NBIO 890 Microscopy Principles and Applications course syllabus | spring 2013 Jan 17th – May 7th Tuesdays, 10 – 11:45 am Thursdays, 10 – 11:45 am Neuroscience Research Building Room 3118 Vladimir Ghukasyan 115 Mason Farm Rd. Bld. 245, Rm. 7109F 919.966.5807 [email protected] This course aims to provide the knowledge one may need to understand the reach of microscopy imaging techniques, to be able to choose the right imaging modality, label the sample, carry out the experiment, analyze data and troubleshoot any pitfalls that may occur. We will start from the principles of microscopy, proceed to the description of conventional and advanced modern techniques, and evaluate advantages and disadvantages of each method. You will understand what studies can be addressed with each technique and what is the level of details that can be expected. Applications of microscopy will be illustrated with the review of recent research with the focus on Neuroscience. This semester special attention will be paid to hands-­‐on demonstrations. W e will spend a lot of time with the imaging instruments and sample preparation equipment. We will also spend some time with data analysis software and practice with data presentation formats Course Outline • Fundamentals of Optics An outline of the principles underlying the field of microscopy – from the properties of light to image formation. • Light-­‐matter interactions What happens when the light hits a sample? Basics of fluorescence and phosphorescence. • Widefield Microscopy Principles of operation and alignment of a widefield microscope. Discussion of limitations • Confocal Microscopy Principles, advantages, limitations, and anatomy of a confocal microscope. Pitfalls and remedies. • Multiphoton Microscopy Advantages, basic setups and related instrumentation. Basics of multiphoton imaging. • Labeling and Sample Preparation Labels for live and fixed samples. How to prepare samples and label them. What are the problems to expect and how to solve them. • Advanced Microscopy Techniques New developments in microscopy: overview of Forster resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), superresolution techniques (STED, STORM, PALM, SIM), single-­‐molecule techniques, etc. • Biosensors and photomanipulation Introduction to biosensors and lasers application to cell manipulation. • Image Processing and Data Analysis Methods, algorithms and software. • Microscopy Applications A wide scope of biomedical studies with application of different microscopy techniques
Course Schedule s
Tue Mon Thu Wed 17 Fri Sat Sun Optics I. Physics of light; light-­‐matter 18 19 20 Image formation. Diffraction, resolution 25 26 27 1 2 3 8 9 10 January interaction. 21 22 Optics II. Geometrical optics – lenses, 23 24 image formation and aberrations. 28 29 Basics of fluorescence. Jablonski and point-­‐spread function. 30 31 diagram, molecular orbitals, labels. 4 5 Widefield microscopy. Brightfield, components and working principles. 6 7 Confocal microscopy. Basic principle, February darkfield, DIC, phase contrast, etc. 11 12 advantages and limitations. Spinning disk. Multiphoton microscopy. Deep imaging 13 14 Widefield microscopy [Lab: Nikon Eclipse 15 16 17 basics, multiphoton lasers, advantages and limitations. 80i, Zeiss AxioZoom with Apotome (possibly)] L 19 Confocal microscopy [Lab: Zeiss LSM 710 20 21 Multiphoton microscopy [Lab: Zeiss LSM 22 L and Olympus FV1000] A L 7MP] A 23 24 25 26 Quiz | Paper review/report 27 28 Discussion Midterm Exam 1 2 3 4 5 6 7 8 9 10 March 11 Labeling. Genetically encoded fluorescent Advanced imaging techniques I. April 12 Advanced imaging techniques II. Förster resonane energy transfer (FRET), FLIM, Anisotropy imaging, SHG. 13 14 Superresolution techniques (STED, SIM, PALM, etc.) 18 19 Advanced imaging techniques III. Side 15 16 17 plane illumination, Adaptive Optics, Photoacoustics, etc. Biosensors and photomanipulation. 20 21 Quiz |Paper review/report. 22 23 24 (by Dr. Klaus Hahn, Thurman Distinguished Professor, UNC). 25 26 Image processing and data analysis I. Histogram, filters, fitting, deconvolution, etc. 27 28 Image processing and data analysis II. Histogram, filters, fitting, deconvolution, etc. 29 30 31 1 2 L Image processing [Lab: ImageJ, Matlab, 4 Image processing [Lab: ImageJ, Matlab, 5 6 7 Neurolucida, Deconvolution] 3 L 9 Applications I. Imaging applications I. 10 11 12 13 14 8 May A 18 A proteins and antibodies (by Drs. JrGang Cheng and Jason Newbern, UNC) A Basic microscope anatomy. Major Neurolucida, Deconvolution] Review and news. Applications II. Imaging applications I. Review and news. 15 16 Quiz |Paper review/report. 17 18 Discussion 19 20 21 22 23 24 25 Final exam /Assignments/ 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 Final Exam /Theory/ Assignments L Lab Changes to this syllabus Minor changes are possible to be made to the contents of this syllabus. Changes in scheduling are expected. At some point, instructor will need to leave for 1-­‐1.5 weeks around mid-­‐February. Lectures/labs/assignments will be reorganized, and updated calendars will be given to students. Assignments Assignments are an important part of this course. Upon the completion of a certain part of NBIO 890-­‐
001, students will need to put the gained knowledge to practice and perform data acquisition and data analysis/image processing. All details, needed to perform these assignments will be discussed in class and hands-­‐on demos will be performed during the lab sessions. For each assignment, the class will be divided into groups of 3-­‐4 people, all with different objectives. The results will be presented during the class. Please note, in order to let the students spend more time with the microscopes, the assignments may be scheduled not only on Tuesdays and Thursdays but also other weekdays. Part of the images collected will be used during the Image processing/Data analysis lab session and assignments. Exams Midterm exam will be given in the form of a paper – students will need to answer the questions. Both correctness and level of details will affect the grade. This work can be done at home. Final exam will consist of two parts – theory, similar to midterm, and experimental. Attendance Acceptable absences – 2. Grading Midterm exam (100 points). Questions in the paper will give points (70). Number of the points will increase with the question complexity and amount of the text expected. Both clarity and level of details will affect the grading. Assignments will bring you 10 points each. There will be total of 3 assignments for the midterm exam: image acquisition on a widefield (1), confocal (2) and multiphoton (3) system. Same amount of points will be given to all members of the assignment performing group. Attention to details will be evaluated. Paper report (20 points). Same amount will be given to the members of the paper report team (2 people). Final exam (150 points). It will consist of a paper (100 points) and a final assignment (image acquisition and image processing/data analysis) (50 points). Attendance. If more than 3 absences is registered, points will be taken out of your summary score: (-­‐
3) for 3 missed classes/labs, (-­‐5) for 4, (-­‐7) for 5. If more than 5 classes/labs are missed without serious reason, the course has to be retaken. Final grade is calculated the following way: ME – your midterm exam result PR – your paper report score FE – your final exam score AT – attendance (See above) ME*0.3 + PR*0.125 + FE*0.45 – AT Score Grade 95-­‐100 HP 85 – 95 P 65 – 85 LP >65 F