imaging
3D
of high-throughput
CHALLENGES
Overcome the
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Out-of-focus light or haze
Common challenges of high-throughput 3D imaging
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Reliable automated focusing
Difficult light penetration
Long acquisition time
Analysis bottleneck
High-content data mining
Challenge:
Out-of-focus light or “haze” can skew image analysis
Solution:
AgileOptix spinning disk technology optimized for high resolution, tissue penetrance and high-throughput
Widefield + Digital Confocal
Widefield
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Digital Confocal 2D On-the-fly Deconvolution
>30% more nuclei counted
More visible cells in center of spheroid
Clearer edges
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Spinning disk confocal microscopy
Confocal
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Exposure times are too long and better resolution needed
Water Immersion Objectives:
Signal increases of up to 4X Improved z-resolution & spherical aberrations Sharper, crisper images Available in 20X (0.95 NA), 40X (1.15 NA), and 60X (1.2 NA)
• • • •
20X Plan Apo (0.75 NA)
20X Water Immersion (0.95 NA)
Water immersion objective: Improves image analysis
20X Plan Fluor Air 0.45 NA
20X Plan Apo Air 0.75 NA
20X Plan Apo Water 0.95 NA
Water immersion objective: Increases signal intensity
20x Plan Apo (0.75 NA)
20x Water Immersion (0.95 NA)
Water immersion objective: Improves Z-resolution
20x 0.45 NA Air ELWD
20x 0.75 NA Air Plan Apo
20x 0.95 NA Water Immersion
Water immersion objective: Improves ellipticity to retain sample shape / morphology in 3D
Increases signal to background Reduces exposure time and photobleaching Reduce scan times
• • •
Water immersion objective: Signal increases up to 4X
60x Water Immersion Objective
*All images captured with Confocal 60um and solid state light source
Click to toggle exposure times
Exposure time: 700 ms
60x Air Objective
60x Plan Apo Confocal 60 µm LED Light Source DAPI – 2000 ms FITC – 2000 ms TxRed – 2000 ms
Exposure time: 2000 ms
Water immersion objective
Standard Light Standard Spinning DiskAir ELWD
Laser Light Deep Tissue Penetrating, Confocal Spinning DiskAir ELWD
Deep tissue penetrating confocal disk module
Light penetrance into thick 3D tissue samples
Specialized deep tissue confocal disk module with a geometry designed to:
Improve suppression of out-of-focus light Reduce haze (pinhole crosstalk) Penetrate deeper into thick tissue samples for sharper images
Deep Tissue
Standard
Deep Tissue Confocal Disk Module
60 micron Pinhole Confocal Module
Plate acquisition time is long due to long exposure times for higher quality images
8-channel high-intensity laser light source:
Get sharper images with higher signal-to-background Generate up to a 2x boost in scan speed attributed to reduced exposure times Run FRET experiments using lasers for CFP and YFP
Solid State light source = 1000 ms Max intensity = 5903
Laser light source = 300 ms Max intensity = 13480
Laser provides comparable image quality and higher image intensities with much lower exposure time
Images courtesy of AxoSim, Inc.
LED
Laser
High intensity lasers: Offers speed boost with reduced exposure times
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Solid State light source = 5000 ms
Laser light source = 845 ms
Laser light source S/B ratio 4:1
Increase signal to background Detect low signals in samples Increase Image analysis accuracy
Confocal Laser Light Source U2OS FITC 300 msec, MitoTracker Deep Red (DAPI 690/Mito-Cy5 520/Phall-FITC 300)
Confocal Standard U2OS FITC 300 msec, MitoTracker Deep Red (DAPI 690/Mito-Cy5 520/Phall-FITC 300)
Tap to toggle Laser light source
High intensity lasers: improve signal / background
20x Plan Apo, Confocal 60 micron pinhole (Digital Zoom)
High intensity lasers: Improve contrast, offering greater cellular detail
High intensity lasers
Reliable automated focusing on 3D plate types (Organ-on-a-chip, U-bottom)
Robust High Speed laser hardware AND image-based autofocus
Flexibility – plate compatibility Open interface – modify for customization Image-based focus – greater sample compatibility and variability
Laser auto-focus
Software auto-focus
High-speed laser autofocus
Data sets are often large and complex. Large data sets can take hours to analyze, becoming a bottleneck.
Accelerate analysis speeds by up to 40X with multi-threaded, parallel processing with MetaXpress® PowerCore™ Software. Take your 3D analysis from hours to minutes, reducing any bottlenecks.
Multi-threaded, parallel processing
Powerful analytics combined with an intuitive user interface simplify workflows for advanced phenotypic classification and/or 3D image analysis. Advanced features provide the functionality you need to analyze data at scale and deliver real-time insights without the need for complex pre- or post-processing operations. Put machine learning to work for you and perform complex classification automatically with the optional Phenoglyphs™ module.
In Carta Image Analysis Software Featuring Machine Learning Capabilities
IN Carta Image Analysis Software
Data sets are often large and complex. Stand-alone 3D imaging software is not designed for multi-well, multi-plate data visualization and high content data mining
Review your results with ease, minimizing the need to switch between applications by simultaneously viewing image thumbnails and plate heatmaps. Analyze, interpret, and mine data further with AcuityXpress™ High-Content Informatics software
Data Mining and Visualization
