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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

MetaMorph Workshop

Ed RaderSeptember 12th-14th, 2012


Agenda

Agenda

  • Images & Image Display

  • Acquisition

    • Acquire

    • Scan Slide

    • Calibrate Distance

    • MDA

  • Working With Stacks

  • Image Processing

  • Image Analysis

  • Presenting Images

  • Automation


Goals

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


How to get help

How to get help

  • Phone

    • 1-800-635-5577

      • Press 3 for Tech Support

      • Press 2 for Cellular Imaging

      • Press 2 for MetaMorph

  • Email

    • [email protected]

  • Web

    • http://support.meta.moleculardevices.com/

    • http://www.meta.moleculardevices.com/authorize/


Image tools display

Image Tools & Display

Ed RaderSeptember 12th-14th, 2012


Image display

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


Image display1

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


Image info

Image Info

  • Edit Menu

  • Besides the pixel data, other information is stored with images when they are saved


Image histogram

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


What s wrong here

What’s wrong here?

  • These two images are of the same field of view


Scale image

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


Scale image1

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


Scale image2

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


Image window tools

Image Window Tools

  • Zoom – makes the image larger / smaller

    • You can also use the wheel on the mouse

  • Look Up Table

  • Scale Image


Color image

Color Image

  • What’s the difference between monochrome and color images?

  • How can you tell?

  • Why should you care?


Color image1

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


Acquiring images

Acquiring Images


Acquire

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


Acquire1

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


Acquire2

Acquire

  • Autoexpose is good when there is “strong” signal

  • Watch your target intensity (Acquire Tab)


Acquire3

Acquire

  • Binning

  • Sum adjacent pixel intensities together to form new pixels

  • Binning of 2x2, 4x4, 8x8, are common in detectors


Acquire4

Acquire

  • Why Bin

    • Faster Frame times

    • Shorter Exposure times

    • Smaller files

  • Giving up

    • Spatial details


Acquire5

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


Acquire6

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


Acquire special tab

Acquire – Special Tab


Special tab common settings

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


Live replay

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


Live replay1

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


Acquire under the hood

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?


Advanced acquisition topics

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


Stream acquisition

Stream Acquisition

  • Optimized mode of camera operation

  • Acquire Images as fast as possible

  • Typically, Streaming & Live use the same mode of camera operation


Stream acquisition1

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


Device streaming

Device Streaming

  • Software Synchronizing of Acquisition with microscope peripherals

  • Patented Process

  • Multiple High speed devices coordinated together

  • Devices not asked for position information


Device streaming1

Device Streaming

  • Piezo, Galvanometer, Monochromator, AOTF, DAQ

  • Typically devices move within 1-2ms

  • Fixed acquisition times for all images

  • Devices move during readout


Overlapped non overlapped mode

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.


Streaming to hard drive

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


Stream preview

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.


Timelapse acquisition

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


Scan slide

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


Scan slide1

Scan Slide

  • Apps Menu

  • Need motorized stage

  • Work from top down

  • Similar controls for Live and Snap


Scan slide2

Scan Slide

  • Main tab for selecting magnification

  • Description and file saving

  • Save all settings to a file for later use


Scan slide3

Scan Slide

  • Select Camera Binning and Number of Wavelengths

  • One Wavelength tab for each

  • Shading Correction allows for correction of uneven illumination


Scan slide4

Scan Slide

  • Select Illumination Setting

  • Select Exposure duration

  • Image Autofocusing available in case sample is not level on stage


Scan slide5

Scan Slide

  • Journals can be run at different times during the acquisition sequence

  • Check the event and browse for the journal


Scan slide6

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


Scan slide7

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


Scan slide8

Scan Slide

  • Show low resolution montage of images

  • Draw a region on montage

  • Press Show Image to see high resolution

  • Stitched result


Calibrate distance

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


Calibrate distance1

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


Calibrate distance2

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


Calibrate distance3

Calibrate Distance


Calibrate distance4

Calibrate Distance

  • Quick estimate of the calibration in your images?

  • Size of pixels on camera / total system magnification

  • Example:

    • 6.45 um per pixel / 60x objective (no tube lens magnification) = ~.1um per pixel


Multi dimensional acquisition

Multi Dimensional Acquisition


Multidimensional acquisition

Multidimensional Acquisition

  • What can you do with MDA?

    • Timelapse

    • Multiple Stage Positions

    • Multiple Wavelengths

    • Z Series

    • Streaming

    • Run Journals

  • It is possible to do all of the above at the same time


Multidimensional acquisition1

Multidimensional Acquisition

  • Saving

    • Describe experiment

    • Select a directory

    • Name the experiment


Multidimensional acquisition2

Multidimensional Acquisition

  • Timelapse

    • Number of time points you want

    • Length of experiment you want

    • Elapsed time between time points you want

      • MetaMorph will suggest a minimum interval that you cannot go below


Multidimensional acquisition3

Multidimensional Acquisition

  • Multiple Stage Positions

    • Unlimited number of positions can be acquired

    • Move the stage with your sample on it to each position you want imaged

    • Push Plus or Right Arrow to add positions to the list

    • Use the red “X” to delete positions

    • Use the up and down arrows to move positions up and down in the list


Multidimensional acquisition4

Multidimensional Acquisition

  • Multiple Wavelengths

    • Up to 8 wavelengths can be acquired

    • As you add wavelengths, the number of tabs down the side of the wizard will increase accordingly


Multidimensional acquisition5

Multidimensional Acquisition

  • Multiple Wavelengths

    • Choose your wavelength in the illumination dropdown menu

    • The most important element is the exposure time for each wavelength


Multidimensional acquisition6

Multidimensional Acquisition

  • Z series

  • Two ways to acquire a Z series

    • From user-defined known positions of Top and Bottom

    • Range around current: the Z series will go, in this example, 15 steps above and below the current Z.

  • The step size recommended will depend on your objective’s magnification and NA


  • Multidimensional acquisition7

    Multidimensional Acquisition

    • Streaming

      • Select the length of time to stream

      • Select the location to stream to

      • Select overlapped or non-overlapped camera state

    • MetaMorph will calculate and let you know what memory you have and what’s required

      • If you don’t have enough memory the acquisition won’t start


    Multidimensional acquisition8

    Multidimensional Acquisition

    • Journal

      • At a given time point

      • For a given wavelength

      • At “special” times during the experiment


    Multidimensional acquisition9

    Multidimensional Acquisition

    • When all parameters are set to your specifications, click the Acquire button to start the acquisition

    • Also, once you have all your parameters set within the MDA dialog box, you can save them as a state to be recalled later for another experiment


    Multidimensional acquisition10

    Multidimensional Acquisition

    • Snaps a single image

    • Starts the live


    Multidimensional acquisition11

    Multidimensional Acquisition

    • Don’t have to acquire every wavelength at every timepoint

    • Beginning, End, Every N timepoints

    • Periodic Transmitted light reference image


    Multidimensional acquisition12

    Multidimensional Acquisition

    • Image Autofocusing to help correct for drift, sample depth, stage level

    • Find the best contrast

    • 3 Search Methods

      • Step

      • Binary

      • Advanced Binary

    • Configure Steps sizes and range

    • Align Wavelengths to correct for Registration & aberrations


    Multidimensional acquisition13

    Multidimensional Acquisition

    • Multiple Z Axis control

    • Offset positions for auto focus stability


    Review multi dimensional data

    Review Multi Dimensional Data


    Review multidimensional data

    Review Multidimensional Data

    • What can you do with RMD?

