stating processduring stating process
• Optically clear, substitute for cover-Optically clear, substitute for cover-slips (need to be witting correction slips (need to be witting correction range)range)
• Chemically inertChemically inert
Continuous Staining ProcessContinuous Staining Process
• Tight control of process parameters Tight control of process parameters (temperature, flow rate, chemical (temperature, flow rate, chemical concentrations, etc.)concentrations, etc.)
• Clean-room environment: dust-free, Clean-room environment: dust-free, high purity, no manual stepshigh purity, no manual steps
• Uniform, predictable distortionsUniform, predictable distortions
Computational InfrastructureComputational Infrastructure
• Instrument controlInstrument control
• Front-end signal processingFront-end signal processing
Control System OverviewControl System Overview
• Linux basedLinux based
• 3 Functional 3 Functional Blocks:Blocks:– System ControlerSystem Controler
– Signal ProcessingSignal Processing
– File system / File system / DatabaseDatabase
Image processingImage processing
• Deconvolution (CAT scan, MRI)Deconvolution (CAT scan, MRI)• Super-Resolution [Cheeseman et al]Super-Resolution [Cheeseman et al]
• 3D reconstruction3D reconstruction
• Image fusionImage fusion
• 3D Compression3D Compression
Circuit Extraction AlgorithmCircuit Extraction Algorithm
• Linear scan of the data setLinear scan of the data set• Connectivity function integrated Connectivity function integrated
with Bayesian super-resolution with Bayesian super-resolution algorithmalgorithm
Summary of Instrument Summary of Instrument CapabilitiesCapabilities• 6 band (3 backscatter + 3 6 band (3 backscatter + 3
fluorescent labeled) confocal fluorescent labeled) confocal microscopemicroscope
• 0.20.2m (or better) 3D resolutionm (or better) 3D resolution
• Integrated sectioning and stainingIntegrated sectioning and staining
• Image acquisition and processing Image acquisition and processing speed sufficient for large volume speed sufficient for large volume scanningscanning
SummarySummary
• Large scale, high resolution scanning of Large scale, high resolution scanning of biological specimen will become biological specimen will become practicalpractical
• Automated tracing of neurological Automated tracing of neurological systems is conceivablesystems is conceivable
• Provides a clear, long term research Provides a clear, long term research focusfocus
• Significant Research potentialSignificant Research potential
• Intermediate, practical spin-off potentialIntermediate, practical spin-off potential
Top Related