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Live Cell Imaging - Practical Issues

Silver Spring & San Diego, June 2005

Live Cell Practical Considerations

When selecting which system to use for imaging living cells, one should consider three things: – Sensitivity of Detection– Speed of Acquisition – Viability of the Specimen

See “Light Microscopy Techniques for Live Cell Imaging”, David J. Stephens et al, Science, Vol. 300, pp. 82-86, 4 April 2003

What Do I Need to Control?

– Camera (Gain, Binning & Averaging)– Speed of Acquisition (Exp. Time, Time

Intervals, ROIs)– Dimensions of Acquisition (X,Y,Z,

Time, Wavelength, Location)– Sample Environment (Temperature,

Gases, etc.)

Controlling the Sensitivity of Detection

Use a Combination of Gain, Binning and Averaging to control the Sensitivity of Detection.

Controlling the Speed of Acquisition

Region of Interest

Use a Combination of ROI, Binning, Exposure Time and Frame Rate to control the Speed of Acquisition.

Control the Dimensions of Acquisition

Use a Combination of XY, Z, Color to control the Dimensions of the Live Cell Acquisition.

3D

XY

Color

Control the Viability of the Specimen

Use a Combination of Trend Charting, Signals & Actions and Vital Systems Control to control the Viability of the Specimen.

Trend

Charting

Vital Systems Control

Signals & Actions

Setup of Live Cell Imaging in 5 Dimensions

Long Time Lapse (24h single channel)

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Select “Start” to acquire Long Time Lapse

High Speed Time Lapse (1000 I/30 sec)

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Calculate # of Images

4. Select “Burst” to acquire at High Speeds

High Speed Ratio Time Lapse

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Define Ratio Channels

4. Calculate # of Images

5. Select “Burst” to acquire at High Speeds

3D Time Lapse

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Define # of Planes

4. Define Z Spacing

5. Select 3D Time Lapse

6. Select “Start” to acquire 3D Time Lapse

Multiple Log-points Time Lapse

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Define # of Log Points

4. Select Time Lapse

5. Select “Start Multi Acquire” to acquire Multiple Log-points Time Lapse

Multi Dimensional Live Cell Analysis

Live Ratio Analysis

1. Define Sensitivity of Detection

2. Define Speed of Acquisition

3. Select Time Lapse

4. Define Multiple ROIs

5. Select Trend Feature

6. Display Live or Compute on Movie

Ion Channel Imaging

1. Define Background Correction

2. Define Intensity Calibration

3. Define Psuedo Color Display

4. Define Multiple ROIs

FRET Imaging

1. Define Background Correction

2. Define Channels

3. Calculate FRET Image

Tracking of objects

1. Define Tracking Measurements

2. Define Tracking Options

3. Define Multiple ROIs

4. Filter Data

3D Deconvolution

1. Verify Imported Deconvolution Parameters

2. Define Deconvolution Options

3. Deconvolve

3D Reconstruction and Measurements

1. Verify Imported 3D Parameters

2. Define 3D Options

3. Measure 3D Colocalization

Image Pro 3D Suite Automatic Volumetric Measurements Over Time4D Tracking of objects!!

1. Verify Imported 3D and Time Parameters

2. Define 3D Options

3. Define Tracking Options

4. Track Objects

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