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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

*Course Evaluation Form*


Course Schedule [PDF]

Course Instructors [PDF]

Imaging Rotation Groups [PDF]

COVID Testing Instructions [PDF]

Join the conversation on Twitter: 

@AICjanelia | #IALSJanelia



Download the full size group photo here [JPG]

Students can share photos from the course via Google Drive



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] 

From Hypothesis To Results Dataset

6.45 GB of images [download .zip]

Temporary Imaris License

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

SR-Tesseler (for PALM/STORM Data Analysis)

Click here to download for Windows

"Big Data" Demo Materials

Click here to download and unzip to your laptop

2022-07-11 IALS Group Photo - Small Banner.jpg

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

L03 Fundamentals of Digital Images

Basic concepts of digital images, file format, color scheme

L04 Fundamentals of Image Processing

Histograms, displays, pixel adjustment, filters, kernels 

L05 Fourier Transformation in Image Processing and Optics

Image processing in Fourier space

L06 Object Segmentation

Turning pixel map into discrete objects

L07 Instruments for the Cellular Scale Imaging Labs

For imaging lab W2

L08 Fluorescent Molecules

Importance, pitfalls, and applications

L09 Object-based Co-localization Analysis

Quantifying overlapping objects

L10 Pixel-based Co-localization and Intensity Analysis

Colocalization coefficients, ratiometric imaging 

L11 Object Segmentation with Machine Learning

Introduction to machine learning and trainable segmentation models

L12 Analysis of Biological Movement

Fluorescence recovery rate, particle tracking, motion analysis

L13 Instruments for the Subcellular Scale Imaging Labs

For imaging lab W4

L14 Instruments for the Super-Resolution Imaging Labs


L15 Accurate and Sufficient Scientific Reporting

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

L16 Image Processing for Super-Resolution Microscopy

For imaging lab W5

L17 Data Analysis for Super-Resolution Images

Filtering, point statistics, co-localization

L18 Introduction to Week 2 Instruments

For imaging lab W7

L19 Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy

Potential pitfalls to consider

L20 Open-Source Gems

Image restoration with machine learning

Expansion Microscopy: Super-Resolution for All Occassions

Lecture by Paul Tillberg

Multiscale Biological Imaging at High Spatiotemporal Resolution

Lecture by Hari Shroff


Lab and Analysis Sessions




W1 Co-localization Imaging Lab


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


W6 Expansion Microscopy (ExM) Imaging Lab


W7 Organism-scale Imaging Lab



A1 Analysis Lab: Cell Migration

Segmentation and Tracking

A2 Hypothesis Formulation and Experimental Design

Making experimental decisions

A3 Analysis Lab: Subcellular Scale Imaging

FRAP measurements

A4 Co-localization Analysis

Analyzing the data from W1

A5 Analysis Lab: Super-Resolution Imaging


A6 From Hypothesis to Results

Materials will be provided during the session

A7 Hypothesis Formulation: Organism-scale Imaging

Zebrafish Analysis


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


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

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

Lee, R.M., Eisenman, L.R., Khuon, S., Aaron, J.S. and Chew, T.-L.

J. Cell Sci. 2024 137 (1): jcs261567

doi: 10.1242/jcs.261567

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