Optical Imaging Across Biological Length Scales
From Nanometers to Millimeters
July 19-31, 2026
HHMI Janelia Research Campus
Course Directors:
Teng-Leong Chew and Rachel M. Lee (HHMI Janelia)
Jump to: Lecture Notes | Labs and Analysis Sessions | Further Reading
COURSE INFORMATION
Course Schedule [PDF]
Course Instructors [PDF]
Imaging Rotation Groups [PDF]
Microscope Map [PDF]
Directions to laundry [PDF]
IALS 2026 Group Photo and Bonus Image
Share other photos of the course on Google Drive
Join the conversation on Bluesky:
@AICjanelia | #IALSJanelia
Additional Resources
Free Janelia Fluor dyes are available at dyes.janelia.org.
Plasmids from the Integrative Imaging team are on Addgene.
DATA & DOWNLOADS
Instructions for preparing your laptop and Fiji for IALS
Introduction Slide - Due to aic@janelia.hhmi.org by Wed July 15
Download template [60 KB .pptx]
Fiji Program
Use the IALS version of Fiji for all bootcamp activities.
Do not update Fiji after downloading
IALS Fiji for Mac [download 685 MB .zip]
IALS Fiji for Windows [download 630 MB .zip]
Sample Images for Fiji Lectures
1.5 GB of images [download 883 MB .zip]
Data for From Hypothesis to Results Activity
32 MB of images [download 16 MB .zip]
Accessing prfs\hackathondata
Instructions for accessing the Hackathon data share will be provided on the first Monday of the course.
Lecture Notes
L01 Hypothesis Driven Experimental Design
Including an introduction to the workshop
L02 From Nanometers to Millimeters
An introduction to the techniques and experiments for bootcamp
L03 Fundamentals of Digital Images
Basic concepts of digital images, file format, color scheme
L04 Introduction to Microscopy
Basic optical principles in microscopy
L05 Live Cell Fluorescence Imaging
Importance, pitfalls, and applications
L06 Introduction to Fluorescence Microscopy and Optical Sectioning
Widefield, TIRF, confocal, two-photon
L07 Introduction to the Co-localization Class Challenge
Exploring co-localization through imaging and analysis
L08 Fundamentals of Image Processing
Histograms, displays, pixel adjustment, filters, kernels
L09 Fourier Transformation in Image Processing and Optics
Image processing in Fourier space
Turning pixel map into discrete objects
The solution to the object segmentation homework will be posted on Wed
Shape analysis and object-based co-localization
L12 Analysis of Biological Movement I: Structural Motion
Kymographs, fluorescence recovery rate
L13 Analysis of Biological Movement II: Translational Motion
Particle tracking, motion analysis
L14 Introduction to Enhanced Resolution Imaging
SIM, pixel reassignment
L15 Object Segmentation with Machine Learning
Introduction to machine learning and trainable segmentation models
L16 Introduction to Multispectral Imaging
MSI fundamentals and best practices
L17 Pixel-based Co-localization and Intensity Analysis
Co-localization coefficients and ratiometric imaging
Co-localization of multispectral images
L19 Achieving Depth with Sample Prep
Clearing and Expansion Microscopy
L20 Introduction to Super-Resolution Imaging
STED, PALM/STORM
L21 Accurate and Sufficient Scientific Reporting
Effects of inaccurate and insufficient documentation and what constitutes good scientific reporting
L22 Image Processing for Super-Resolution Microscopy
Processing of STED, SIM, and PALM/STORM data
L23 Data Analysis for Localization Microscopy
Filtering, point statistics, segmentation
Types of light sheet systems and practical considerations
Blog post on understanding objective angles and the code the AIC uses for deskewing.
L25 Machine Learning Resources
Image restoration, neural networks, and other machine learning tools
L26 From Hypothesis to Results: Activity Discussion
Follow up after analysis lab A5
Approaches for increasing imaging depth, including aberration correction
Data handling and visualization
Tools for tracking big data
Lab and Analysis Sessions
LAB SESSIONS
W1 Co-localization Imaging Lab
Mitochondrial Membranes
Mitochondria fixation protocol
Single-Cell Migration
W3 Subcellular Scale Imaging Lab
Actin and Myosin
Protocols for live and fixed imaging
ExM and clearing
Additional slides on sample mounting
Clearing protocol | ExM protocol
W5 Super-resolution Imaging Lab
Focal Adhesions
Fixation and staining protocols
Zebrafish Wound Response
ANALYSIS AND DISCUSSION SESSIONS
A1 Analysis Lab: Cell Migration
Segmentation and tracking
Group Presentations | Google Colab Notebook
A2 Analysis Lab: Subcellular Scale Imaging
FRAP measurements
Pearson and Manders coefficients
A4 Hypothesis Formulation and Experimental Design
Making experimental decisions
Materials will be provided during the session
A6 Hypothesis Formulation: Organism-scale Imaging
Tracking wound responses
Group Presentations | Google Colab Notebook
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
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
Analysis of Image Similarity and Relationship
Jesse S. Aaron, Teng-Leong Chew
In: Jerome W., Price R. (eds) Basic Confocal Microscopy.
Springer, Cham
doi:10.1007/978-3-319-97454-5_11
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
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
Practical considerations for quantitative light sheet fluorescence microscopy
Hobson, C.M., Guo, M., Vishwasrao, H.D., Wu, Y., Shroff, H., Chew, T.-L.
Nat. Meth. 2022 19, 1538-1549
doi: 10.1038/s41592-022-01632-x
Combining multiple fluorescence imaging techniques in biology: when one microscope is not enough
Hobson, C.M. and Aaron, J.S.
MBoC 2022 33 (6)
doi: 10.1091/mbc.E21-10-0506
Ellen Quarles, Leanna Eisenman, Mai Rahmoon, and Teng-Leong Chew
EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization
Yuhan Wang, et. al.
Cell 2021 184 (26)
doi: 10.1016/j.cell.2021.11.024
Deep-tissue transcriptomics and subcellular imaging at high spatial resolution
Valentina Gandin, et. al.
Science 2025 388 (6744)
doi: 10.1126/science.adq2084
SuperPlots: Communicating reproducibility and variability in cell biology
Samuel J. Lord, Katrina B. Velle, R. Dyche Mullins, and Lillian K. Fritz-Laylin
Journal of Cell Biology 2020 219 (6): e202001064
doi: 10.1083/jcb.202001064
Redefining colocalization analysis with a novel Phasor Mixing Coefficient
Puls, O.F., Aaron, J.S., Quarles, E.K., Khuon, S., Eisenman, L.R., Kamaid, A., Malacrida, L., Chew, T.-L.
Journal of Cell Science 2025
doi: 10.1242/jcs.264388
MethodsJ2: a software tool to capture metadata and generate comprehensive microscopy methods text.
Ryan, J., Pengo, T., Sanders, M.A., & Brown, C.M.
Nature Methods 2021
doi: 10.1038/s41592-021-01290-5
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