Fiji使用手册.pdf

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About this Manual

Introduction

A Note on Image Analysis

Distribution

Authors

Requirements

Initial Setup

Image Contributors

Introduction to Fiji

Introduction

Where to get Fiji

Configuration

Memory and Processor Threads

Proxy Settings

Updates

Monitoring and Clearing up Excess Memory Usage

Monitoring Memory

Basic Module

Aim

Opening Files

Zooming In and Out

Panning the Image

Duplicating an Image

Regions of Interest (ROI)

Drawing ROIs

Moving ROIs

Resizing ROIs

Using the ROI Manager to add Multiple ROIs

Aim

Adjusting Brightness and Contrast – Colour Images

Adjusting the Colour Balance – Colour Images

Adjusting the Brightness and Contrast – Monochrome Images

Aim

Calibrating an Image

Adding a Scale Bar

Aim

Changing Bit Depth – 24bit RGB

Adding Colour – Using Lookup Tables

Single Colour LUTs

Multicolour LUTs

Intensity Distribution

Merging Images

Merging Up To 7 Channels and DIC/Phase/Brightfield – Standard Colours

Merging Up To 7 Channels and DIC/Phase/Brightfield – Non-standard Colours

Merging 8 or more colour channels

Simulating Colour Blindness

Aim

LOCI Tools

Initial File Opening

Displaying Metadata

Default Opening States

Opening Confocal Data

Leica – LIF Format

Nikon – ICS/IDS File Format

Nikon – ND2 File Format

Single Data Set

Multiple Data Set

Olympus – OIB and OIF File Format

Method 1 – Reassign LUTs

Method 2 – Applying Colour at Opening

Zeiss – LSM File Format

Opening DeltaVision Data

Aim

Visualising 3D Data

Z Projection

Orthogonal Projection

Slice Montage

3D Volume – Simple

Making Movies

Aim

Measuring the Area and Intensity of Stain - Fluorescence

Separating Merged Multichannel Images

Threshold Image

Automatic Threshold Algorithms

Measure Thresholded Area

Measuring the Area of Stain – Brightfield

Make a Brightfield Image Look like a Fluorescent One

Aim

Initial Binary Creation

Counting Cells – Basic Count

Counting Cells – Detailed Information

Aim

Creating Whole Cell Binary Masks

Analysing the Image

Checking the Effectiveness of Segmentation

Method 1 – ROI Manager

Method 2 – Create Colour Overlays

Complex Segmentation

Refinement of Compartment Masks

Removing Extra Nuclear Masks

Working with Tricker Stains

Aim

Single Point Analysis

Multi Point Analysis – Manual ROI Entry

Multi Point Analysis – Automatic ROI Entry

Aim

Fixed Sample Analysis – Fluorescence

Selecting the Scratch

Measuring the Scratch and Logging the Data

Removing Unwanted Area from the Analysis

Method 1 – Size exclusion

Method 2 – Using Image Filters

Create an Outline of the Result

Fixed Sample Analysis – Brightfield

Live Sample Analysis

Selecting the Scratch

Measuring Scratch Area

Creating Overview Movie of Result

Aim

Tips for Capturing Your Images

Manually Creating a Montage

Automatically Creating a Montage – Brightfield

Automatically Creating a Montage – Fluorescence

Aim

Auto Alignment of Confocal Data

Auto Alignment of Serial Sections

Aim

Extended Depth of Focus – Macro Images

Extended Depth of Focus – Microscope Images

Aim

3D Visualisation

Visualisation

Making Movies

Predefined

Manually Set

3D Measurement

Measuring Objects in 3D

Generating 3D models of the Results

Aim

Training the Module

Running the Analysis

Reviewing the Data

Analysing the Data

Advanced Module

Aim

The Macro Recorder

Recording your First Macro – Threshold Measurement

Re-running the Macro

Recording your Second Macro – Cell Counting

Aim

The Macro Editor

Variables

Writing your first Macro – Automatically Merge Images

Merge DAPI, FITC and DIC.ijm – Final Code

Writing Your Second Macro – Channel Separate and Threshold Measurement

Vessel and Hypoxia Measurment.ijm – Final Code

Outputting a Result Overview

Vessel and Hypoxia Measurement (with Result Output).ijm – Final Code

Aim

Batch Processing Concepts

Batch Process - Cell Counting

Cell Count (Batch).ijm – Final Code

Batch Process - Cell Counting – Contaminated Directory

Cell Count (Batch – Tiff Only).ijm – Final Code

Batch Processing and Outputting Data

Vessel and Hypoxia Measurement (with Result Output).ijm – Final Code

Aim

Creating a Custom Log Table

First Steps – Batch Code

Custom Log Creation

The Analysis Code

Ki67 and PH3 Count.ijm – Final Code

Adding Refinements – Positive Cell Selection

Ki67 and PH3 Count (Refined).ijm – Final Code

Outputting Results Images to a Macro Created Folder

Ki67 and PH3 Count (Refined with Output).ijm – Final Code

Aim

Global Variables

Vessel and Hypoxia Measurement

Directory Selection and Array Function

Split Images Function

Measure Function

Save Overview Images Function

Vessel and Hypoxia Measurement (with Result Output – functions).ijm – Final Code

Ki67 and PH3 Count

Global Variables

File Function

Make Table Function

Channel Function

Count DAPI Function

Count Ki67 Function

Count PH3 Function

Calculate Percentage Function

Log Data Function

Save Image Output Function

Ki67 and PH3 Count (Refined with Output – functions).ijm – Final Code

Macro Functions

Macro Functions List

Contents About this Manual .................................................................................................................................. 1 Introduction ........................................................................................................................................ 1 A Note on Image Analysis ................................................................................................................... 1 Distribution ......................................................................................................................................... 1 Authors ................................................................................................................................................ 2 Requirements ...................................................................................................................................... 2 Initial Setup ......................................................................................................................................... 2 Image Contributors ............................................................................................................................. 4 Introduction to Fiji .................................................................................................................................. 5 Introduction ........................................................................................................................................ 5 Where to get Fiji ................................................................................................................................. 5 Configuration ...................................................................................................................................... 