1 / 295

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.

tex
Download Presentation

MetaMorph Workshop

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related