Live Cell Fluorescence Imaging Presented by: Nicholas Beavers – Media Cybernetics, Inc. Welcome to Sponsored by Starting Soon…

Goals Understand how to… 1.Select and Set up a Camera 2.Set up the Optics and peripheral Hardware

Goals cont… 3.Use Software Options utilizing Media Cybernetics InVivo 4.Acquire Live Cell Examples

Goals cont… Live Cell Examples: Time Lapse and Burst Movies 3D Image Stacks Multi-Position Images using your: Slide Well Plate Cell Chamber Z

Goals cont… Live Cell Examples: Multi-Channel Images Triggering and Signaling Merged using ImagePro Plus

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-e- CCD

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.

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 =

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.

1) Setup the Camera cont… Camera Setup

2) Set up the Optics & Hardware Important to Note: Hardware controlled via the software means rapid 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.

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…

2) Setup the Optics & Hardware cont… 1) Delta T Dish2) Bioptechs FCS2

2) Setup the Optics & Hardware cont… 4) Stable Z3) Lens Warmer

2) Setup the Optics & Hardware cont… Setup Optics & Hardware

3) Use Software Options There are universal software tools to aid Live 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.

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

3) Software Options cont… Acquisition Types to choose from: 1.Time Lapse and Burst 2.3D Stacks 3.Multi-Position o Slide o Well Plate o Cell Chamber Z

3) Software Options cont… Acquisition Types to choose from: 4.Multi-Channel 5.Triggering and Signaling Demo in Image Pro

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

4) Acquire Live Cell Examples cont… 5D acquisition o 3D stack, Multicolor, Time-lapse Slide & Well plate Navigation Triggered acquisition dependent on changes in intensities

4) Acquire Live Cell Examples cont… Examples

Review 1.Setup the Camera 1.Sensitivity 2.Linearity 3.Dynamic range 4.Low Noise 5.External Triggering 6.Full support by Software

Review cont… 2.Setup the Optics & Hardware 1.Delta T Open and Closed Dish 2.Bioptechs FCS2 & 3 3.Objective Warmer 4.Stable Z for Petri Plates

Review cont… 3.Use Software Options 1.Divide by Flat field 2.Time Lapse and Burst Movies 3.3D Stacks 4.Multi position Acquisitions 5.Multi Channel 6.Triggering and Signaling

Review cont… 4.Acquire Live Cell Images 1.High Speed Single-probe and multiple-probe fluorescence imaging 2.3D stack acquisition 3.3D time-lapse of a single fluorescent probe 4.3D time-lapse of multiple fluorescent probes 5.5D acquisition 6.Slide & Well plate Navigation 7.Triggered acquisition dependent on changes in intensities

Live Cell Fluorescence Imaging Thank you for attending! Sponsored by For more information, contact: Presented by: Nicholas Beavers- Media Cybernetics, Inc.