6 Memory and Processor Threads ..................................................................................................... 6 Proxy Settings .................................................................................................................................. 8 Updates ........................................................................................................................................... 9 Monitoring and Clearing up Excess Memory Usage ......................................................................... 11 Monitoring Memory ..................................................................................................................... 11 Basic Module ......................................................................................................................................... 13 Basic Navigation in Fiji .......................................................................................................................... 14 Aim .................................................................................................................................................... 14 Opening Files ..................................................................................................................................... 14 Zooming In and Out .......................................................................................................................... 15 Panning the Image ............................................................................................................................ 15 Duplicating an Image ........................................................................................................................ 16 Regions of Interest (ROI) ................................................................................................................... 17 Drawing ROIs ................................................................................................................................. 17 Moving ROIs .................................................................................................................................. 18 Resizing ROIs ................................................................................................................................. 18 Using the ROI Manager to add Multiple ROIs ............................................................................... 18 Adjusting Brightness and Contrast ........................................................................................................ 20

Aim .................................................................................................................................................... 20 Adjusting Brightness and Contrast – Colour Images ......................................................................... 20 Adjusting the Colour Balance – Colour Images ................................................................................. 22 Adjusting the Brightness and Contrast – Monochrome Images ....................................................... 24 Calibrating and Adding a Scale Bar ....................................................................................................... 27 Aim .................................................................................................................................................... 27 Calibrating an Image ......................................................................................................................... 27 Adding a Scale Bar ............................................................................................................................. 29 Fluorescent Images – Changing Bit Depth, Adding Colour, Merging Images and Simulating Colour Blindness ............................................................................................................................................... 30 Aim .................................................................................................................................................... 30 Changing Bit Depth – 24bit RGB ....................................................................................................... 31 Adding Colour – Using Lookup Tables ............................................................................................... 32 Single Colour LUTs ......................................................................................................................... 32 Multicolour LUTs ........................................................................................................................... 33 Intensity Distribution .................................................................................................................... 34 Merging Images ................................................................................................................................ 36 Merging Up To 7 Channels and DIC/Phase/Brightfield – Standard Colours ................................. 36 Merging Up To 7 Channels and DIC/Phase/Brightfield – Non-standard Colours ......................... 38 Merging 8 or more colour channels .............................................................................................. 41 Simulating Colour Blindness ............................................................................................................. 44 Opening Confocal and Deconvolution Data Sets .................................................................................. 46 Aim .................................................................................................................................................... 46 LOCI Tools ......................................................................................................................................... 46 Initial File Opening ........................................................................................................................ 46 Displaying Metadata ..................................................................................................................... 47 Default Opening States ................................................................................................................. 47 Opening Confocal Data ..................................................................................................................... 49 Leica – LIF Format ......................................................................................................................... 49 Nikon – ICS/IDS File Format .......................................................................................................... 54 Nikon – ND2 File Format ............................................................................................................... 56

