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
COURSE INFORMATION
Course Schedule [PDF]
Course Instructors [PDF]
Imaging Rotation Groups [PDF]
Microscope Map [PDF]
Directions to laundry [PDF]
Course Evaluation: https://www.surveymonkey.com/r/GYGQQW6
Share your photos here: Google Drive Folder
Join the conversation on Twitter:
@AICjanelia | #IALSJanelia
Listen to music across global length scales:
Submit a song here | Listen to the playlist here
Participant-created song: Recording & Lyrics
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.
Announcement of AIC Proposal Call.
DATA & DOWNLOADS
Accessing Data on PRFS\Hackathon
Remote access to the Hackathon folder is no longer available.
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
Sample Images for Fiji Lectures
1.92 GB of images [download .zip]
Co-localization Data
PoCA (Program for PALM/STORM Data Analysis)
If you have a Windows machine, download the latest release's zip file here.
Data for From Hypothesis to Results Activity
32 MB of images [download .zip]
IALS 2024 Team Photo
To "decode" this image, look for the FFT image in the "PhotoSharing" folder in hackathon!
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 Fundamentals of Image Processing
Histograms, displays, pixel adjustment, filters, kernels
L05 Fourier Transformation in Image Processing and Optics
Image processing in Fourier space
Turning pixel map into discrete objects
Solution to the object segmentation homework
L07 Introduction to Fluorescence Microscopy and Optical Sectioning
Widefield, TIRF, confocal, two-photon
L08 Live Cell Fluorescence Imaging
Importance, pitfalls, and applications
L09 Object-based Co-localization Analysis
Quantifying overlapping objects
L10 Pixel-based Co-localization and Intensity Analysis
Colocalization coefficients and ratiometric imaging
L11 Analysis of Biological Movement I: Structural Motion
Kymographs, fluorescence recovery rate
L12 Analysis of Biological Movement II: Translational Motion
Particle tracking, motion analysis
L13 Introduction to Enhanced Resolution Imaging
SIM, pixel reassignment
L14 Introduction to Super-Resolution Imaging
STED, PALM/STORM
L15 Accurate and Sufficient Scientific Reporting
Effects of inaccurate and insufficient documentation and what constitutes good scientific reporting
L16 Object Segmentation with Machine Learning
Introduction to machine learning and trainable segmentation models
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).
L18 Data Analysis for Localization Microscopy
Filtering, point statistics, segmentation
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
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.
Data handling and visualization
L22 Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy
Potential pitfalls to consider
L23 Machine Learning Resources
Image restoration and other machine learning tools
L24 Stitching and Tracking Large Datasets
Approaches to stitching images and tools for tracking big data
Lab and Analysis Sessions
LAB SESSIONS
W1 Co-localization Imaging Lab
Myosin
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
ANALYSIS AND DISCUSSION SESSIONS
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
Pearson and Manders coefficients
A5 Analysis Lab: Super-Resolution Imaging
Resolution vs acquisition choices
Materials will be provided during the session
A7 Hypothesis Formulation: Organism-scale Imaging
Tracking wound responses
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
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