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Course Schedule [PDF]

Course Instructors [PDF]

Imaging Rotation Groups [PDF]

End-of-Course Survey

Job Posting: Advanced Microscopy Imaging Fellow at the Advanced Imaging Center

Join the conversation on Twitter:

#OkinawaMirscopy2024 | @AICjanelia@OISTedu | Twitter List


Introduction Slide

Please click here to download a slide you will use to introduce yourself to the group. Please stay within the space allotted for each question; you will only have one minute to present your slide to the group. For the image, you may add a microscope image you have acquired or another image that is reflective of your research. Please return your completed slide to by Monday, Sept 12 with the subject line ILA 2022 Introduction Slide.

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

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


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


Lecture Notes

Workshop Introduction

Driving principles and goals of the workshop

Digital Images as Quantitative Data Sources

Basic concepts of digital images, file format, color scheme

Introduction to Optics I

Image formation, magnification, resolution, objective lenses

Introduction to Optics 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

Object Segmentation with Machine Learning

Introduction to machine learning and trainable segmentation models

Super-resolution Techniques

SIM, Airyscan, STED and SMLM

FIJI: Object-based Co-localization

Quantifying overlapping objects

FIJI: Pixel-based Co-localization 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 (ML)

ML techniques for improving SNR, deblurring, etc.

Bias and Sufficient Scientific Reporting

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

Colocalization Lab Primer

Introduction to Analysis Lab II


Lab and Analysis Sessions

Imaging Lab1: Basic Microscopy Techniques

Kohler illumination, brightfield microscopy, phase contrast, DIC, image formation demonstration, dark field

Imaging Lab 2: Advanced Microscopy Techniques

Point spread functions, fixed slide imaging, Fourier optics demonstration

Imaging Lab 3: Super-resolution Techniques

Live cell imaging of actin and lysosomes

Imaging Lab 4: Live Cell Imaging
Live cell imaging of actin and lysosomes

Class Challenge: Hypothesis Formation and Experimental Design

Hypothesis-driven experimental design

Analysis Lab I: Live Cell Imaging

Tracking, FRAP, kymographs. Download the Example Data here.


Analysis Lab II: Colocalization

Co-localization Analysis. Download the Example Data here.


Further Reading


Okinawa Microscopy Workshop 2024

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

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


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


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.


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

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


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.


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

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

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