      • Enables you to view, filter, organize, and analyze MetaMorph images from a Multi Dimensional Data Set (*.nd) and generate new images based on your viewing criteria


    Review multidimensional data1

    Review Multidimensional Data

    • Select Base File

      • Opens the Multi Dimensional Data Set Utilities dialog box. Use this dialog to select the file that you want to use as the base file for your multi dimensional data. Choose the file from the list of the data sets in the Data Sets (*.nd) box or click Select Directory to select a data set from a different directory.

        Note: After you select or change your base file selection, you must click View to implement your selection and return to the Review Multi Dimensional Data dialog box.


    Review multidimensional data2

    Review Multidimensional Data

    • Enable Montage

      • Activates and deactivates the Montage controls and creates a montage image of thumbnails for each time point in a wavelength.

        • If multiple wavelengths are selected, a montage image is created for each wavelength.

        • After the montage image is displayed, click a thumbnail in the montage image to open a full image. You must have at least one wavelength selected to use this feature.

        • If you click Enable Montage before selecting any wavelengths, a montage for a specific wavelength will be generated when you select the wavelength.


    Review multidimensional data3

    Review Multidimensional Data

    • Load Images

      • Loads selected images into MetaMorph as new images and displays the images. The image can be saved to a file using the Set Up Sequential File Name dialog box.

    • Select Best Focus

      • Selects the best focused image from the Z-planes at each time point.

    • Clear

      • Clears all the image selections from the image grid.


    Review multidimensional data4

    Review Multidimensional Data

    • Full Image

      • Displays an entire image without regard to active regions.

    • Active Region

      • Displays only the active region of an image.

    • Color Composite

      • Combine the defined and selected image sources into a single image, assigning the selected primary color to each of the respective images.

    • Source R:

      • Assigns the primary color Red to the wavelength that you select as source.

    • Source G:

      • Assigns the primary color Green to the wavelength that you select as source.

    • Source B:

      • Assigns the primary color Blue to the wavelength that you select as source.

        • After you assign a wavelength to these sources, you can turn the image selection off and on in the Image Window.


    Review multidimensional data5

    Review Multidimensional Data

    • Run Journal Loop

    • Post acquisition Processing / Analysis


    Review multidimensional data6

    Review Multidimensional Data

    • Run Journal Loop

    • Post acquisition Processing / Analysis


    Multidimensional data utilities

    Multidimensional Data Utilities

    • Experiment Set Maintenance

    • Thumbnails are for montage

    • Preference to turn off Thumbnails by default


    Working with stacks

    Working with Stacks


    Stacks

    Stacks

    • What are stacks?

    • A set of images organized in a series, displayed one image at a time in an image window

    • Time Series

    • Z Series

    • Wavelength Series

    • Many people think of stacks as movies


    Stacks1

    Stacks

    • Usually saved in one file

      • 2.0 – 7.1 an .stk file

      • 7.5 or greater a multi-image tiff

    • Stack Player controls

    • “Thumb” for picking a single plane

    • Play Preferences on Stack Menu


    4d viewer

    4D Viewer

    • File Menu

    • 2D and 3D Display

    • Different wavelengths overlaid simultaneously

    • Original and volume displays available

    • Similar controls as regular image window


    4d viewer1

    4D Viewer

    • Use the mouse to rotate and spin the 3D projection

    • Make movies of rotations, time series or both in 3D

      • Select a Codec - Cinepak

    • Surface projection to identify discrete volumes and make measurements

      • Volume size and intensity

      • Distances

      • Colocalization


    4d viewer2

    4D Viewer

    • Scaling

    • Switch between 2D and 3D views

    • Select wavelengths

    • Select rendering option and level of detail


    4d viewer3

    4D Viewer

    • Set the X & Y calibration

      • Or set the Z calibration from Edit menu

    • “High performance” display card required to see finest detail

    • Large data sets are problematic

    • As much physical RAM as possible for best performance


    4d viewer4

    4D Viewer

    • IsoSurface enabled

    • Select individual objects with the mouse, double-click

    • Object volume measurements available


    4d viewer5

    4D Viewer

    • Distance measurements

    • Colocalization available when multiple wavelengths are in the same data set


    Keep planes

    Keep Planes

    • Stack Menu

    • Used to copy a portion or crop a stack

    • Can sub-sample a data set with the Step Size setting


    Keep planes1

    Keep Planes

    • Specify a range of planes to select

    • Press Select Planes in range

    • Scroll list to see check marks

    • Pick the action

    • Click Apply


    Montage

    Montage

    • Stack Menu

    • Arrange planes side by side to create a single image of the stack

    • Rows and columns can be configured

    • Result can be zoomed or stitched


    Montage1

    Montage

    • Select the fill order

    • Select the rows and columns if you don’t like the defaults

    • Zoom the image if necessary

    • Stitch the result if source is an X,Y Stage Scan

    • Press OK


    Image processing

    Image Processing


    Image filtering

    Image Filtering

    • Filtering helps prepare images for segmentation and/or analysis

    • Filtering accomplishes:

      • Noise removal

      • Correction of uneven background

      • Elimination of image details other than the objects of interest

    • No image filter will work perfectly on all images


    Basic filters

    Basic Filters

    • Process Menu

    • Weighted filtering and rank filtering

    • Examining each pixel and its neighborhood of adjacent pixels

    • The size of the neighborhood is called the “kernel”


    Basic filters1

    Basic Filters

    • The center of the kernel defines the pixel location being processed

    • Larger size kernels enhance the effect of any specific filter

    • Kernels are placed at each pixel in the image and the filter operation is performed

    • Watch for edge effects


    Basic filters2

    Basic Filters

    • Low Pass

      • suppresses high frequencies by replacing each pixel intensity with the local average intensity

      • blurs edges

    • Sharpen

      • enhances edges by applying a high pass filter

      • multiplication of each kernel’s value with pixels and summing results


    Basic filters3

    Basic Filters

    • Median

      • removes noise by replacing each pixel intensity with the local median intensity

      • good at noise removal while preserving edges

    • Rank

      • removes noise by replacing each pixel intensity with a local rank statistic intensity such as minimum, maximum, or median


    Basic filters4

    Basic Filters

    • Low Pass: suppresses high frequencies by replacing each pixel intensity with the local average intensity

    • Median: removes noise by replacing each pixel intensity with the local median intensity

    • Rank: removes noise by replacing each pixel intensity with a local rank statistic intensity such as minimum, maximum or median

    • Remove haze: subtracts out of focus blur estimated from neighboring planes

    • Sharpen: enhances detail by applying a high pass filter

    • Unsharp mask: subtracts out of focus blur estimated by applying a low pass filter


    Stack arithmetic

    Stack Arithmetic

    • Stack Arithmetic

    • Compress the data from an entire stack into one image

    • Can be used:

