www.magworldwide.com live cell fluorescence imaging presented by: nicholas beavers – media...
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www.magworldwide.com
Live Cell Fluorescence Imaging
Presented by: Nicholas Beavers – Media Cybernetics, Inc.
Welcome to
Sponsored by
Starting Soon…
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Goals
Understand how to…
1. Select and Set up a Camera
2. Set up the Optics and peripheral Hardware
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Goals cont…
3. Use Software Options utilizing Media Cybernetics InVivo
4. Acquire Live Cell Examples
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Goals cont…
Live Cell Examples:
• Time Lapse and Burst Movies
• 3D Image Stacks
• Multi-Position Images using your:• Slide• Well Plate• Cell Chamber
Z
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Goals cont…
Live Cell Examples:
• Multi-Channel Images
• Triggering and Signaling
Merged using ImagePro Plus
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1) Setup the Camera
When selecting a camera for Live Cell Imaging be sure to check for…
1. Sensitivity: Quantum Efficiency at the Spectral Range (Emissions) you will be using.
• QE: Photon to Electron Conversion
• Spectral Range
e-
CCD
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1) Setup the Camera cont…
2. Linearity: Converting CAPTURED photons into MEASURED gray level.
3. Dynamic range: The number of gray levels in an image or the potential number capable of being recorded by a camera.
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1) Setup the Camera cont…
4. Low Noise: System noise refers to electrons in the CCD signal that have their origins in sources other than the object photons.
Signal-to-Noise Ratio
Object Signal
Noise of Surrounding Background
=
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1) Setup the Camera cont…
5. External triggering: Important for synchronizing your camera with an external device.
6. Full support by the host software: Before purchasing a camera be sure your software package will support it.
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1) Setup the Camera cont…
Camera Setup
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2) Set up the Optics & Hardware
Important to Note:
• Hardware controlled via the software meansrapid transition between settings.
• i.e., Motorized filter wheels controlled via InVivo will lower exposure time and save your specimen from harmful light.
• Keep filter wheel positions close.
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2) Setup the Optics & Hardware cont…
Micro-Environmental Hardware:
•A must in Live Cell Imaging.
•Compatible with the microscope optics.
•Provide the physiological needs of the specimen.
•A few recommendations for micro-environmental control…
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2) Setup the Optics & Hardware cont…
1) Delta T Dish 2) Bioptechs FCS2
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2) Setup the Optics & Hardware cont…
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4) Stable Z3) Lens Warmer
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2) Setup the Optics & Hardware cont…
Setup Optics & Hardware
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3) Use Software Options
There are universal software tools to aidLive Cell Imaging…
•Flat Field Correction
o Used on an image with shading error across the image background.
o The flat field image (dark image) is taken from a featureless background region in the specimen.
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3) Software Options cont…
1. (Raw – Dark) = Corrected Raw(Flat – Dark) = Corrected Flat
(Corrected Raw) / (Corrected Flat)= Resultant Image
2. (Raw/Flat) x (Average of Flat) = Resultant Image
o Result: Even illumination across the image.
Demo in Image Pro
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3) Software Options cont…
Acquisition Types to choose from:
1. Time Lapse and Burst
2. 3D Stacks
3. Multi-Positiono Slideo Well Plateo Cell Chamber
Z
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3) Software Options cont…
Acquisition Types to choose from:
4. Multi-Channel
5. Triggering and Signaling
Demo in Image Pro
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4) Acquire Live Cell Examples
• High Speed Single-probe and multiple-probe fluorescence imaging
• 3D stack acquisition
• 3D time-lapse of a single fluorescent probe
• 3D time-lapse of multiple fluorescent probes
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4) Acquire Live Cell Examples cont…
• 5D acquisitiono 3D stack, Multicolor, Time-lapse
• Slide & Well plate Navigation
• Triggered acquisition dependent on changes in intensities
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4) Acquire Live Cell Examples cont…
Examples
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Review
1. Setup the Camera1. Sensitivity2. Linearity3. Dynamic range4. Low Noise5. External Triggering6. Full support by Software
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Review cont…
3. Setup the Optics & Hardware1. Delta T Open and Closed Dish2. Bioptechs FCS2 & 33. Objective Warmer4. Stable Z for Petri Plates
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Review cont…
3. Use Software Options1. Divide by Flat field2. Time Lapse and Burst Movies3. 3D Stacks4. Multi position Acquisitions5. Multi Channel6. Triggering and Signaling
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Review cont…
4. Acquire Live Cell Images1. High Speed Single-probe and multiple-probe
fluorescence imaging2. 3D stack acquisition3. 3D time-lapse of a single fluorescent probe4. 3D time-lapse of multiple fluorescent probes5. 5D acquisition6. Slide & Well plate Navigation7. Triggered acquisition dependent on changes in
intensities
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Live Cell Fluorescence Imaging
Thank you for attending!
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Presented by: Nicholas Beavers- Media Cybernetics, Inc.