1. FROM IMAGES TO ANSWERS Live Cell Imaging - Practical Issues Silver Spring & San Diego, June 2005

2. 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

3. 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.)

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

5. 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.

6. Control the Dimensions of Acquisition Use a Combination of XY, Z, Color to control the Dimensions of the Live Cell Acquisition. 3D XY Color

7. 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

8. Setup of Live Cell Imaging in 5 Dimensions

9. Long Time Lapse (24h single channel) 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Select “Start” to acquire Long Time Lapse

10. 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

11. 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

12. 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

13. 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

14. Multi Dimensional Live Cell Analysis

15. 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

16. Ion Channel Imaging 1.Define Background Correction 2.Define Intensity Calibration 3.Define Psuedo Color Display 4.Define Multiple ROIs

17. FRET Imaging 1.Define Background Correction 2.Define Channels 3.Calculate FRET Image

18. Tracking of objects 1.Define Tracking Measurements 2.Define Tracking Options 3.Define Multiple ROIs 4.Filter Data

19. 3D Deconvolution 1.Verify Imported Deconvolution Parameters 2.Define Deconvolution Options 3.Deconvolve

20. 3D Reconstruction and Measurements 1.Verify Imported 3D Parameters 2.Define 3D Options 3.Measure 3D Colocalization

21. Image Pro 3D Suite Automatic Volumetric Measurements Over Time 4D 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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