      • to improve signal to noise

      • compress stack of images into single plane

      • show the path of motion in a single plane


    Morphology filters

    Morphology Filters

    • Process Menu

    • Spatial and intensity-based filtering

    • Settings for basic shape, size, and area

    • Most intuitive results on binary images, but can operate on grayscale images


    Morphology filters1

    Morphology Filters

    • Dilate – grow bright things

    • Erode – shrink bright things

    • Close – dilate then erode

    • Open – erode then dilate

    • Open and Close are equivalent to where does the shape fit


    Morphology filters2

    Morphology Filters

    Close to fill small black holes in white

    Open filters out small white in a black background


    Morphology filters3

    Morphology Filters

    • Top Hat – Residue of Open Filter

      • Keeps the things that the open filter would have removed

      • Top Hat to find small features

    • Bottom Hat – Residue of Close

    • H-Dome – Local intensity peaks

    • H-Basin – Local intensity troughs

    • Top Hat to find small features

    • H-Dome to flatten an uneven background


    Morphology filters4

    Morphology Filters

    • Gradient – Difference between Dilate and Erode

    • Watershed Lines – Helps with splitting touching features


    Segmentation

    Segmentation

    Ed RaderSeptember 12th-14th, 2012


    Segmentation1

    Segmentation

    • Differentiate between what to and what not to measure

    • Regions of Interest

    • Thresholding

    • Morphology

    • Morphometry

    • Application Modules


    Regions of interest

    Regions of Interest

    • Regions Menu

    • Rectangle

    • Ellipse

    • Polygon

    • Line

    • Multi-Line

    • Multi-Line Trace

    • Region Properties


    Regions of interest1

    Regions of Interest

    • Active region

    • Region tooltips for position and size (in calibrated units)

    • Some filtering tools operate in active region


    Regions of interest2

    Regions of Interest

    • Double-click on line regions to edit positions of points

    • Use Rectangle region and Ctrl-D to crop an image or Ctrl-Shift-D to crop a stack

    • Tooltips are displayed when regions are being created


    Regions of interest3

    Regions of Interest

    • Right-click menu: copy, paste, delete regions

    • Save and load regions to a file

    • Transfer regions to another image

    • Create Region function


    Thresholding

    Thresholding

    • Measure Menu and on image window toolbar

    • Select pixels in image based on a range of intensities

    • Three states

      • Off

      • Inclusive Threshold

      • Exclusive Threshold

    • Subjective to user


    Thresholding1

    Thresholding

    • Colored overlay visible on image

    • Even illumination/background necessary for good thresholding

    • Pixels are either thresholded or not so binary images can be created

    • A prerequisite step for some analysis functions


    Thresholding2

    Thresholding

    • Blue Wedges on image windows can be used to adjust threshold

    • Autothreshold to find either bright or dark objects in image

    • Preference to select between two different algorithms


    Roi thresholding

    ROI + Thresholding

    • You can combine using Regions of Interest and Thresholding

    • Threshold Image

    • From the Regions menu select Create Regions around Objects


    Roi thresholding1

    ROI + Thresholding

    • Each discrete object will be traced with a region

    • Must be at least two adjacent pixels to create a region


    Combining tools

    Combining Tools

    • Threshold Image

    • Create Binary Image

    • Open Filter

    • Erode Filter


    Combining tools1

    Combining Tools

    • Threshold Image

    • Create Binary Image

    • Open Filter

    • Erode Filter

    • Watershed Lines Filter (using Binary and Erode images)

    • Invert Watershed

    • Logical AND between Binary and Invert Result


    Combining tools2

    Combining Tools

    • Notice some of the new lines separating the nuclei

    • Use the Arithmetic function to create a final image using the original image and this filtered image to get the original image intensities combined with the Watershed Segmentation applied


    Image analysis

    Image Analysis


    Image analysis1

    Image Analysis

    • What questions can MetaMorph answer about images?

      • How Many

      • How Bright

      • How Big

      • How Long

      • How Far

      • How much Overlap

    • Quantify Results


    Image analysis2

    Image Analysis

    • All Analysis function can send data to a text file or Microsoft Excel; also known as “Logging”

    • Simplest measurement is a pixel intensity with the mouse


    Line scan

    Line Scan

    • Measure Menu

    • Intensity profile along a line region

    • Results shown in graph

    • Good for checking the flatness of the background

    • One line at a time


    Show region statistics

    Show Region Statistics

    • Measure Menu

    • Measurements of a single region

    • Measurements on a single image

    • Intensity and spatial measurements


    Show region statistics1

    Show Region Statistics

    • Pick the active region or the entire image

    • Check Use Threshold to measure on thresholded pixels in the region

    • Updates even if measuring on the Live Image


    Region measurements

    Region Measurements

    • Measure Menu

    • Measure many regions all at once

    • Measure single plane or all planes in a stack

    • Data in table or graph

    • Summary data available


    Region measurements1

    Region Measurements

    • Graph measurements for all the planes in a stack

    • Pick the X axis as Plane Number, Elapsed Time, Z, or Wavelength

    • Y axis is the available list of measurements enabled


    Colocalization

    Colocalization

    • What is colocalization? Assume we are overlaying two separate images

      • Provides quantitative data regarding the amount of overlap of two fluorescent probes

      • Aggregate measurements

    red & green probes colocalized


    Measure colocalization

    Measure Colocalization

    • Apps Menu

    • Threshold images

    • Measures the amount of overlap in wavelengths


    Measure colocalization1

    Measure Colocalization

    A

    B


    Measure colocalization2

    Measure Colocalization

    A

    B


    Integrated morphometry analysis

    Integrated Morphometry Analysis

    • Measure Menu

    • Measures attributes of objects like intensity, size, shape, and count

    • Presents data in different tables and graphs

    • Filter objects that you don’t want to measure


    Integrated morphometry analysis1

    Integrated Morphometry Analysis

    • To measure with the IMA the image must be thresholded

    • After being measured, a green object overlay is displayed on the image

    • Toggle the overlay on/off

    • The object overlay can become a new binary image


    Integrated morphometry analysis2

    Integrated Morphometry Analysis

    • The list of measurement parameters to display and or filter

    • Check the box to display, filter or both

    • To filter, enter the range of values to include in the measurement


    Integrated morphometry analysis3

    Integrated Morphometry Analysis

    • Pick the measurements that you want to either view or filter on the image

    • Expand the + to see the available measurements in each group

    • Check the measurements to make


    Integrated morphometry analysis4

    Integrated Morphometry Analysis

    • Summary statistics for each measurement parameter

    • Count = how many objects were measured


    Integrated morphometry analysis5

    Integrated Morphometry Analysis

    • Histogram

    • Bar graph of distribution of measurements of the objects

    • Can filter parameter by calipers on graph


    Integrated morphometry analysis6

    Integrated Morphometry Analysis

    • Scatter Plot graphs the relationship between two measurements

    • Correlation coefficient is calculated

    • Pick which measurements to graph

    • Can filter objects here also with calipers on graph


    Integrated morphometry analysis7

    Integrated Morphometry Analysis

    • Preferences

    • Mouse clicks on image

      • find measurements

      • exclude/include object

      • Define filter range for acceptable objects

    • Object overlay drawing

    • Measure all regions


    Integrated morphometry analysis8

    Integrated Morphometry Analysis

    • The data display tables and graphs can be:

      • Docked

      • In a separate window

      • On a tab in the IMA

      • Not displayed

    • If you close docked window, it displays on a tab

    • Configure data display on View tab


    Integrated morphometry analysis9

    Integrated Morphometry Analysis

    • Reset the filters to their defaults

    • Save/Load IMA configuration to/from a file

    • Reset measurements

    • Create object mask


    Presentation images

    Presentation Images


    Duplicate as displayed

    Duplicate as Displayed

    • Want to show regions and other overlays on image

    • Create a “snapshot” of the image and all the items on it

    • The new image can be saved and opened in other applications


    Duplicate as displayed1

    Duplicate as Displayed

    • Edit – Duplicate – Duplicate as Displayed

    • Dropin

    • Representative of the original data, but not identical


    Color combine

    Color Combine


    Color combine1

    Color Combine

    • Display Menu

    • Combine one to three images together to make a color result

    • Only does red, green, and blue

    • Does not handle transmitted light images (brightfield, DIC, phase contrast)


    Color separate

    Color Separate

    • Display Menu

    • Separates color images into their component amounts


    Overlay images

    Overlay Images

    • Display Menu

    • Create a color image combining/overlaying up to 7 different images together

    • Select 5 predefined colors or use the hue selection

    • Pick the images corresponding to each hue


    Overlay images1

    Overlay Images

    • Automatic balancing

    • Adjusting scaling in images also effects result

    • Preview result


    Make movie

    Make Movie

    • Stack Menu

    • Make an AVI or Quick Time move

    • Duration of each frame; e.g., 30 -30ths = 1 second in movie

    • Pick a file name and location

    • Select a compression Codec


    3d deconvolution

    3D Deconvolution


    Why deconvolution

    Why Deconvolution?

