Optical Imaging Across Biological Length Scales
From Nanometers to Millimeters
July 28 - August 9, 2024
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​
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COURSE INFORMATION
Course Schedule [PDF]
Course Instructors [PDF]
Imaging Rotation Groups [PDF]
Microscope Map [PDF]
Directions to laundry [PDF]
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Course Evaluation: https://www.surveymonkey.com/r/GYGQQW6
Share your photos here: Google Drive Folder​
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​Join the conversation on Twitter:
@AICjanelia | #IALSJanelia​​​​​​​​​​
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​Listen to music across global length scales:
Submit a song here | Listen to the playlist here​​
Participant-created song: Recording & Lyrics
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​Link to Thorlabs Fourier Kit
Free​ Janelia Fluor dyes at dyes.janelia.org.
Plasmids from the Integrative Imaging team on Addgene.
Information for Udvar-Hazy trip.
Pecan and Cacao Nib cookie recipe.
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Announcement of AIC Proposal Call.
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DATA & DOWNLOADS
Accessing Data on PRFS\Hackathon
Remote access to the Hackathon folder is no longer available.
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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
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Sample Images for Fiji Lectures
1.92 GB of images [download .zip]
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Co-localization Data
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​PoCA (Program for PALM/STORM Data Analysis)
If you have a Windows machine, download the latest release's zip file here.
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Data for From Hypothesis to Results Activity
32 MB of images [download .zip]
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IALS 2024 Team Photo
To "decode" this image, look for the FFT image in the "PhotoSharing" folder in hackathon!
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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
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L03 Fundamentals of Digital Images
Basic concepts of digital images, file format, color scheme
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L04 Fundamentals of Image Processing
Histograms, displays, pixel adjustment, filters, kernels
L05 Fourier Transformation in Image Processing and Optics
Image processing in Fourier space
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Turning pixel map into discrete objects
Solution to the object segmentation homework
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L07 Introduction to Fluorescence Microscopy and Optical Sectioning
Widefield, TIRF, confocal, two-photon
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L08 Live Cell Fluorescence Imaging
Importance, pitfalls, and applications
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L09 Object-based Co-localization Analysis
Quantifying overlapping objects
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L10 Pixel-based Co-localization and Intensity Analysis
Colocalization coefficients and ratiometric imaging
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L11 Analysis of Biological Movement I: Structural Motion
Kymographs, fluorescence recovery rate
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L12 Analysis of Biological Movement II: Translational Motion
Particle tracking, motion analysis
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L13 Introduction to Enhanced Resolution Imaging
SIM, pixel reassignment
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L14 Introduction to Super-Resolution Imaging
STED, PALM/STORM
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L15 Accurate and Sufficient Scientific Reporting
Effects of inaccurate and insufficient documentation and what constitutes good scientific reporting
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L16 Object Segmentation with Machine Learning
Introduction to machine learning and trainable segmentation models​​​​​​​
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L17 Image Processing for Super-Resolution Microscopy
Processing of STED, SIM, and PALM/STORM data
FairSIM: An open source SIM reconstruction plugin for FIJI
SIM Check: An open source FIJI plugin to diagnose SIM reconstruction issues (Note: this plugin is installed via the FIJI updater. Do NOT do this with your Bootcamp version of FIJI).
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L18 Data Analysis for Localization Microscopy
Filtering, point statistics, segmentation
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L19 From Hypothesis to Results: Activity Discussion
Follow up after analysis lab A6
Reference for understanding biological replicates: doi:10.1083/jcb.202001064
Reference for determining good sample size: doi:10.1083/jcb.202401074
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L20 Light Sheet Imaging Modalities and Adaptive Optics
Types of light sheet systems and aberration correction
Blog post on understanding objective angles and the code the AIC uses for deskewing.
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Data handling and visualization
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L22 Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy
Potential pitfalls to consider​
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L23 Machine Learning Resources
Image restoration and other machine learning tools
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L24 Stitching and Tracking Large Datasets
Approaches to stitching images and tools for tracking big data
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Lab and Analysis Sessions
LAB SESSIONS
W1 Co-localization Imaging Lab
Myosin
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Single-Cell Migration
Overnight Collective Migration Assay
W4 Subcellular Scale Imaging Lab
Actin and Myosin
W5 Super-resolution Imaging Lab
Focal Adhesions
W6 Expansion Microscopy (ExM) Imaging Lab
Zebrafish Wound Response
Additional slides on sample mounting
Zebrafish Wound Response
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ANALYSIS AND DISCUSSION SESSIONS
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A1 Analysis Lab: Cell Migration
Segmentation and tracking
A2 Hypothesis Formulation and Experimental Design
Making experimental decisions
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A3 Analysis Lab: Subcellular Scale Imaging
FRAP measurements
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Pearson and Manders coefficients
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A5 Analysis Lab: Super-Resolution Imaging
Resolution vs acquisition choices
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Materials will be provided during the session
A7 Hypothesis Formulation: Organism-scale Imaging
Tracking wound responses
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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
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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
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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
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Eric C. Wait, Michael A. Reiche, Teng-Leong Chew
J Cell Sci 2020 133.
doi:10.1242/jcs.250027
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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
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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
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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
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Imaging methods are vastly underrepresented biomedical research
Guillermo Marques, Thomas Pengo, Mark A. Sanders
eLife 2020;9:e55133
doi: 10.7554/eLife.55133
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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
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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
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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
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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
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Accuracy and precision in quantitative fluorescence microscopy
Jennifer C. Waters
J. Cell Biol. 2009 187: 1135-1148
doi: 10.1083/jcb.200903097
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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
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What If Scientists Shared Their Reagents for Free?
Amanda Heidt
The Scientist, July 2022 Issue 2
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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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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
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