CB 399 Quantitative Fluorescence Microscopy Nanocourse Part I & II Spring 2010

Intellectual Unit: Techniques

Quantitative Fluorescence Microscopy Nanocourse Part I & II

Course Lecturers: Jennifer Waters and Khuloud Jaqaman

Curriculum fellow: Anu Seshan, anupama_seshan@hms.harvard

Obtaining quantitative data from live cell images is the key to testing mechanistic hypotheses of molecular and cellular processes. This generally requires (1) using light microscopy to probe the quantity, dynamics and/or interactions of proteins and other molecules within their native environment in the cell, and (2) using computer vision methods to analyze the acquired images and quantitatively measure dynamics and spatiotemporal regulation. For this approach to succeed, experiments must be designed such that digital images acquired with the light microscope are amenable to automated image analysis, while computer vision tools must be optimized to reliably extract complete information from the digital images as pertinent to the hypotheses being tested.

The following two nanocourses are an introduction to using the light microscope as a quantitative instrument, from image acquisition through image analysis. These courses were designed to complement one another, and therefore students who wish to take the courses for credit are required to register for both (1 nanocourse credit will be given for each course for a total of 2 nanocourse credits).

Quantitative Microscopy Part I: Image Acquisition

Jennifer C. Waters, Ph.D.

The accuracy and precision of quantitative light microscopy measurements depends on the quality of the acquired digital images. Obtaining digital images suitable for quantitation requires careful consideration of specimen preparation, the mode of microscopy, and the optics, filters, and detector. In Part I, we will (1) learn how to judge image quality; (2) discuss how the various choices of equipment and imaging parameters affect image quality; and (3) learn how to best make the compromises necessary in live cell experiments to balance image quality and acquisition speed while minimizing photo-toxicity. On the second day, we will critically discuss recent papers that use quantitative microscopy techniques.

Quantitative Microscopy Part II: Image Analysis

Khuloud Jaqaman, Ph.D.

Live-cell images often consist of punctate features representing single fluorophores tagging single molecules, sub-resolution fluorophore clusters associated with small molecular assemblies or fluorophore blobs associated with vesicles or more extended organelles. In Part 2, we will discuss basic principles of computer vision as applied to live-cell images, with an emphasis on the detection and tracking of punctate image features due to their abundance in cell biological applications. Topics covered include: (1) detection/segmentation; (2) localization; (3) resolution; (4) particle tracking/trajectory construction; and (5) image analysis software benchmarking. The second day of the course will take place in the Countway Library computer lab, where students will obtain hands-on experience with image analysis.


NOTE: students are required to attend all four sessions to be eligible to receive credit. Students will receive credit for TWO nanocourses. This nanocourse is limited to 15 students.


First Meeting Part I (Lecture): Wednesday, February 24 1:30 -5 pm

Location: Cannon room

First Meeting Part II (Lecture): Friday, February 26 1:30 -5 pm

Location: Cannon room

Second Meeting Part II: Wednesday, March 3, 2 – 4:30 pm

Location: Countway Library Electronic Classroom, 403

Second Meeting Part I: Friday, March 5, 12:30 – 3 pm

Location: TMEC 309

Links to videotaped lectures

Lecture 1, Feb 24th: http://mycourses.med.harvard.edu/vmms_preview.asp?id=Quantitative-Microcopy-Part-I_Quantitative-Microscopy-Part-1_02252010_rm_SS_1

Lecture 2, Feb 26th: http://mycourses.med.harvard.edu/vmms_preview.asp?id=Quantitative-Microcopy-Part-I_Quantitative-Microscopy-Part-II_02262010_rm_SS_1

Assigned Readings

Day 1, Part I, February 24th, 1:30-5:00: Please read the following papers in preparation for this lecture (attached below):
Waters J. Accuracy and precision in quantitative fluorescence microscopy. JCB 2009, 185(7): 1135-1148.

Pawley J. The 39 Steps: A Cautionary Tale of Quantitative 3-D Fluorescence Microscopy. BioTechniques 2000, 28(5): 884-887.

North A. Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. JCB 2006, 172(1): 9-18.

Day 1, Part II, February 26th, 1:30-5:00: Please read the following chapter in preparation for this lecture (attached below):
Jaqaman K and Danuser G. Computational image analysis of cellular dynamics: A case study based on particle tracking. Chapter 15 in Live Cell Imaging: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 2010.

Day 2, Part II, March 3rd, 2:00-4:30: Please read the following article and be familiar with the analysis methods as you will be using these in your hands-on session. (You can download this from the Attachments box below).

Jaqaman K et al. Robust single-particle tracking in live-cell time-lapse sequences. Nature Methods 2008, 8: 695-702

Day 2, Part I, March 5, 12:30-3:00: Please read the following two articles and be prepared to present the figures. (You can download these from the Attachments box below).

Wu JQ and Pollard TD. Counting Cytokinesis Proteins Globally and Locally in Fission Yeast. Science 2005, 310: 310-314.    

Wan X et al. Protein Architecture of the Human Kinetochore Microtubule Attachment Site. Cell 2009, 137: 672-684.