    • Imaging a point source of light doesn’t give you a point in your image – you get an Airy Disk

    • Tries to take the information in the 3D image dataset and restore the original specimen

    • Improved Contrast & Resolution laterally and more so Axially


    Point spread function

    Point Spread Function

    • Point Spread Function

    • PSF is the 3D representation of the diffraction pattern of light in the microscope

    • Use either a mathematical or measured PSF as information on how the optical system is distorting the object into the image


    Deconvolution

    Deconvolution

    • Process Menu

    • MetaMorph Licenses algorithms from Autoquant

    • Uncheck “Faster Processing…”

    • Number of Iterations to converge to original specimen

    • Expert Settings are good default


    Deconvolution1

    Deconvolution

    • Enter information on the optical system

    • Fully automated and calibrated systems get information from Images


    Deconvolution2

    Deconvolution

    • Select either the Theoretical (Blind) or Measured PSF algorithm

    • Full frame stacks takes on the order of 10’s of minutes to process


    Deconvolution3

    Deconvolution

    • Optical Sectioning Spacing & Range?

    • Implication on Live Cells

    • No Recipe for the best Deconvolution


    Deconvolution results

    Deconvolution Results

    • Typical Contrast Results

    • Similar Analysis for contrast and resolution can be done axially


    Tracking

    Tracking


    Stack arithmetic1

    Stack Arithmetic

    • Stack Arithmetic is used to find object tracks or a background for the image series.

    • For fluorescence or luminescence use stack maximum for object paths and stack minimum for a background.

    • For transmitted light methods use stack average or maximum for the background.


    Stack arithmetic2

    Stack Arithmetic

    Original Image

    Background Image after Stack Arithmetic, Average

    Result Image after subtracting background from every plane.


    Stack arithmetic3

    Stack Arithmetic

    • Using Stack Arithmetic, Minimum, the path of the object can be identified.


    Difference image

    Difference Image

    • Advantages

      • Simple to do in a journal.

      • Very graphical, shows extension as well as retraction. ) Areas moving into appear black, areas moving out of appear white and areas that stay the same appear gray)

      • Easy to quantitate how much (m2/s) has moved.

    • Disadvantages

      • Little positional information.


    Difference image1

    Difference Image

    • Do Tracking Exercises 1-4


    Kymograph

    Kymograph

    Original usage: A device which graphically records motion or pressure.

    The Kymograph Image shows a linescan verses time. Stationary objects appear as vertical lines. Objects in motion appear as diagonal lines.


    Kymograph1

    Kymograph

    Advantages

    • Provides a pictorial representation of motion verses time.

    • Changes in velocity and duration of movements are readily apparent.

    • Multiple objects can be followed. Objects can cross paths, or be identical.

    • Can analyze some objects that are not easily discernible.

      Disadvantages

    • Only analyzes linear motion. Movement perpendicular to the axis of analysis is not taken into account.

    • Calibrations are not automatically taken into account


    Track points

    Track Points

    The Track Points command allows you to track one or more selected points through each frame in a series of images and to derive measurements of the paths, positions, and velocities of the points.

    Advantages

    • Fairly fast

    • Work from OMDR, stack or live / updating image

    • The best (most flexible) at not losing the target

      Disadvantages

    • Requires constant manual interaction

    • Accuracy limited to a few pixels

    • Only one object can be tracked at a time


    Track points1

    Track Points

    Click on Add Track


    Track points2

    Track Points

    Data Table displays selected Data Type


    Track points3

    Track Points

    You can Print or Log the data table to a spreadsheet


    Track points4

    Track Points

    Configures the settings associated with the time intervals between planes in a stack.


    Track points5

    Track Points

    Opens the Track Points Overlay Options dialog box. Use this dialog box to change the default settings of the Track Points marker and path displays.


    Track points6

    Track Points

    The default origin of the coordinate system used in image windows is in upper left corner of the window (or lower left, if you chose Y-Coordinate Increases up the Screen in the Set Graphics dialog box). Set Origin allows you to obtain point coordinates that are defined in terms of a particular location in the image that you select as a different point of origin.


    Track points7

    Track Points

    Creates a blank image with a copy of the track overlay, as currently displayed on the tracking image.


    Track points8

    Track Points

    Displays object data over time or plane number in graphical format. You can select the Data type to display


    Track objects

    Track Objects

    Tracks one or more selected objects through each image in an image stack or a sequential series of single images. You can derive measurements of the paths, positions, and velocities of the points.

    Advantages

    • Can require little user intervention

    • Multiple targets can be tracked with subpixel accuracy

    • Very reproducible

    • Many display/analysis tools available

      Disadvantages

    • Requires certain targetable features

    • Does not work if targets bump

    • Can be slow


    Track objects1

    Track Objects

    Different Source Image options


    Track objects configuration

    Track Objects- Configuration

    Determines the Algorithm MetaMorph uses to find the objects as it moves


    Track objects configuration1

    Track Objects- Configuration

    Configures the Time interval, Display options and where the origin will be.


    Track objects placing regions to track objects

    Track Objects- Placing Regions to track Objects

    Pressing Track button opens Select Objects dialog


    Track objects displaying and editing data

    Track Objects- Displaying and Editing Data

    Displays the statistical data for each object's track.


    Track objects displaying and editing data1

    Track Objects- Displaying and Editing Data

    Displays the point-by-point measurement data for individual objects.


    Track objects displaying and editing data2

    Track Objects- Displaying and Editing Data

    Displays object data over time or plane number in graphical format.


    Track objects displaying and editing data3

    Track Objects- Displaying and Editing Data

    Allows you to edit specific points in the data set.


    Track objects displaying and editing data4

    Track Objects- Displaying and Editing Data

    Creates a blank image with a copy of the track overlay, as currently displayed on the tracking image.


    Multi dimensional motion analysis

    Multi Dimensional Motion Analysis


    Track objects2

    Track Objects

    • Do Tracking Exercise 5-8


    Motion analysis

    Motion analysis

    • When would you use Kymograph, Track Points, Track Objects or nD Motion?

      • Kymograph

        • Visualization of time vs. space can aid in locating faint objects

        • Useful for objects traveling along known one-dimensional paths (e.g. leukocyte rolling along vessel wall)

      • Track Points

        • Manual for difficult samples and/or greatest accuracy

        • Validation of automated methods

      • Track Objects

        • Automated search

          • Saves time

          • Avoids human bias

      • Multi-dimensional Motion Analysis

        • Automated object identification

        • nD data sets


    Advanced segmentation analysis

    Advanced segmentation & analysis


    What are application modules

    What are Application Modules?

