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 Evaluation Form*
COURSE INFORMATION
Course Schedule [PDF]
Course Instructors [PDF]
Imaging Rotation Groups [PDF]
COVID Testing Instructions [PDF]
Join the conversation on Twitter:
Download the full size group photo here [JPG]
Students can share photos from the course via Google Drive
DOWNLOADS
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
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
Turning pixel map into discrete objects
L07 Instruments for the Cellular Scale Imaging Labs
For imaging lab W2
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
STED & iPALM
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
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
LAB SESSIONS
W1 Co-localization Imaging Lab
Myosin
Cell Migration
Overnight wound healing assay
W4 Subcellular Scale Imaging Lab
Actin and Myosin
W5 Super-resolution Imaging Lab
Paxillin
W6 Expansion Microscopy (ExM) Imaging Lab
Zebrafish
Zebrafish
ANALYSIS AND DISCUSSION SECTIONS
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
Analyzing the data from W1
A5 Analysis Lab: Super-Resolution Imaging
Segmentation
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
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
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
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