User ManualUser Manual

MIQuant

Semi-automated histological quantification of myocardial

i f iinfarct size

IntroductionExperimental rodent models of myocardial infarction (MI) are extensively used toExperimental rodent models of myocardial infarction (MI) are extensively used to

investigate molecular, cellular and histological alterations following MI. Moreover, thesemodels have steadily been applied to access the potential of cell-based and/orpharmacological strategies aiming at the functional restoration of the damaged myocardium.The infarct size, defined as the percentage of the left ventricle (LV) affected by coronary arteryocclusion, is typically calculated on transverse histological sections stained with Masson´sTrichrome (MT). The lost myocardial tissue is partly replaced by collagen fibers that can bevisualized in blue following MT stain as early as 7 days post MI The final assessment of MIvisualized in blue following MT stain as early as 7 days post-MI. The final assessment of MIinfarct size is an average of the values obtained for all representative cross-sections of the LV.Determination of the infarct extension is time-consuming, laborious and prone to bias becauseit requires the manual definition of the infarcted and of the non-infarcted tissue in each one ofthe representative sections. The MIQuant software is a user-friendly semi-automatic tool thatfacilitates the infarct size calculation in a single cross-section or in multiple sections, retrievingthe final average value and the respective intermediate values in an excel file-format.

Schematic representation ofcross-section sampling of an

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infarcted heart collected 21days post-MI. Twelverepresentative sections wereobtained and stained with MT.Infarcted regions are limited by300µm

300µm300µm300µm300µm300µm

20X

15X

15X

15X

15X

20X Infarcted regions are limited bya yellow line.

300µm300µm

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IntroductionThe MIQuant software was developed to allow the quantification of the infarct size,

expressed as percentage, with two well-validated methods: the area measurement and themidline length measurement. The area measurement is based on the ratio between theinfarcted and normal LV area, while the midline length measurement is based on a line drawnin the center between the epicardial and the endocardial surfaces. The infarct size iscalculated dividing the length of the infarcted myocardium defined as the region with >50% ofcalculated dividing the length of the infarcted myocardium, defined as the region with >50% ofthe thickness of the myocardial wall with collagen deposition, with the LV length.

Area Measurement Midline Length MeasurementMI

midline

TM stain (21days post-MI)

LV

LVlumen

MILV

midline

MIQuant installation

1. The MIQuant software was developed in MatLab; itrequires the installation of the MCRInstaller exe (inrequires the installation of the MCRInstaller.exe (incase MatLab is not already installed in the destinationcomputer) prior to use.p ) p

2. To use MIQuant, the user must copy all files of the, pyapplication to a desired folder and then open theexecutable file MIQuant.exe (double-click).

User interface overview

Quick user guide

1. Follow instruction at the footnote bar –thi ill i i hi t h t l MIQ t

The basic steps to calculate MI size using MIQuant:

2 3this will give you main hints how to explore MIQuant.

2. Upload image/multiple images – MIQuantallows the quantification of MI size of a single imageor calculates the average value for several loadedimages

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images.

3. Edit image (if necessary) – remove the rightventricle, undesired staining artifacts or blood from theLV lumen which will otherwise interfere with tissuesectioning During the editing it is possible to enlarge

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sectioning. During the editing, it is possible to enlargethe image.

4. Segment normal-tissue – select the “normaltissue” button and double-click over a viablemyocardium region in the uploaded image

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6

6Previous N t myocardium region in the uploaded image.

5. Adjust threshold, if necessary, and repeatsegmentation.

6. Segment the LV lumen (if present) and adjust

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710

6Previous image

Nextimage

Image in analysis

threshold, if necessary.

7. Calculate MI size8. Color pallet assists on the image segmentation.

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9. Save image and/or save data in excelformat.

10. Proceed to the next image, if applicable.

Detailed user guide

MIQuant evaluates the infarct extension on transverse-

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histological sections of rodent hearts based on thedeposition of collagen (blue) in the ischemic region ashighlighted by the MT stain.