    • Intuitive, dedicated dialog boxes for biology-specific applications, consistent basic set of input parameters, interactive data and graphics

    • Adaptive Background Correction™: Improved segmentation through adaptation to local content

    • Field and cell-by-cell data logging

    • “Canned,” walk-away automation

    • Validated results in-house and with third-party

    • Can be incorporated into a journal for increased customization and further automation of your analysis


    Available modules for version 7 7

    Available modules for version 7.7

    • Angiogenesis Tube Formation

    • Cell Cycle

    • Cell Health*

    • Cell Scoring

    • Count Nuclei

    • Granularity

    • Live/Dead

    • Mitotic Index

    • Monopole Detection

    • Multi Wavelength Cell Scoring

    • Neurite Outgrowth

    • Micronuclei


    Adaptive background correction robust algorithms built in

    Adaptive Background Correction: robust algorithms built-in

    • Splitting of touching cells

    • Detection even in noisy and poorly stained images

    • Stable parameters perform consistently across entire experiment set

    Analysis automatically adjusts for uneven image background


    Running the application modules

    Running the Application Modules

    • Modules require grayscale 16 bit fluorescent* images (one for each wavelength analyzed)

    • Modules require a set of size and contrast parameters to adjust the processing the image(s)

      • Approximate object size range

      • Intensity above local background levels

    • The measurements typically include

      • Object counts and normalized counts

      • Areas

      • Intensity, average and total

      • Cell classifications

    *some modules can be used with transmitted light also


    Running the application modules preview each wavelength

    Running the Application Modules: preview each wavelength

    Consistent set of segmentation parameters: size range and staining intensity

    Each wavelength configured independently with quick Preview of results


    Running the application modules determining size parameters

    Running the Application Modules: determining size parameters

    • Select the Line tool from the Region toolbar

    • Move the mouse pointer the edge of a small object and click

    • A tool-tip appears showing the current X and Y values of the pointer, as well as the length

    • Move the mouse pointer to the opposite edge of the object and note the Length value

      • This number represents the cell width in pixels

      • If the image is calibrated, the length is in both pixels and calibrated units

    • Type or select the value in microns as “Approximate min width”

    • Repeat steps for a large object—values are approximate to control filtering during segmentation


    Running the application modules intensity parameters

    Running the Application Modules: intensity parameters

    • The “Intensity above local background” field is common for all application modules

      • Part of automatic Adaptive Background Correction system

      • Specifies the intensity threshold relative to nearby background values

      • Controls the sensitivity of object detection

    • Estimate the difference between the object’s signal and background

      • Hint: gray-level values of image pixels under the mouse cursor are shown in the main MetaMorph status bar (bottom of main window)

    • Enter this number in the “Intensity above local background” field


    Running the application modules logging measurements

    Running the Application Modules: logging measurements

    Summary Log Parameters Data Log (Cell) Parameters

    Neurite OutgrowthNeurite Outgrowth


    Running the application modules interactive graphics

    Running the Application Modules: interactive graphics

    Graphic overlays of segmentation results interact with cell-by-cell spreadsheet views of measurement data


    Running the application modules interactive data table

    Running the Application Modules: interactive data table

    Cellular Results Table: click on objects in the table and the corresponding cells in the source images are highlighted in yellow. Click on a cell in the source images and the corresponding data is highlighted in the Cellular Results Table.


    Count nuclei application module

    Count Nuclei Application Module

    • Single-wavelength segmentation and counting

      • Building block for multi-wavelength applications

    • Applications

      • Cell Proliferation

      • Cell Counting

      • Cell Migration

    • Note: Grey value in result image = cell assigned label #


    Angiogenesis tube formation application module

    Angiogenesis Tube Formation Application Module

    • Applications

      • Measuring and characterizing endothelial tube formation (a model system for angiogenesis)

      • Research focused on the promotion or inhibition of blood vessel formation

        Important in cancer, diabetes and other vascular disease research

      • Specific kits available from leading manufacturers

      • Area of research increasing in popularity with success of drugs such as Genentech’s Avastin which blocks VEGF activity

        • Reference and list available of other similar drugs in clinical trials

    • Features

      • Unique ability to acquire data sets with Z series (~1mm) to as tubes do not grow in a flat plane in the Matrigel™ matrix

      • In vitro models in combination with automated image acquisition and analysis are cost effective

    Top: best focus image bottom: White: tubes, green: nodes. BD BioCoat™ Angiogenesis System: Angiogenesis Endothelial Cell Tube Formation Assay. Courtesy of Min Wu, formerly BD Biosciences.


    Cell cycle application module

    Cell Cycle Application Module

    • Applications

      • Cancer research

    • Features

      • Interactive color coded graphs for data display and setting classification cutoffs

      • Flexible configuration

        • Single wavelength mode: Nuclear stain provides DNA content and average intensity indicates mitosis—5 classifications of cell cycle stage

        • Optional Mitosis-specific staining

        • Optional Apoptosis staining (6th cell classification)


    Cell health application module

    Cell Health Application Module

    • Three wavelength analysis

      • Nuclear stain, apoptotic stain, dead stain

    • Applications

      • Apoptosis

        • Classification of cells into four classes: viable, early apoptotic, late apoptotic, necrotic

    • Validated with common flow cytometry kits

      • Vybrant #7 from Molecular Probes

      • Annexin V

      • JC-1 with Hoechst: used to study loss of mitochondrial potential

    Cells analyzed by the module. Green: viable, blue: early apoptotic, purple: late apoptotic, red: necrotic.


    Cell scoring application module

    Cell Scoring Application Module

    • Applications: 2 wavelengths

      • Identifying subpopulations of cells tagged with a second fluorescent probe

        • Probe 1: nuclear marker

        • Probe 2: anything

      • General and flexible

        • Examining transfection efficiencies

        • Kinase activation – pathway analysis

        • Adipogenesis

    Top: Acquired image, two wavelengths overlay. Bottom: Analyzed image shows the identification of two probes.


    Granularity application module

    Granularity Application Module

    • Identification of punctate staining

      • Robust detection of granules even in noisy and uneven image backgrounds

      • Optional nuclear marker for normalized counts

    • Applications

      • GPCR internalization

      • Assays of clustering target molecules


    Monopole detection application module

    Monopole Detection Application Module

    • Applications

      • Cancer research

        • Disruption of normal bipolar spindle formation

        • Disruption of centrosome separation (e.g. monastrol)

    • Features

      • Classification of cells as interphase, bipole or monopole

    Left: 3T3-L1 mouse fibroblast cells treated with monastrol and stained with mouse anti-beta tubulin primary antibody detected with a FITC conjugated goat antimouse secondary antibody. Nuclei are stained with Hoechst 33342. Right: segmented image shows interphase cells (red), bipolar spindles (blue) and monopole (green).


    Multi wavelength cell scoring application module

    Multi Wavelength Cell Scoring Application Module

    • Applications

      • Your research—configure your own custom module easily without the need for programming or macros

      • Configurable from 1 to 7 wavelengths

      • Cell-by-cell and summary scoring profiles reported across wavelengths

    • Features

      • Independently preview individual wavelength settings

      • Customize wavelength and profile naming to match your research

      • Interactive graphics link scoring data between individual wavelengths and full multi-wavelength profiles


    Neurite outgrowth application module

    Neurite Outgrowth Application Module

    • Applications

      • Measuring and characterizing outgrowths (length and branching)

      • Used in the study of

        • Neurodegenerative disease such as Alzheimer’s and Parkinson’s

        • Neuroregenerative research: Spinal Cord Repair

        • Cell Differentiation (Stem Cell Research)

    • Features

      • Complements imaging system -this assay is impossible to perform without imaging techniques

      • Consistent and faster results over manual tracing which is a prevalent technique in academia

    Top: image acquisition. Right: Each filament is assigned to a cell body. All the filaments and cell bodies are then measured. Images courtesy of Kris Poulsen and DavideFoletti, Rinat Neuroscience Corporation.


