Quantitative MicroscopyQuantitative Microscopy

Morphogenesis using Nikon Morphogenesis using Nikon NIS, ImageProNIS, ImagePro

Stella Breslin Biosc3 Spring Stella Breslin Biosc3 Spring 20102010

Nikon Manual CountNikon Manual Count

Run the [View > Analysis Controls > Annotations and Measurements] command. Run the [View > Analysis Controls > Annotations and Measurements] command. The manual measurement control window appearsThe manual measurement control window appears..

The Auto Detect filled tool. Detect hollows using threshold techniques. Click to the image to place the probe, the detected area is drawn. You can adjust the thresholding range by using the mouse wheel or by pressing the +, - keys. [Ctrl+B]

Nikon Manual CountNikon Manual Count

Nikon Automated Nikon Automated MeasurementMeasurement

• Optical system calibration. Optical system calibration. • Image acquisition.Image acquisition.• Defining threshold, creating the Defining threshold, creating the

binary layer. binary layer. • Performing the automated Performing the automated

measurement.measurement.• Results presentation.Results presentation.

Binary LayerBinary Layer

Binary layer] - is typically used for shape and Binary layer] - is typically used for shape and size measurements (area, perimeter, surface size measurements (area, perimeter, surface fraction).fraction).

Binary Layer - ManipulationBinary Layer - Manipulation

Automated MeasurementsAutomated Measurements

[View > Analysis Controls > Automated Measurement]

Object CountObject Count

Nikon ThresholdNikon Threshold

By Reference Points – 1 Point, 3 Points (circle), 6 PointsReset, Undo, Redo,

RestrictionsRestrictions

• • Right click to the restrictions field to select one or more of Right click to the restrictions field to select one or more of the available measurement features.the available measurement features.

• • Select the restriction feature you would like to define.Select the restriction feature you would like to define.

• Move sliders below the histogram to set the limits. The limit values are indicated next to the feature name, and can be modified by double clicking the indicated value.• Decide whether the defined interval will be excluded or included - this is done by setting the [Inside/Outside] option next to the feature name.• The nearby check box indicates whether the restriction is currently being applied or not. If applied, the histogram below is color, otherwise it is gray.

Separating ObjectsSeparating Objects

Measurement MenuMeasurement Menu

Measurement Menu >

Perform Measurement

Object Features

…

Options

Nikon1 Object Count – No Calibration Needed Since nd2 FileNikon1 Object Count – No Calibration Needed Since nd2 File

NumberObjects 23AreaFraction 0.027StgPosX 35205.8StgPosY 11610.2Feature Mean St.Dev Minimum MaximumArea 175.03 61.43 32.23 318.4EqDiameter 14.64 2.92 6.41 20.13Circularity 0.911 0.072 0.686 0.994MeanIntensity 1325.68 239.11 817.72 1746.14

Sort By Area to determine Restriction By Area Size, etc

Measurement ControlsMeasurement Controls

• Results of all automated Results of all automated measurements are presented and measurements are presented and can be exported from the [View > can be exported from the [View > Analysis Controls >Automated Analysis Controls >Automated Measurement Results] control Measurement Results] control window.window.

Results – Can be Exported to Excel or TextResults – Can be Exported to Excel or Text

[ Total Area] is the area of the whole image in px2 [ Area Fraction] is the ratio between [Total Area] area of the thresholded objects. [ Number of objects] -after restrictions. [ Table of results] - measurement results [ #] - number of selected objects

Counting In Image ProCounting In Image Pro

The basic steps in counting The basic steps in counting

1. Set the range of 1. Set the range of intensities intensities (monochrome images) (monochrome images) or the colors (color or the colors (color images) that identify images) that identify the object you want to the object you want to count using the tools count using the tools in the Intensity Range in the Intensity Range Selection group boxSelection group box..

Manual Intensities – Select Ranges Manual Intensities – Select Ranges • Use this dialog box Use this dialog box

to segment out the to segment out the objects that are to objects that are to be counted and/or be counted and/or measured.measured.