Olympus – OIB and OIF File Format .............................................................................................. 60 Zeiss – LSM File Format ................................................................................................................. 62 Opening DeltaVision Data ................................................................................................................. 63 Visualising 3D and Live Data ................................................................................................................. 65 Aim .................................................................................................................................................... 65 Visualising 3D Data............................................................................................................................ 65 Z Projection ................................................................................................................................... 65 Orthogonal Projection................................................................................................................... 67 Slice Montage................................................................................................................................ 68 3D Volume – Simple ...................................................................................................................... 70 Making Movies .................................................................................................................................. 72 Simple Analysis – Area and Intensity of Stain ....................................................................................... 74 Aim .................................................................................................................................................... 74 Measuring the Area and Intensity of Stain - Fluorescence ............................................................... 74 Separating Merged Multichannel Images ..................................................................................... 74 Threshold Image ........................................................................................................................... 75 Automatic Threshold Algorithms .................................................................................................. 77 Measure Thresholded Area........................................................................................................... 78 Measuring the Area of Stain – Brightfield......................................................................................... 81 Make a Brightfield Image Look like a Fluorescent One ................................................................ 83 Cell Counting ......................................................................................................................................... 85 Aim .................................................................................................................................................... 85 Initial Binary Creation ....................................................................................................................... 85 Counting Cells – Basic Count ............................................................................................................. 88 Counting Cells – Detailed Information .............................................................................................. 91 Cell Segmentation and Analysis ............................................................................................................ 95 Aim .................................................................................................................................................... 95 Creating Whole Cell Binary Masks .................................................................................................... 95 Analysing the Image ........................................................................................................................ 103 Checking the Effectiveness of Segmentation .................................................................................. 105 Method 1 – ROI Manager ........................................................................................................... 105

Method 2 – Create Colour Overlays ........................................................................................... 108 Complex Segmentation ................................................................................................................... 111 Refinement of Compartment Masks........................................................................................... 113 Working with Tricker Stains ............................................................................................................ 115 Intensity over Time ............................................................................................................................. 118 Aim .................................................................................................................................................. 118 Single Point Analysis ....................................................................................................................... 118 Multi Point Analysis – Manual ROI Entry ........................................................................................ 121 Multi Point Analysis – Automatic ROI Entry .................................................................................... 125 Scratch Wound Assay Analysis (Fixed and Live) .................................................................................. 130 Aim .................................................................................................................................................. 130 Fixed Sample Analysis – Fluorescence ............................................................................................ 130 Selecting the Scratch ................................................................................................................... 130 Measuring the Scratch and Logging the Data ............................................................................. 132 Removing Unwanted Area from the Analysis ............................................................................. 134 Method 1 – Size exclusion .......................................................................................................... 134 Method 2 – Using Image Filters .................................................................................................. 136 Create an Outline of the Result .................................................................................................. 139 Fixed Sample Analysis – Brightfield ................................................................................................ 144 Live Sample Analysis ....................................................................................................................... 148 Selecting the Scratch ................................................................................................................... 148 Measuring Scratch Area .............................................................................................................. 152 Creating Overview Movie of Result ............................................................................................ 154 Image Stitching ................................................................................................................................... 157 Aim .................................................................................................................................................. 157 Tips for Capturing Your Images ....................................................................................................... 157 Manually Creating a Montage ........................................................................................................ 157 Automatically Creating a Montage – Brightfield ............................................................................ 161 Automatically Creating a Montage – Fluorescence ........................................................................ 164 Image Stack Alignment ....................................................................................................................... 167 Aim .................................................................................................................................................. 167