    Micronuclei application module

    Micronuclei Application Module

    • Applications

      • Counting of micronuclei

      • Counting of mono-, bi- and multinucleated cells

      • Classification of cells by health, interphase, number of nuclei, presence of micronuclei

      • Used in the study of

        • Genotoxicity and chromosomal damage studies

        • Mitotic abnormalities

    • Features

      • Complements imaging system -this assay is impossible to perform without imaging techniques

      • Faster results over manual scoring which is a prevalent technique in industry


    Running the application modules micronuclei

    Running the Application Modules: Micronuclei

    Nuclei and Micronuclei settings

    Additional probes for cell health and others


    Automation

    Automation


    Metamorph journals

    MetaMorph Journals

    • What are journals?

      • Specific sequence of steps

      • Macros that automate repetitive tasks

    • Journals can be created via:

      • Recording a series of mouse clicks

      • Using the Journal Editor 

    • Journals can be run from:

      • The Journal Menu

      • Within another journal

      • Specific dialogs

      • Taskbars

      • Toolbars

    • The Journal Editor

      • Used to edit existing journals or create new journals


    An example maximum projection

    An Example: Maximum Projection

    • A Maximum Projection

      • Collapses a volume of fluorescence thru-focus slices into a single image

      • A very useful tool for anyone visualizing fluorescence structures in 3D

      • Fast and easy to do


    An example maximum projection1

    An Example: Maximum Projection

    • The Goal

      • Make it an easy “one button” task to make a Maximum Projection

    • The Method

      • Record the Maximum command into a Journal

      • Create a taskbar

      • Put the journal on the taskbar


    Recording the journal

    Recording the Journal

    Example: Maximum Projection


    Step 1 open an image

    Step 1: Open an image

    • Choose Open (File Menu)

    • Open the nerve.stk sample image

    Note: The Open is not recorded into the journal. Instead we are opening a sample image stack (nerve, in this case) before making the journal. The journal we make will work on any grayscale stack that is loaded in MetaMorph.


    Step 2 start recording

    Step 2: Start recording

    • Choose Start Recording (Journal Menu)

      or

    • Press the Start/Stop Recording icon on the Journal toolbar


    Step 3 verify that recording is on

    Step 3: Verify that recording is on

    • You can verify that recording is on by:

      • The phrase “[Recording]” appears in the title bar

      • The “Start/Stop Recording” icon changes from to


    Step 4 choose the command s to record

    Step 4: Choose the command(s) to record

    • Choose Stack Arithmetic (Process Menu)

    • The Stack Arithmetic dialog will open

    • While recording a journal, any button that records its action to the journal will be highlighted in blue

    • With Stack Arithmetic, the Apply button is highlighted in blue

    • Set the parameters (in this case choose the stack name to work on, “nerve”, and the operation, “Maximum”)

    • Press Apply


    Step 5 results of the command s chosen

    Step 5: Results of the command(s) chosen

    • After pressing Apply, a new result image called Maximum will be created and displayed


    Step 6 stop recording

    Step 6: Stop recording

    • Choose Stop Recording (Journal Menu)

      or

    • Press the black “Start/Stop Recording” icon in the Journal toolbar


    Step 7 name the journal

    Step 7: Name the Journal

    • When you stop recording you will be asked to save the journal

    • Choose a name (in this example choose “maximum” since the journal is computing the stack maximum)

    • Press Save once you have entered the name


    Step 8 verify that recording is off

    Step 8: Verify that recording is off

    • You can verify that Recording is Off by:

      • The phrase “[Recording]” no longer appears in the title bar

      • The “Start/Stop Recording” icon changes back to


    How to run the journal

    How to run the Journal

    • There are several ways to run the journal

      • Run Journal (Journal menu)

      • Run Journal icon on the Journal toolbar

      • From a taskbar

    • Using the menu or icon is generally useful only for journals that are run occasionally, as you have to select the journal by name from the file list

    • Using a taskbar is the most convenient method as you can create a “one button” solution where clicking the button (whose name you can choose) runs the journal

    • The first 10 buttons on a taskbar also have a “hotkey” associated with them – so in addition to “one button” the journal can be run with one key-press


    Creating the taskbar

    Creating the Taskbar


    Taskbars

    Taskbars

    • What is a taskbar?

      • A taskbar gives “one-click” access to journals, functions, and other taskbars

      • A taskbar is customizable

      • Taskbars can be resized to modify the width of the buttons, change the number of columns, and change the number of rows

      • The active taskbar is saved when you exit MetaMorph

      • When you start MetaMorph the next time, the taskbar you were last using will be displayed again

      • Only one taskbar can be used at a time

    • Creating a taskbar

      • Journal Menu > Taskbars > Create Taskbar

      • The Taskbar Editor dialog box will open


    Creating a taskbar step by step

    Creating a taskbar: step-by-step

    • Drag items from the list in the Taskbar Editor dialog box onto the new taskbar

    • There are three categories of items:

      • MetaMorph functions, journals, or other taskbars


    Creating a taskbar step by step1

    Creating a taskbar: step-by-step

    • After you have dragged a function over to the New Taskbar button, you can modify the button name


    Creating a taskbar step by step2

    Creating a taskbar: step-by-step

    • Renaming the taskbar button


    Creating a taskbar step by step3

    Creating a taskbar: step-by-step

    • Click on one of the Category radio buttons to select whether to put Menu Functions, Journals, or other Taskbars on the Taskbar


    Creating a taskbar step by step4

    Creating a taskbar: step-by-step

    • Click the Rename Taskbar button to rename your taskbar

    • The Rename Taskbar dialog box opens

    • Once you have typed a Taskbar Title, click OK


    Creating a taskbar step by step5

    Creating a taskbar: step-by-step

    • Once the taskbar is created, click “Save As” to save your taskbar

    • Name your taskbar and click OK

    • Click Close on the Taskbar Editor to close the dialog box


    Creating a taskbar step by step6

    Creating a taskbar: step-by-step

    • To load the taskbar use Journal menu > Taskbars > Load Taskbar


    Creating a taskbar step by step7

    Creating a taskbar: step-by-step

    • Select the taskbar to open


    Creating a taskbar step by step8

    Creating a taskbar: step-by-step


    Advanced journaling

    Advanced Journaling

    Image Selectors, Multiple Commands in a Journal, Journal Editor


    How does the journal know what image to work on

    How does the Journal know what image to work on?

    • Many images can be present within MetaMorph

    • Why is this important?

      • Since one command in a journal often works off the results of the previous one, it is important to know which image to work on

    • Each command in a journal can work on:

      • The image that was active when the journal started. This is called the “current at start” image.

      • The image that was created by the previous step in the journal. This is called the “last result” image.

      • The image that was created by a specific step in the journal. This image has a number prefixed, such as “1:” for the first image created.

      • An image with a specific name, such as “nerve”. This is called a “specified(name)” image.

