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Optical Imaging Across Biological Length Scales
From Nanometers to Millimeters

July 10-22, 2022

HHMI Janelia Research Campus

Course Directors:

Teng-Leong Chew (HHMI Janelia)

Kate McDole (MRC Lab. of Mol. Bio.)

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Jump to: Lecture Notes | Labs and Analysis Sections | Further Reading

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*Course Evaluation Form*

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COURSE INFORMATION

Course Schedule [PDF]

Course Instructors [PDF]

Imaging Rotation Groups [PDF]

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COVID Testing Instructions [PDF]

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Join the conversation on Twitter: 

@AICjanelia | #IALSJanelia

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Download the full size group photo here [JPG]

Students can share photos from the course via Google Drive

 

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DOWNLOADS

Fiji Program

You should use this version even if you already have Fiji installed on your laptop.

Do not update Fiji after installation.

Click here to download for Mac
Click here to download for Windows
Click here to download for Linux

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Sample Images for Fiji Lectures
1.15 GB of images [download .zip] 

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From Hypothesis To Results Dataset

6.45 GB of images [download .zip]

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Temporary Imaris License

A temporary Imaris license file will be provided during bootcamp. You can download the program and installation instructions here.

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SR-Tesseler (for PALM/STORM Data Analysis)

Click here to download for Windows

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"Big Data" Demo Materials

Click here to download and unzip to your laptop

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Lecture Notes

Lecture Notes

L01 Hypothesis Driven Experimental Design

Including an introduction to the workshop

 

L02 Introduction to Week 1 Instruments

Diffraction-limited, enhanced-resolution, and super-resolution microscopy

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L03 Fundamentals of Digital Images

Basic concepts of digital images, file format, color scheme

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L04 Fundamentals of Image Processing

Histograms, displays, pixel adjustment, filters, kernels 
 

L05 Fourier Transformation in Image Processing and Optics

Image processing in Fourier space

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L06 Object Segmentation

Turning pixel map into discrete objects

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L07 Instruments for the Cellular Scale Imaging Labs

For imaging lab W2

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L08 Fluorescent Molecules

Importance, pitfalls, and applications

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L09 Object-based Co-localization Analysis

Quantifying overlapping objects

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L10 Pixel-based Co-localization and Intensity Analysis

Colocalization coefficients, ratiometric imaging 

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L11 Object Segmentation with Machine Learning

Introduction to machine learning and trainable segmentation models

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L12 Analysis of Biological Movement

Fluorescence recovery rate, particle tracking, motion analysis

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L13 Instruments for the Subcellular Scale Imaging Labs

For imaging lab W4

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L14 Instruments for the Super-Resolution Imaging Labs

STED & iPALM

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L15 Accurate and Sufficient Scientific Reporting

The effects of inaccurate and insufficient documentation, and what constitutes good scientific reporting

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L16 Image Processing for Super-Resolution Microscopy

For imaging lab W5

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L17 Data Analysis for Super-Resolution Images

Filtering, point statistics, co-localization

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L18 Introduction to Week 2 Instruments

For imaging lab W7

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L19 Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy

Potential pitfalls to consider

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L20 Open-Source Gems

Image restoration with machine learning

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Expansion Microscopy: Super-Resolution for All Occassions

Lecture by Paul Tillberg

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Multiscale Biological Imaging at High Spatiotemporal Resolution

Lecture by Hari Shroff

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Lab and Analysis Sessions

Labs

LAB SESSIONS

 

W1 Co-localization Imaging Lab

Myosin

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W2 Cellular Scale Imaging Lab

Cell Migration


W3 Multicellular Imaging Lab

Overnight wound healing assay


W4 Subcellular Scale Imaging Lab

Actin and Myosin


W5 Super-resolution Imaging Lab

Paxillin


W6 Expansion Microscopy (ExM) Imaging Lab

Zebrafish


W7 Organism-scale Imaging Lab

Zebrafish
 

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ANALYSIS AND DISCUSSION SECTIONS

