Okinawa Microscopy Workshop 2024 (Extremely Useful Materials)
Okinawa Microscopy Workshop 2024
I had the incredible opportunity to attend the microscopy workshop at the Okinawa Institute of Science and Technology. I was immersed in a wealth of microscopy knowledge and bioimage data analysis. The instructors from AIC Janelia HHMI, the Neuroscience Microscopy Core Facility at UNC, and the Image Facility at OIST were true experts, enthusiastically sharing their experiences in microscopy, bioimage analysis, and scientific integrity. They even encouraged us to share the teaching materials to help grow the bioimage community within Southeast Asia! I feel so fortunate to have been selected as a participant – I hear the competition was fierce!
Workshop participants came from all across Southeast Asia (Malaysia, Cambodia, Indonesia, Singapore, Thailand, Vietnam, and the Philippines) and nearly every region of Japan! The atmosphere was dynamic, productive, and occasionally a bit stressful as we worked to complete our assignments.
Workshop Introduction
Driving principles and goals of the workshop
Fundamentals of Digital Images
Basic concepts of digital images, file format, color scheme
Introduction to Microscopy I
Image formation, magnification, resolution, objective lenses
Introduction to Microscopy II
Kohler illumination, microscope configurations, contrast in microscopy
Basics of Fluorescence
Histograms, displays, pixel adjustment, filters, kernels
FIJI: Image Processing
Histograms, displays, pixel adjustment, filters, kernels
FIJI: Fourier Transformation in Image Processing
Image processing in Fourier space
Fluorescence Microscopy Modalities
Widefield, TIRF, confocal, two-photon, image scanning microscopy
FIJI: Object Segmentation
Turning pixel map into discrete objects
FIJI: Object Segmentation with Machine Learning
Introduction to machine learning and trainable segmentation models
Super-Resolution Microscopy
SIM, Airyscan, STED and SMLM
FIJI: Object-based Colocalization
Quantifying overlapping objects
FIJI: Pixel-based Colocalization and Intensity Analysis
Colocalization coefficients, ratiometric imaging
Advanced Fluorescence Techniques
FRET, FRAP, FLIP, photomanipulation
Live Cell Imaging
Environmental control, photon budget, phototoxicity
FIJI: Measurement of Dynamic Changes
Fluorescence recovery rate, particle tracking, motion analysis
Image Restoration
Open-source ML techniques for improving SNR, deblurring, etc.
Bias and Reporting
The effects of inaccurate and insufficient documentation, and what constitutes good scientific reporting
Zeiss talk
Exploring the Potential of Image Analysis & AI for Automated Microscopy Workflows
Lab and Analysis Sessions:
Imaging Lab1: Basic Microscopy Techniques
Köhler Illumination, Brightfield Microscopy, Dark Field Microscopy, Phase Contrast Microscopy, Differential Interference Contrast (DIC) Microscopy, Widefield Fluorescence Microscopy
Imaging Lab 2: Advanced Microscopy Techniques
Laser Scanning Confocal Microscopy, Spinning Disk Confocal Microscopy, Light-Sheet Microscopy, Two-Photon Microscopy
Imaging Lab 3: Super-Resolution Techniques
Airyscan, Structured Illumination Microscopy (SIM), Single-Molecule Localization Microscopy (SMLM), Stimulated Emission Depletion (STED) Microscopy
Imaging Lab 4: Live Cell Imaging
Fluorescence Recovery After Photobleaching (FRAP) and Lysosome Tracking
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
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.
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
Aaron J., Chew TL. (2018) Analysis of Image Similarity and Relationship. In: Jerome W., Price R. (eds) Basic Confocal Microscopy. Springer, Cham (PDF)
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
Transfection of Cultured Primary Neurons
Annalisa Rossi, Ralf Dahm, and Paolo Macchi
Stem Cell Technologies in Neuroscience, 2017
doi: 10.1007/978-1-4939-7024-7_4
Phototoxicity in live fluorescence microscopy, and how to avoid it
Jaroslav Icha, Michael Weber, Jennifer C. Waters, and Caren Norden
BioEssays, 2017
doi: 10.1002/bies.201700003
Believing is seeing – the deceptive influence of bias in quantitative microscopy
Rachel M. Lee, Leanna R. Eisenman, Satya Khuon, Jesse S. Aaron, Teng-Leong Chew
J. Cell Sci. 2024
doi: 10.1242/jcs.261567
A list of useful microscopy resources can be found at:
https://www.janelia.org/support-team/integrative-imaging/additional-information
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