      • An image that you choose when the journal runs. This is called the “Select on Playback” image.


    How does the journal know what image to work on1

    How does the Journal know what image to work on?

    • Use the Image Selector to choose which image to work on

    The Normal Image Selector (not recording a journal)

    The Image Selector when recording a journal

    The Image Selector when editing a journal


    An example sharpen and then best focus

    An Example: Sharpen and then Best Focus

    • Open the Nerve Stack

    • Start Recording (Journal Menu)

    • Basic Filters (Process Menu)

      • Choose “Nerve” “All Planes” using the source imageImage Selector

      • Choose Sharpen as the operation

      • Choose Low as the filter

    • (This will create a result image called Sharpen)

    • (…with the Sharpen image still active…)

    • Stack Arithmetic (Process Menu)

      • Choose “Sharpen” using the stack name Image Selector

      • Choose Best Focus as the operation

    • Stop Recording (Journal Menu)


    An example sharpen and then best focus1

    An Example: Sharpen and then Best Focus

    • Stop Recording (Journal Menu)

      • Name the journal “sharpen and best focus.jnl”

    • Because your “My Taskbar” taskbar is already open it will ask:

    • Press “Yes” to add your journal to the taskbar

    • Drag the “sharpen and best focus” journal from the journal list to an available button on the taskbar

    • Save the taskbar


    Editing a journal

    Editing a Journal

    • Change the Sharpen effect from a Low to a Medium sharpen

    • Solution:

      • Edit “sharpen and best focus”

      • Modify the existing Sharpen step

    • Close the intermediate image (the one called “sharpen”) and only have as the result the image which is the best focus of the sharpened version of the original

    • Solution:

      • Edit “sharpen and best focus”

      • Add a “Close” command

        • Make sure the Close command is closing the intermediate “sharpen” image and not the final “best focus” image!


    Editing a journal1

    Editing a Journal

    • Choose “Edit Journal” (Journal Menu)

      or

    • If the journal you want to edit is already on a taskbar button, then hold down SHIFT and click on that journal’s taskbar button – instead of running the journal, it will open that journal in the Journal Editor


    The journal editor

    The Journal Editor

    These tabs control whether the list below shows all the menu items, other journals, or actions like “if … then”

    The name of the journal being edited

    The list of commands recorded into the journal


    Interpreting the commands in the journal editor

    Interpreting the commands in the Journal Editor

    • The image that is “Current at Start” when the journal is first run, will be Sharpened using the Low filter setting, producing an intermediate image called “Sharpen”

    • The “Last Result” (in other words, the result of the sharpening) will have the Stack Arithmetic “best focus” applied to it, producing a result image called “Best Focus”


    Editing an existing command

    Editing an existing command

    • To edit an existing command in the journal

      • Highlight that line in the journal

      • Double-click

      • The dialog corresponding to that line will open

      • Make changes as necessary in the dialog – example: change Sharpening Filter to “Medium” (instead of “Low”)

      • Press the action button (Apply, etc) which will be colored blue

    double-click


    Adding to the journal

    Adding to the Journal

    • Open the journal in the journal editor

    • Drag commands from the left side of the editor into the journal

      • You can drag menu commands, other journals, or actions

    • Drop the command where you want it in the journal

    • Once the command is in the right spot in the journal, Drop it

      • The command’s configuration dialog will appear

      • Set the configuration appropriately

    • Commands in the journal can be dragged up or down as needed

    • Save the edited journal


    Example close the sharpen image

    Example: Close the Sharpen image

    • Open the “sharpen and best focus” journal in the Journal Editor

    • Find the command “close” in the list of commands

    • Drag and drop the command “close” at the end of the journal

    Drop

    Drag


    Example close the sharpen image1

    Example: Close the Sharpen image

    Make sure to select the proper image to close – in this case you want to close the result of command “1” in the journal – the Sharpen image


    Protecting from invalid actions

    Protecting from invalid actions

    • What happens if you run the journal on the “best focus” image ?

    Error!

    It is an invalid action because the “Best focus” image is not a stack, therefore Stack Arithmetic will not work on it


    Protecting from invalid actions1

    Protecting from invalid actions

    • Need to do an “if … then” to protect against the invalid action

      • IF “the image is a stack” THEN

        • Do the Sharpen and Best Focus

      • ELSE

        • Display a friendly message that this only works on Stacks

      • END IF

    • Use the action called: IF…THEN…ELSE

    • Test for the Number of Planes to determine whether the image is a stack

      • Greater than 1 plane means it is a stack

      • Operator for “Greater than” is the symbol “>”


    Protecting from invalid actions2

    Select the “Actions” tab in the Journal Editor

    Choose the “If/Then/Else” action, and drag it into the journal

    Note that it appears RED because it is waiting for the condition (what it needs to test for) to be entered

    Protecting from invalid actions


    Protecting from invalid actions3

    Protecting from invalid actions

    • Press the button with the 3 dots to access the Variables dialog so that the condition can be entered


    Protecting from invalid actions4

    Protecting from invalid actions

    • The Condition to test for is “is the image a stack”. In other words, “IF the image is a stack THEN do the various things”.

    • To see if the Image is a stack, look at the number of planes that the image has. If it is a stack, it will have more than 1 plane.


    Protecting from invalid actions5

    Protecting from invalid actions

    Select the “Image” category and then find the variable “Image.NumPlanes” and highlight it. Press the “>” button to add it into the Expression box.


    Protecting from invalid actions6

    Protecting from invalid actions

    Within the Expression entry area, type in “> 1” after the Image.NumPlanes


    Protecting from invalid actions7

    Protecting from invalid actions

    • The RED goes away from the IF…THEN because the condition has been entered.


    Protecting from invalid actions8

    Protecting from invalid actions

    • Highlight the original parts of the journal (the 3 lines that do the Sharpen, Best Focus, and Close) and drag them up and between the IF and the ELSE


    Protecting from invalid actions9

    Protecting from invalid actions

    • If the starting image is not a stack, then it would be nice to display an appropriate message.

    • To do this, find the “Show Message and Wait” command in the alphabetic list of functions and drag it between the ELSE and the END IF.

    • The settings for Show Message and Wait allow you to specify the message.


    Protecting from invalid actions10

    Protecting from invalid actions


    Advanced journaling1

    Advanced Journaling

    Loop/Repeating Journals


    Start up journal

    Start-up Journal

    Start-up Journal

    • Use this command to configure a journal of your choice to run automatically every time you start MetaMorph.

    • This can be useful for performing a sequence of configurations that set up MetaMorph to your specific needs, such as:

      • opening a particular combination of windows or dialog boxes

      • loading a state file

      • moving a stage to a specific position

      • selecting a video device and channel.


    Journaling looping

    Journaling Looping

    Loop A Journal

    • Use this command when you want to repeat a journal a specified number of times.

    • Specify the number of time to loop (30,000 max)

    • Can prompt on journal playback the number of times to loop


    Journaling looping1

    Journaling Looping

    Loop for all Planes

    • Use this command when you want to repeat a journal for each plane of the selected stack.

    • You can record the Loop for All Planes command as part of another journal.

    • You can use variables for the same functionality without the need for a second journal.


    Journaling looping2

    Journaling Looping

    Loop for all Regions

    • Use this command when you want to repeat a journal for each region of interest that is defined in the selected image.

    • You can record the Loop for All Regions command as part of a journal.

    • You can use variables for the same functionality without the need for a second journal.