Auto Alignment of Confocal Data ................................................................................................... 167 Auto Alignment of Serial Sections................................................................................................... 169 Extended Depth of Focus .................................................................................................................... 171 Aim .................................................................................................................................................. 171 Extended Depth of Focus – Macro Images ..................................................................................... 171 Extended Depth of Focus – Microscope Images ............................................................................. 174 3D Visualisation and Measurement .................................................................................................... 177 Aim .................................................................................................................................................. 177 3D Visualisation ............................................................................................................................... 177 Visualisation ................................................................................................................................ 177 Making Movies ............................................................................................................................ 180 3D Measurement ............................................................................................................................ 184 Measuring Objects in 3D ............................................................................................................. 184 Generating 3D models of the Results ......................................................................................... 188 Advanced Image Segmentation .......................................................................................................... 191 Aim .................................................................................................................................................. 191 Training the Module ....................................................................................................................... 191 Running the Analysis ....................................................................................................................... 199 Reviewing the Data ......................................................................................................................... 202 Analysing the Data .......................................................................................................................... 206 Advanced Module ............................................................................................................................... 207 Basic Macro Recording ........................................................................................................................ 209 Aim .................................................................................................................................................. 209 The Macro Recorder ....................................................................................................................... 209 Recording your First Macro – Threshold Measurement ................................................................. 210 Re-running the Macro ................................................................................................................. 214 Recording your Second Macro – Cell Counting .............................................................................. 216 Writing Macros and an Introduction to Variables .............................................................................. 220 Aim .................................................................................................................................................. 220 The Macro Editor ............................................................................................................................ 221 Variables ......................................................................................................................................... 221 Writing your first Macro – Automatically Merge Images ............................................................... 222 Merge DAPI, FITC and DIC.ijm – Final Code ................................................................................ 229

Writing Your Second Macro – Channel Separate and Threshold Measurement ............................ 230 Vessel and Hypoxia Measurment.ijm – Final Code ..................................................................... 234 Outputting a Result Overview ......................................................................................................... 235 Vessel and Hypoxia Measurement (with Result Output).ijm – Final Code ................................. 237 Batch Processing ................................................................................................................................. 239 Aim .................................................................................................................................................. 239 Batch Processing Concepts ............................................................................................................. 239 Batch Process - Cell Counting.......................................................................................................... 239 Cell Count (Batch).ijm – Final Code ............................................................................................. 244 Batch Process - Cell Counting – Contaminated Directory ............................................................... 245 Cell Count (Batch – Tiff Only).ijm – Final Code ........................................................................... 246 Batch Processing and Outputting Data ........................................................................................... 247 Vessel and Hypoxia Measurement (with Result Output).ijm – Final Code ................................. 249 Custom Data Logging .......................................................................................................................... 251 Aim .................................................................................................................................................. 251 Creating a Custom Log Table .......................................................................................................... 251 First Steps – Batch Code.................................................................................................................. 252 Custom Log Creation ....................................................................................................................... 252 The Analysis Code ........................................................................................................................... 254 Ki67 and PH3 Count.ijm – Final Code .......................................................................................... 258 Adding Refinements – Positive Cell Selection ................................................................................. 260 Ki67 and PH3 Count (Refined).ijm – Final Code .......................................................................... 261 Outputting Results Images to a Macro Created Folder .................................................................. 263 Ki67 and PH3 Count (Refined with Output).ijm – Final Code ..................................................... 265 Functions (sub-processes/sub-routines) ............................................................................................. 268 Aim .................................................................................................................................................. 268 Global Variables .............................................................................................................................. 268 Vessel and Hypoxia Measurement ................................................................................................. 269 Directory Selection and Array Function ...................................................................................... 269 Split Images Function .................................................................................................................. 272 Measure Function ....................................................................................................................... 274

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