1. IMAGE INFORMATICS SOLUTIONS Live Cell Imaging

2. All About Live Cell Imaging Study & understanding the structure, function, and behavior of living organisms, using evolving advances in technology, such as the development of imaging systems and fluorescent probes at the cellular and sub cellular levels. E.g. Dynamic Fluorescence Cell Motility Analysis Automated Multiwell plate scanning for increased throughput FRET analysis of molecular interaction Mr. Wim van Egmond Rotterdam, The Netherlands Specimen: Living Volvox colony (green algae) Technique: Differential interference contrast, 100x Objective

3. All About Live Cell Imaging Traditionally we have been using biochemical and genetic approaches from in vitro reconstitution of cellular processes. However, these techniques only provide a static, snapshot view of cells. coronal section of rat hippocampus tissue

4. All About Live Cell Imaging Being able to observe processes as they happen within the cell by light microscopy adds a vital extra dimension to our understanding of cell function. BEFORE AFTER ? MISSING LINK

5. All About Live Cell Imaging Live cell imaging goes far beyond techniques: Widefield, confocal, deconvolution, 2-photon and fluorescent proteins. Molecular interactions. Molecular control of cellular events. Cell interactions (Tumor/T cell). Rapid throughput, cellular assays. Complex multi parametric questions.

6. Live Cell Imaging Systems When selecting which system to use for imaging living cells, one should consider … –Sensitivity of detection required –Speed of acquisition –Viability of the specimen –System Integration: A live cell imaging system should consist of components that ensure compatibility with each other – allowing you to focus on research and not on integration and system configuration –Flexibility: flexibility should be available to take advantage of new technologies, hardware and experimental techniques –Support: Availability of technical, applications, web support with a large worldwide user group.

7. Live Cell Imaging Systems The collaboration provides systems for a wide range of live cell imaging experiments: - High-speed acquisition of images over time (time lapse) - Ratio imaging - FRET - Fast deconvolution of image stacks - Multiple dimension acquisition (X, Y, Z, time, λ, scan area, stage position) - Cell motility - TIRFM, DSU, 3D Construction and Image Analysis

8. Image-Pro Family Software Series Perfect for Basic Imaging or Capture Stations More Advanced than Image- Pro Express, Includes Support for Plug-Ins The Ultimate Imaging Package, Includes Support for Macros and Plug-Ins

9. Image-Pro Family Bundles Image-Pro Advanced Microscopy Suite This complete solution for high-level microscopy analysis and acquisition combines Image-Pro Plus 5.1 with powerful tools for multi-dimensional microscope control, image deconvolution, and 3D rendering.Image-Pro Plus 5.1 Image-Pro Multi-Dimensional Acquisition Ideal for automated microscopy work, this package combines Image-Pro Plus 5.1 with tools for multi-dimensional microscope control and acquisition.Image-Pro Plus 5.1

10. Image-Pro Family Bundles Image-Pro 3D Suite Ideal for deconvolution and interactive control and analysis of volume stacks, this package combines Image-Pro Plus 5.1 with deconvolution and rendering tools.Image-Pro Plus 5.1 Image-Pro Microscope Control The Image-Pro Microscope Control (MC) package combines Image-Pro Plus Version 5.1 with tools to control and program all major automated microscopes.Image-Pro Plus Version 5.1

11. Image-Pro Family Bundles Image-Pro 3D Discovery Perfect for confocal microscopy, the Image-Pro 3D Discovery package combines Image-Pro Discovery 5.1 with tools to provide full interaction and analysis of all 3D volumes. It includes major confocal microscope support. Image-Pro Analyzer Ideal for networked users who want advanced analysis tools but do not need image capture functionality at each work station. Image-Pro Analyzer offers all of the powerful analysis features of Image- Pro Plus 5.1, but does not include support for acquisition.Image- Pro Plus 5.1

12. Live Cell Imaging Systems Automated Stage Thermal Regulation High Speed Shutters

13. Live Cell Imaging Systems Example of Time Lapse GFP Imaging Experiments could involve fluorescence imaging, study of morphology. It may extend over a period of time but experiment must not kill the specimens even with multiple exposures, thus: – shuttered illumination – sensitive detectors/cameras – complex experiments involve computer control – Image sets may be tens of gigabytes Dr Andrew Paterson, Institute for Molecular Biosciences, University of Queensland, Australia. CHO cells spreading, with GFP-tubulin being expressed. Olympus IX81microscope

