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MetaMorph Workshop

MetaMorph Workshop. Ed Rader September 12 th -14 th , 2012. Agenda. Images & Image Display Acquisition Acquire Scan Slide Calibrate Distance MDA Working With Stacks Image Processing Image Analysis Presenting Images Automation. Goals.

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MetaMorph Workshop

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  1. MetaMorph Workshop Ed RaderSeptember 12th-14th, 2012

  2. Agenda • Images & Image Display • Acquisition • Acquire • Scan Slide • Calibrate Distance • MDA • Working With Stacks • Image Processing • Image Analysis • Presenting Images • Automation

  3. Goals • Familiarize you with MetaMorph’s tools and where to find them • Provide a basic understanding of each tool’s use • Not covering every button and setting in every function

  4. How to get help • Phone • 1-800-635-5577 • Press 3 for Tech Support • Press 2 for Cellular Imaging • Press 2 for MetaMorph • Email • Support.dtn@moldev.com • Web • http://support.meta.moleculardevices.com/ • http://www.meta.moleculardevices.com/authorize/

  5. Image Tools & Display Ed RaderSeptember 12th-14th, 2012

  6. Image Display • Can you guess what this image looks like? • Our perception is trained to interpret light intensity from the eye to images • Our perception is not adapted well to convert numbers to brightness in an image

  7. Image Display • MetaMorph uses a mapping of intensities to produce an image • Contrast • Definition of an image • Two dimensional grid of pixels • Each pixel contains an intensity value • Other information in images • Annotation and properties

  8. Image Info • Edit Menu • Besides the pixel data, other information is stored with images when they are saved

  9. Image Histogram • Measure Menu • A graph of the distribution of the number of pixels at each intensity in the image • Why do we care about this histogram? • Tells us how effectively we are utilizing the detector

  10. What’s wrong here? • These two images are of the same field of view

  11. Scale Image • Display Menu • Use the High Wedge to set the White • Use the Low Wedge to set the Black • Changes display, does NOT alter the data, only how it appears to our eyes

  12. Scale Image • Pixels equal and above the High intensity value are White • Pixels equal and below the low intensity value are Black • Changes display, does NOT alter the data, only how it appears to our eyes

  13. Scale Image • AutoScale • Automatically finds the lowest and highest intensity in the image and sets the scaling there • Wedges can then be changed to select % of pixels below and above wedges

  14. Image Window Tools • Zoom – makes the image larger / smaller • You can also use the wheel on the mouse • Look Up Table • Scale Image

  15. Color Image • What’s the difference between monochrome and color images? • How can you tell? • Why should you care?

  16. Color Image • No Scale Image tool • Pick Color Channel • Three intensities per pixel instead of one • Easier to get images into other programs • Create color images with overlays for presentation

  17. Acquiring Images

  18. Acquire • Acquire Menu • Acquire snaps an image and displays it • Autoexpose not good if imaging beads or low signal • Show live displays a continuously updating image

  19. Acquire • The duration of the collection of photons to create the image • How much area of the camera should be used to capture images • Shows the camera’s temperature or some warnings here

  20. Acquire • Autoexpose is good when there is “strong” signal • Watch your target intensity (Acquire Tab)

  21. Acquire • Binning • Sum adjacent pixel intensities together to form new pixels • Binning of 2x2, 4x4, 8x8, are common in detectors

  22. Acquire • Why Bin • Faster Frame times • Shorter Exposure times • Smaller files • Giving up • Spatial details

  23. Acquire • Display tab mostly used for setting the scaling • Acquire tab mostly used for synchronizing shutters & Autoexpose settings • Correct tab for background subtraction and flatfield correction

  24. Acquire • Annotate tab sets what settings to save with the image • Special tab is for device specific settings • Live Replay is like a MetaMorph DVR

  25. Acquire – Special Tab

  26. Special Tab – Common Settings • Speed - Digitizer • Gain – Amplify signal and intensities • Average Frames • To minimize noise • Driver Information • Triggering • Controlling or being controlled by external timing signals

  27. Live Replay • Live Replay is like a MetaMorph DVR. • Use is based on visually identifying an expected event • Specify the number of images before and after the event to capture • Memory is allocated to hold Images

  28. Live Replay • Start Live to begin capturing pre-images • Press F11 to lock pre-images and begin capturing post-images • Result stack displayed, current plane is the “marked” timepoint

  29. Acquire – Under the hood • Send Acquisition parameters to camera • Open Shutter / Set Illumination to Current • Expose CCD (Exposure Time) • Transfer Image to MetaMorph • Display Image in Window • How can we optimize this for faster performance?