Manual Intensities – Select Ranges Manual Intensities – Select Ranges

2. Select the kinds of 2. Select the kinds of measurement you measurement you want to record in want to record in the Count/Size : the Count/Size : Measure menu’s Measure menu’s Select Select Measurements Measurements command.command.

Count / Size OptionsCount / Size Options

Set Outline Set Outline Style – Fill, Style – Fill, Outline, etcOutline, etc

Label StyleLabel Style Label ColorLabel Color Fill HolesFill Holes Clean Border Clean Border

optionsoptions

Object Options:Object Options:..

Clean Border: Clean Border: Select an option from the drop-down list Select an option from the drop-down list to exclude from the count any objects that intersect to exclude from the count any objects that intersect the designated edges of your image or AOI.the designated edges of your image or AOI.

Pre-filter: Pre-filter: Select this option to include in the Select this option to include in the Total Total CountCount display, only those objects that are within the display, only those objects that are within the specified range. specified range.

When When Pre-filter Pre-filter is not checked, the is not checked, the Total Count Total Count value value represents all objects within the AOI or image, not just represents all objects within the AOI or image, not just those meeting the range criteria.those meeting the range criteria.

Fill Holes: Fill Holes: Select this option to exclude from the count Select this option to exclude from the count any objects that are embedded within other objects.any objects that are embedded within other objects.

Convex hull: Convex hull: Select this option to create a smooth area Select this option to create a smooth area aroundthe perimeter of irregularly shaped objectsaroundthe perimeter of irregularly shaped objects

ImagePro CountImagePro Count

3. Count and 3. Count and measure the measure the objects using the objects using the Count buttonCount button

ImagePro CountImagePro Count

Measure Objects – Measure Objects – record record measurements measurements with Countswith Counts

Apply RangesApply Ranges

Clear CountsClear Counts

Edit Menu –Split ObjectsEdit Menu –Split Objects

Split Manually – line, Split Manually – line, freeformfreeform

Auto-SplitAuto-Split

Watershed SplitWatershed Split

Limited Watershed SplitLimited Watershed Split

Draw /Merge CommandDraw /Merge Command

Draw AOIDraw AOI

Convert AOI to ObjectConvert AOI to Object

Edit MenuEdit Menu

Toggle Objects On/Off: Toggle Objects On/Off:

Use this command to show or Use this command to show or hide objectsin your image. hide objectsin your image.

When you left-click the When you left-click the mouse cursor on an object, mouse cursor on an object, its overlay is hidden, so its overlay is hidden, so that object will not be that object will not be taken into account in any taken into account in any measurements or measurements or statistics. statistics.

Left-click the mouse cursor Left-click the mouse cursor again to display the object.again to display the object.

Delete Hidden Objects: Delete Hidden Objects:

Use this command to remove Use this command to remove objects that you have objects that you have hidden, or that are out of hidden, or that are out of the selected range. the selected range.

Click Yes to remove the Click Yes to remove the objects. The remaining objects. The remaining objects in the image will be objects in the image will be renumbered.renumbered.

Apply to Multichannel Image usingApply to Multichannel Image usingFile>Save OutlinesFile>Save OutlinesFile> Load OutlinesFile> Load Outlines

Measure DataMeasure Data

Sort

Locate the Object – highlights selected object in image

Measure Data - SortMeasure Data - Sort

Count / View MenuCount / View Menu

Locate the Object – highlights selected object in image

Exported TableExported Table#Total Count: 21 Obj# Area

1 0.21522 0.25583 0.19164 0.1925 0.16756 0.22737 0.23128 0.24059 0.1958

10 0.182711 0.211312 0.356813 0.372814 0.116315 0.233616 0.253917 0.193118 0.237619 0.162420 0.229221 0.1537

File Menu

•Data to File•Append Data to file•Data to Clipboard•Table to Clipboard•Export Data