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.)


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

Course Schedule [PDF]

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]


Lecture Notes


L01 Hypothesis Driven Experimental Design

Including an introduction to the workshop


L02 Introduction to Week 1 Instruments

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

L08 Photobleaching, Photoactivation, and Photoconversion


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

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 Instruments for the Expansion Microscopy Labs

L17 Image Processing for Super-Resolution Microscopy

L18 Data Analysis for Super-Resolution Images

Filtering, point statistics, co-localization

L19 Introduction to Week 2 Instruments

L20 Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy

Potential pitfalls to consider

L21 Open-Source Gems

Image restoration with machine learning


Lab and Analysis Sessions


Lab 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


Analysis and Discussion Sections

A1 Analysis Lab: Cell Migration

Segmentation and Tracking

A2 Hypothesis Formulation and Experimental Design

A3 Analysis Lab: Subcellular Scale Imaging

FRAP measurements

A4 Co-localization Analysis

A5 Analysis Lab: Super-Resolution Imaging

A6 From Hypothesis to Results

Materials will be provided during the session

A7 Hypothesis Formulation: Organism-scale Imaging


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.

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

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