  30. Advanced Acquisition Topics • Streaming • Captures images at the maximum rate provided by the hardware • Device Streaming • Allows sequencing of wavelength or z-series while streaming, useful for “real time” thru-focal sections • Synchronizing image capture with fast devices • Streaming to Hard Drive • When streaming for long time periods, such as minutes, the data can be accumulated on a fast hard drive. Virtual Stack memory allows the large dataset to be viewed in MetaMorph. • Stream Preview • During streaming, a pseudo-live image is displayed, allowing for interactive change in focus or adjustment of sample conditions during the acquisition

  31. Stream Acquisition • Optimized mode of camera operation • Acquire Images as fast as possible • Typically, Streaming & Live use the same mode of camera operation

  32. Stream Acquisition • Captures images at the maximum rate provided by the hardware • Only half the available memory can be used • Integrated as a tab / window for MDA

  33. Device Streaming • Software Synchronizing of Acquisition with microscope peripherals • Patented Process • Multiple High speed devices coordinated together • Devices not asked for position information

  34. Device Streaming • Piezo, Galvanometer, Monochromator, AOTF, DAQ • Typically devices move within 1-2ms • Fixed acquisition times for all images • Devices move during readout

  35. Overlapped / non-Overlapped Mode • Non-overlapped: Expose then readout • Overlapped: Expose and readout at the same time (sort of) • Effects sampling frequency (live cell specimens) • Synchronize with relatively slower devices (on the order of 10s of Milliseconds.

  36. Streaming to Hard Drive • Streaming to RAM is limited to the amount of RAM • Hard drive space is much more plentiful • Slower to write images to the hard drive vs RAM

  37. Stream Preview • How do I know that my Stream Acquisition is still collecting valuable data • Every Nth frame displayed to user in an Image window.

  38. Timelapse Acquisition • How do I improve the frame rate of my timelapse? • MDA Automatically Saves images • CPU Compatibility Mode Preference – MetaMorph has more processor time • Minimize image window • Annotations

  39. Scan Slide • Sample bigger than the field of view • A set of images collected at different fields of view • Montage together to present image of entire sample (low resolution) • High Resolution detail of any specific region

  40. Scan Slide • Apps Menu • Need motorized stage • Work from top down • Similar controls for Live and Snap

  41. Scan Slide • Main tab for selecting magnification • Description and file saving • Save all settings to a file for later use

  42. Scan Slide • Select Camera Binning and Number of Wavelengths • One Wavelength tab for each • Shading Correction allows for correction of uneven illumination

  43. Scan Slide • Select Illumination Setting • Select Exposure duration • Image Autofocusing available in case sample is not level on stage

  44. Scan Slide • Journals can be run at different times during the acquisition sequence • Check the event and browse for the journal

  45. Scan Slide • Calibration step required to map the size of the field of view and correlate the movement of the stage to the field of view

  46. Scan Slide • Select the bounding corners of the sample location to image • Typically use the Live image and the stage joystick to set the position • Press Scan to start imaging

  47. Scan Slide • Show low resolution montage of images • Draw a region on montage • Press Show Image to see high resolution • Stitched result

  48. Calibrate Distance • Measure Menu • Set the relationship of pixel size to a real physical dimension • Each pixel is how many microns • Calibrations typically setup for each magnification

  49. Calibrate Distance • List of calibrations available in table • Edit changes in the table for existing calibrations • Save calibrations to a file • Define new calibrations and measure with regions

  50. Calibrate Distance • Calibrate by region, image of stage micrometer • Select region type and resize region across the image to known marker dimensions • Enter the name (typically the magnification) and calibrated length • Associate the calibration to magnification and acquisition hardware

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