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A1 Analysis Lab: Cell Migration

Segmentation and Tracking


A2 Hypothesis Formulation and Experimental Design

Making experimental decisions

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A3 Analysis Lab: Subcellular Scale Imaging

FRAP measurements

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A4 Co-localization Analysis

Analyzing the data from W1


A5 Analysis Lab: Super-Resolution Imaging

Segmentation

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A6 From Hypothesis to Results

Materials will be provided during the session


A7 Hypothesis Formulation: Organism-scale Imaging

Zebrafish Analysis
 

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Further Reading

Reading

When Light Meets Biology: How the Specimen Affects Quantitative Microscopy

Michael Reiche, Jesse Aaron, Ulrike Böhm, Michael DeSantis, Chad Hobson, Satya Khuon, Rachel Lee, and Teng-Leong Chew

J. Cell Sci. 2022

doi:10.1242/jcs.259656

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A guide to accurate reporting in digital image acquisition - can anyone replicate your microscopy?

John M. Heddleston, Jesse S. Aaron, Satya Khuon, Teng-Leong Chew

J Cell Sci 2021

doi: 10.1242/jcs.254144

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A guide to accurate reporting in digital image processing - can anyone reproduce your microscopy?

Jesse S. Aaron, Teng-Leong Chew

J Cell Sci 2021

doi:10.1242/jcs.254151

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Hypothesis-driven quantitative fluorescence microscopy - the importance of reverse-thinking in experimental design

Eric C. Wait, Michael A. Reiche, Teng-Leong Chew

J Cell Sci 2020 133.

doi:10.1242/jcs.250027

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Practical considerations in particle and object Tracking and Analysis

Jesse S. Aaron, Eric Wait, Michael DeSantis, Teng-Leong Chew

Curr Prot Cell Biol 2019 e88.

doi: 10.1002/cpcb.88

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Image co-localization – co-occurrence versus correlation

Jesse S. Aaron, Aaron B. Taylor, Teng-Leong Chew

J Cell Sci 2018 131: jcs211847

doi: 10.1242/jcs.211847

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Imaging methods are vastly underrepresented biomedical research

Guillermo Marques, Thomas Pengo, Mark A. Sanders

eLife 2020;9:e55133

doi: 10.7554/eLife.55133

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Model-free quantification and visualization of colocalization in fluorescence images

Aaron B. Taylor, Maria S. Ioannou, Jesse S. Aaron, Teng-Leong Chew

Cytometry Part A 2018 

doi: 10.1002/cyto.a.23356

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Perceptually accurate display of two greyscale images as a single colour image

Aaron.B. Taylor, Maria.S. Ioannou, Takashi Watanabe, Klaus Hahn, Teng-Leong Chew

J. Microscopy 2018 268: jmi.12588

doi:10.1111/jmi.12588 

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Automatic and quantitative measurement of protein-protein colocalization in live cells

Costes, S. V., Daelemans, D., Cho, E. H., Dobbin, Z., Pavlakis, G. and Lockett, S.

Biophys. J 2004 86: 3993-4003.

doi: https://doi.org/10.1529/biophysj.103.038422

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Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

Alison J. North

J. Cell Biol. 2006 172: 9-18

doi: 10.1083/jcb.200507103

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Accuracy and precision in quantitative fluorescence microscopy

Jennifer C. Waters

J. Cell Biol. 2009 187: 1135-1148

doi: 10.1083/jcb.200903097

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Protein-Retention Expansion Microscopy (ExM): Scalable and Convenient Super-Resolution Microscopy

Paul Tillberg

Methods Mol Biol. 2021;2304:147-156.

doi: 10.1007/978-1-0716-1402-0_7

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What If Scientists Shared Their Reagents for Free?

Amanda Heidt

The Scientist, July 2022 Issue 2

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