14. Major Problems In Live Cell Imaging – Photo toxicity and bleaching – Mechanical and thermal drifts – Temperature control – Culture media exchange – Stage and microscope automation – Vibration interference

15. Major Problems Photo toxicity and bleaching One major Limiting Factor in Live Cell imaging is the by product of Fluorescence causing photo toxicity. – During the excitation of fluorescence molecules with the presence of oxygen two events occur: Bleaching & Generation of free radicals – These free radicals will kill the cells. – Interaction of light with cells produces heat which will damage the cells. All these effect could be minimized when individual images are well separated temporally.

16. Major Problems Limiting Photo toxicity How can we prevent prolonged exposure of the cells to light? Maximize the amount of light we collect ; (1) Using High Numerical Aperture Optics e.g. UPLSAPO 60XO with N.A.1.35 Do note that immersion optics usually leads to thermal drifts or other thermal related issues. Things to consider : The length of the experiment, the frequency of image acquisition.

17. How can we prevent prolonged exposure of the cells to light? Using high sensitivity & QE detectors; (2) Evolution TM QEi Super High Sensitivity FireWire TM Camera. A monochrome digital camera that offers high speed, low noise electronics for rapid image capture. Using 2/3” Sony ICX-285 progressive scan interline CCD Up to 35X Gain Major Problems Limiting Photo toxicity

18. Major Problems Drift Usually there are 2 main causes for drift: (1)Thermal Drift (2)Mechanical Drift Mechanical Drift: IX81 has IX-ZDC Focus drift compensation function for time-lapse experiments

19. Major Problems Drift Most biologic properties must be recorded at 37 o C, how can this be done and at the same time minimize the effects of thermal expansion and contraction on focus? Objective Heater Heated Chambers

20. All About Live Cell Imaging 120 minute time lapse example (Cell division - DIC & GFP)

21. Multi-point time lapse imaging Obj.: 40x Plan Fl DIC NA0.6 dry Sample: HeLa on glass-bottom dish (ex488nm) CO2 5%, 37degree, hum >90% Site 1 Site 2 Site 3

22. Multi-channel time lapse Capturing an image set containing multiple dimensions should be easy to setup. However, being able to view an image set along any one dimension is an important visualization tool. time n time n+1 time n+2 CH1CH2CH3Composite

23. Protein Dynamics- TIRFM Imaged with Olympus IX81 microscope equipped with TIRF, 60X 1.45 TIRF objective, using a 488nm Argon laser. Observation of protein dynamics Dr Andrew Paterson, Institute for Molecular Biosciences, University of Queensland, Australia Olympus IX81 equipped with TIRF, 60X 1.45 TIRF objective

24. Intensity Tracking in a FRAP experiment Protein Dynamics- FRAP

25. FRET occurs - If the emission spectrum of a “donor” molecule overlaps the excitation spectrum of an “acceptor” molecule, and the distance between these two molecules are within 10 to 100 Å, then the excited donor molecule will transfer energy non-radioactively (without emitting of photon) to the acceptor. That is, when a double-labeled sample is excited in the excitation range of the donor, emission can be read in the emission range of the acceptor. 440nm 545nm <70Ǻ Protein Dynamics- FRET

26. FRET Algorithms

27. Deconvolution Dust Mite imaged with Olympus FV1000 Confocal Microscope with UPLAPO40X Objective. Remove haze and improve resolution in two- and three- dimensional image stacks using deconvolution and deblurring algorithms

28. 3D Volume rendering Image-Pro Solution for Advanced 3D Rendering and Measurement: Dust Mite imaged with Olympus FV1000 Confocal Microscope with UPLAPO40X Objective. 3D rendering of a kidney glomerulus

29. Neuron Analyzing

30. IQ Base The Intelligent Image Informatics Solution Fast and Easy-to-Use Archive Automatically from within Image-Pro ® Plus Customize to Fit Your Workflow Automate Repetitive Tasks Save Time with Powerful Search Tools Uncover Insights Hidden Within Your Images

31. IO- Pro Solution for controlling analog and digital outputs and inputs IO-Pro enables Image-Pro Plus users to control: Analog and digital outputs for external equipment control Analog inputs for measuring the status of experimental variables such as temperature and fluid flow Digital inputs for controlling sample triggers, such as foot pedals

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