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Okinawa Microscopy Workshop 2024

An all-expenses-paid Microscopy and Image Analysis Workshop
for life scientists working in Japan and Southeast Asia

LectureNotes
LabSessions

This website will be continuously updated leading up to and throughout the workshop.
Please check back (and refresh the page!) regularly for new uploads and links.

End of Course Survey

To keep in touch with everyone in the future:

Directory of Course Participants and Instructors: Access it here!

Course WhatsApp Group: Join here!

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

COURSE INFORMATION

Course Schedule [PDF]

Course Instructors [PDF]

 

Join the conversation on Twitter:

#OKAscope | @AICjanelia@OISTedu | Twitter List

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 [after download "Ctrl+Click" and select "Open" to run]
Click here to download for Windows

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

PHOTOS

If you would like to share photos of the course, you may add them to this Google Drive folder.

The cohort from OMW 2024! Look at all these smiling faces! And some words of wisdom...

(To "decode" this image, go to the Photos folder above to download it!)

"It is the sample.

Garbage in means garbage out.

Fiji doesn't care.

mCherry, a curse,

Worst of the fluorescent lot,

Haunting your image.

It always depends.

Every photon is sacred.

There is no free lunch.

NA's everything.

It's Fourier Transformation.

Physics, not magic."

                                 --- OMW Teaching Team

 [ ChatGPT contributed too -- guess which one ;) ]

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Lecture Notes

Workshop Introduction

Driving principles and goals of the workshop

Fundamentals of Digital Images

Basic concepts of digital images, file format, color scheme

Introduction to Microscopy I

Image formation, magnification, resolution, objective lenses

Introduction to Microscopy II

Kohler illumination, microscope configurations, contrast in microscopy

Basics of Fluorescence

Histograms, displays, pixel adjustment, filters, kernels 
 

FIJI: Image Processing

Histograms, displays, pixel adjustment, filters, kernels

FIJI: Fourier Transformation in Image Processing

Image processing in Fourier space

Fluorescence Microscopy Modalities

Widefield, TIRF, confocal, two-photon, image scanning microscopy

FIJI: Object Segmentation

Turning pixel map into discrete objects

FIJI: Object Segmentation with Machine Learning

Introduction to machine learning and trainable segmentation models

Super-Resolution Microscopy

SIM, Airyscan, STED and SMLM

FIJI: Object-based Colocalization

Quantifying overlapping objects

FIJI: Pixel-based Colocalization and Intensity Analysis

Colocalization coefficients, ratiometric imaging 

Advanced Fluorescence Techniques

FRET, FRAP, FLIP, photomanipulation

Live Cell Imaging

Environmental control, photon budget, phototoxicity

FIJI: Measurement of Dynamic Changes

Fluorescence recovery rate, particle tracking, motion analysis

Image Restoration

Open-source ML techniques for improving SNR, deblurring, etc.

Bias and Reporting

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

Zeiss talk

Exploring the Potential of Image Analysis & AI for Automated Microscopy Workflows

JAEQ_2018-01-25_0092_edited.jpg

Lab and Analysis Sessions

Imaging Lab1: Basic Microscopy Techniques

Köhler Illumination, Brightfield Microscopy, Dark Field Microscopy, Phase Contrast Microscopy, Differential Interference Contrast (DIC) Microscopy, Widefield Fluorescence Microscopy

Imaging Lab 2: Advanced Microscopy Techniques

Laser Scanning Confocal Microscopy, Spinning Disk Confocal Microscopy, Light-Sheet Microscopy, Two-Photon Microscopy


Imaging Lab 3: Super-Resolution Techniques

Airyscan, Structured Illumination Microscopy (SIM), Single-Molecule Localization Microscopy (SMLM), Stimulated Emission Depletion (STED) Microscopy


Imaging Lab 4: Live Cell Imaging
Fluorescence Recovery After Photobleaching (FRAP) and Lysosome Tracking


Class Challenge: Hypothesis Formation and Experimental Design

Hypothesis-driven experimental design
 

Analysis Lab I: Live Cell Imaging

Tracking, FRAP

Data for Analysis

Colocalization Lab Primer

Introduction to Analysis Lab II

 

Analysis Lab II: Colocalization

Colocalization Analysis

Data for Analysis

FurtherReading
JAPE_2018-01-16_0067.jpg

Further 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

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

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

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

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

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

Aaron J., Chew TL. (2018) Analysis of Image Similarity and Relationship. In: Jerome W., Price R. (eds) Basic Confocal Microscopy. Springer, Cham (PDF)

Imaging methods are vastly underrepresented biomedical research

Guillermo Marques, Thomas Pengo, Mark A. Sanders

eLife 2020;9:e55133

doi: 10.7554/eLife.55133

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

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 

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

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

Accuracy and precision in quantitative fluorescence microscopy

Jennifer C. Waters

J. Cell Biol. 2009 187: 1135-1148

doi: 10.1083/jcb.200903097

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

What If Scientists Shared Their Reagents for Free?

Amanda Heidt

The Scientist, July 2022 Issue 2

Transfection of Cultured Primary Neurons

Annalisa Rossi, Ralf Dahm, and Paolo Macchi

Stem Cell Technologies in Neuroscience, 2017

doi: 10.1007/978-1-4939-7024-7_4

Phototoxicity in live fluorescence microscopy, and how to avoid it

Jaroslav Icha, Michael Weber, Jennifer C. Waters, and Caren Norden

BioEssays, 2017

doi: 10.1002/bies.201700003

Believing is seeing – the deceptive influence of bias in quantitative microscopy

Rachel M. Lee, Leanna R. Eisenman, Satya Khuon, Jesse S. Aaron, Teng-Leong Chew

J. Cell Sci. 2024

doi: 10.1242/jcs.261567

A list of useful microscopy resources can be found at:

https://www.janelia.org/support-team/integrative-imaging/additional-information

Jump to: Top | Lecture Notes | Lab and Analysis Sections | Further Reading

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