    Journaling looping3

    Journaling Looping

    Loop for all Images in Directory

    • Use this command when you want to repeat a journal for each image in a directory. You should first be sure that all images In the directory are appropriate for the journal that is to be applied.

    • Options for what to do with each image after journal has been run.


    Journaling looping4

    Journaling Looping

    Other functions:

    • Several other functions within MetaMorph allow repeating (looping) a journal at specific points during a function’s process.

    • i.e. Acquire Timelapse, Acquire Z-Series, Scan Slide, Multidimensional Acquisition, Multidimensional Data Set Utilities, Measure Grid


    Variables

    Variables


    Variables1

    Variables

    Introduction to MetaMorph Variables

    • Similar to variables in many programming languages

    • allow more direct control of hardware, images, regions of interest and manipulation of measurement values.

    • Variables also allow branching within journals and looping.

      What is variable?

    • In the simplest terms, it’s a placeholder for information.

    • The value (the information) stored in the variable can change.

    • The information can be text (string type variable) or numeric.


    Variables2

    Variables

    Variables can:

    • Manipulate text or numbers

    • Manipulate hardware

    • Change image parameters

    • Reference measurements

    • Manipulate regions of interest

    • Log information to any type of log


    Variables3

    Variables

    Variable Rules:

    • Variables can be text (string) or numbers. The type is specified when you assign a value to the variable. Text (string) are defined in quotations.

    • Variables exist inside and outside of journals.

    • Variables are erased when MetaMorph is closed or they are specifically deleted.

    • Names of variables are not case sensitive and cannot have spaces or special characters.

    • Variable assignments are made using Assign Variable, Enter Variable, Prompt User or Assign to Variable.

    • Variables attributes are Read (R), Write (W) or Executable (X).

    • Logical values (true or false) are stored as 1 or 0.


    Variables4

    Variables

    Assign Variable:

    • create user defined (custom) variables

    • > Button: Add at curser position

    • >> Button: Replaces variable name or expression.


    Variables5

    Variables

    Image Variables:

    • refer to storage information, acquisition information and display parameters for the active plane of the active image window.

    • Are dynamic. Will change as the active image changes.


    Variables6

    Variables

    Region Variables:

    • refer to the active region of interest.

    • Are dynamic. Will change as the active Region changes.

    • Region size variables can be manipulated for rectangle, oval, line and poly-line regions.


    Variables7

    Variables

    Region Variables:

    • Region.Top - Upper left corner, Y coordinate

    • Region.Left - Upper left corner, X coordinate

    • Region.Height - Size in Y dimension

    • Region.Width - Size in X dimension


    Variables8

    Variables

    Measurement Variables:

    • Treat measurements as Read only variables.

    • Values refer to the last logged data. Almost everything that is loggable is accessible.

    • You must log a measurement before you can access it.

    • Some measurement functions measure multiple values. Only the last value is accessible. For example, see IMA object or summary data or many of the application modules


    Variables9

    Variables

    Program Variables:

    • Program Variables refer to installed hardware.

    • Common uses of program variables include changing camera exposure setting before using Acquire, adjusting Z position, moving current stage position, and changing magnification or illumination settings.

    • Other program variables are specifically used with Multi Dimensional Acquistion or Screening modules.


    Variables10

    Variables

    Operators:

    • The Operators tab provides access to Arithmetic, Relational, String, Number, Logical, and Trigonometry functions.


    Metamorph workshop

    • Do Exercises 1 - 3


    Variables11

    Variables

    Enter Variable:

    • assigns a value to a custom variable by prompting the user to enter a variable value.

    • supports Yes/No, Yes/No/Cancel, string input, numerical input (with limits), radio button selection, and multiple selection lists.

    • The Preview button displays a preview of the message box as it has been configured. You can position the previewed message dialog box where you want it placed during journal playback.


    Variables12

    Variables

    Log Variable:

    • Logs a custom variable to any type of open log file.


    Graph variable value

    Graph Variable Value

    Graph Variable Value:

    • Use this command to graph the values of one or more variables in a journal. Up to two variables can be displayed per graph and multiple graphs can be displayed and updated at the same time. This command can display the value of multiple variables in the following ways:

      • Using one graph with separate traces for each variable

      • Opening separate graphs for each variable


    Metamorph workshop

    • Do Exercise 4


    Variables ii

    Variables II


    Variables13

    Variables

    Journal Editor Actions Tab:

    • The options available on the Actions tab of the Journal Editor allow you to incorporate journal flow control directly in the editor.


    Variables14

    Variables

    Journal Editor Actions Tab:

    • Assign to Variable: Creates user-defined (custom) variables, assigns values to variables, and performs logical, string, number, trigonometry, and arithmetic operations.

    • Assign to Variable is similar to the Built-in Function Assign Variable. However, the Action Assign to Variable enables you to make variable assignments directly in the Journal Editor.

    • You can enter the variable assignment directly or click the “…” to access the Enter Variable “variable” and “variable expression” boxes.

    • Note: Assign to Variable cannot be made interactive.


    Variables15

    Variables

    Journal Editor Actions Tab:

    • Comment Field: Annotates your journal by inserting comments into the journal. Comments appear red in the journal editor.

    • To separate sections of your journal into easy-to-read blocks, blank lines can be inserted into the journal by adding a Comment Field and deleting the “Enter Your Comment” text in the comment entry field.


    Variables16

    Variables

    Journal Editor Actions Tab:

    • Delete Variable: Deletes a custom variable.

    • Custom variables exist in MetaMorph until you exit MetaMorph or use the Delete Variable function to delete the custom variable.


    Variables17

    Variables

    Journal Editor Actions Tab:

    • Exit Playback: Terminates the journal at a specified point.


    Variables18

    Variables

    Journal Editor Actions Tab:

    • For/Next: Executes a series of commands for a defined number of times.

    • Variables can be used to specify the value for the starting point, end point, and step size.


    Variables19

    Variables

    Journal Editor Actions Tab:

    • If/Then/Else: Evaluates an expression and takes one of two possible courses of action depending on the result.

    • It is not necessary to include any commands within the Else portion of the If/Then/Else statement.


    Variables20

    Variables

    Journal Editor Actions Tab:

    • Pause Playback: Pauses journal playback and opens the Variable dialog.

    • This is a useful debugging tool as it allows you to evaluate the status of custom and MetaMorph variables.


    Variables21

    Variables

    Journal Editor Actions Tab:

    • Prompt User: Prompts user to enter a variable by asking for a number, radio button choice, string, text list, yes/no message box, or yes/no/cancel message box.

    • This action is similar to the Built-in Function Enter Variable.

    • Both functions can be used to create custom variables.

    • Unlike Enter Variable, Prompt User does not contain a preview button. During journal playback, the Prompt User dialog appears in the upper left corner of the MetaMorph desktop.


    Variables22

    Variables

    Journal Editor Actions Tab:

    • Trace: Prints a statement or variable expression to the History Window.

    • This is another useful debugging tool.


    Variables23

    Variables

    Journal Editor Actions Tab:

    • While: Creates a variable loop that is run as long as the condition is true.


    Advanced journaling topics

    Advanced Journaling Topics

    Write Steps

    Use Descriptive Names

    History Window

    Trace Command

    Comments


    Advanced journaling topics1

    Advanced Journaling Topics

    Variables for Devices

    Overwrite Images when possible


    Exercises

    Exercises

    Exercises

    Graph Variable Value - Intensity overtime

    Batch convert image formats

    Rolling/jumping averages

    Taking the square root of an image


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