The user must follow several steps prior to MI sizel l ti T id th f t t b icalculation. To guide the user, a footnote bar is

displayed with basic instruction on the next steps and/oravailable optionsavailable options.

Detailed user guideThe user may either analyze a single or multiple images. Ifmultiple images are uploaded the MIQuant retrieves the

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multiple images are uploaded the MIQuant retrieves theaverage MI size of all sections and the intermediate resultsobtained for each section.

The “File” menu includes commands to upload the imageand/or multiple images in .jpg or .tiff format.

After image uploading the software automatically adjusts theAfter image uploading, the software automatically adjusts theimage’s quality; this pre-processing step can be removed onthe “Image quality” section.

Detailed user guideThe “edit menu” displays commands for

original image edition

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original image edition

Remove tissue – ignores undesired tissue regions. A RV

line must be defined on the original image, introducing a cut that separates the tissue to be removed. Click to define a line path and double-click/ENTER to complete LV

line

C t ti i j i i li b t

the line drawing.

Before AfterConnect tissue regions – a joining line between identical color image-regions allows the connection of different tissue regions that would otherwise be considered as different parts Click seq entiall onconsidered as different parts. Click sequentially on the two regions of the tissue that are to be connected. This tool can be used to link regions that were destroyed during tissue processingwere destroyed during tissue processing.

Detailed user guideThe “edit menu” displays commands for

original image edition

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Clean LV lumen – erases regions that can Before After

original image edition

interfere with tissue segmentation and replace itwith background (white) image color. To definethe region to be cleared. Click to define the pathand double-click/ENTER at last to complete thearea definition. This can be used to remove bloodfrom the LV lumen. Before After

Zoom-in and zoom-out – select a field of the image to be enlarged or decreased before image edition

Undo – erases all the edited images

image edition.

Detailed user guideg

1. Click on the “Normal tissue” or the “LV Lumen” buttons

Image segmentation:

to identify the region to be selected.

2. Click on the uploaded image over the region tosegment and double click/ENTER to completesegment and double-click/ENTER to completeselection.

3. If the segmentation is not correct a newsegmentation can be performed

LV lumen

segmentation can be performedchoosing new points to re-initialize theregion growing algorithm or by adjustingthe homogeneity aggregation parameter

Normal tissue

the homogeneity aggregation parameter(threshold) so that the region grows in adifferent way. The segmentation can berepeated until the user is satisfied withrepeated until the user is satisfied withthe segmentation results.

4. Repeat steps 1-3 to segment the other region.

5. The color pallet assists the user on the evaluation of the imagesegmentation obtained.

Detailed user guidegAfter performing tissue segmentation the MI size results can

be obtained by clicking on the button

The MI size, calculated by both midline length and areamidline length and area

measurements are shown in the MIQuant interface

Midline Editing - the user may adjustthe midline when wrongly predictedusing the “Edit midline” tool The midlineusing the Edit midline tool. The midlinecan be edited by clicking first on the dotto be corrected and then on the desiredfinal position The tool zoom in and

Before After

final position. The tool zoom-in andzoom-out can be used to enlarge ordecrease the image to edit the midline.The MI size by midline length isThe MI size by midline length isautomatically re-calculated.

Detailed user guideFinal segmented images can be saved

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When working with multiple images:

Final data can be saved in excel-format

When working with multiple images:Press or to go to previous or next image, respectively.

A preview of previous and next image of the image underA preview of previous and next image of the image under

analysis is available.

P t bt i th MI f ll th l d d

Previous image

Next image

Press to obtain the average MI for all the loaded

images.

A E l fil b bt i d ith th fi l d i t di t lt

Image in analysis

An Excel file can be obtained with the final and intermediate results

At any moment the user can abort the calculation of MI size by clicking on

, which clears all the performed work and re-initializes the program.

Contacts

MIQuant applications: Diana S. Nascimento (dsn@ineb.up.pt)

MIQuant bugs: Tiago José Arieira Esteves (meb09026@fe.up.pt)