Principles of Microscopy and Confocal and Fluorescence Microscopy Content This course in Light Microscopy follows the series of successful courses in Light Microscopy, Confocal and Fluorescence Microscopy and Electron Microscopy. This course consists of two modules. It is suitable for beginners in microscopy, and for people who use microscopes in their work and now want to extend their knowledge of basic principles and more specialised techniques. The course is divided into two weeks. The first week, Principles of Microscopy, provides an essential grounding in the basic principles of microscopy, including: the limitations of the eye; resolution, contrast and magnification; refraction; lenses and images; conjugate planes; methods of illumination; diffraction, aperture and resolution; generation of contrast; introduction to bright-field, dark ground, phase contrast, polarised light and differential interference contrast. The second week, Fluorescence and Confocal Microscopy, covers the characteristics of the confocal microscope; introduction to spinning disc and TIRF; the selection of fluorophores and corresponding filter sets; immunofluorescent and fluorescent affinity staining of biological specimens. The principles of FRET and FLAP will be covered and demonstrated in practice. In addition, methods for producing images representing two- and three-dimensional data sets from computer-based data sets and deconvolution techniques will be described and demonstrated in practice. Guidelines regarding participation fee The fee for participation DKK 13.500,- must be paid by bank transfer to CFIM, Copenhagen University Bank information: Reg. no. 3001 Account no. 4115 212125. It is very important that you remember to write your name and CFIM project number # 3104002 in notes when making the bank transfer in order for us to confirm your registration and payment. Hotel accommodation Hotel Kong Arthur Address: Nørre Søgade 11, 1370 Copenhagen K Website: www.kongarthur.dk Price: Single room standard DKK 1225,- Booking: Please contact Hotel Kong Arthur, Mr. Jesper Holmberg, ho@brochner-hotels.dk Hotel CABINN Scandinavia Address: Vodroffsvej 55, 1900 Frederiksberg Website: www.cabinn.com Price: Single room standard/double DKK 535,- /DKK 665,- Booking: Please contact CFIM, Ms. Ragnhild Mostert, rmostert@sund.ku.dk
Hotel Wake up Address: Carsten Niebuhrs Gade 11, 1577 Copenhagen V Website: www.wakeupcopenhagen.dk Price: Will be published at a later state Booking: Will be published at a later state In order to obtain the above mentioned rates at the hotels, please state that you are participating in the CFIM PhD course and attach your registration confirmation upon request. Principles of Microscopy Monday 6 August 2012 Time Topic Lecturer 09:00 09:30 Introduction KQ 09:30 10:15 The story of the microscope PJE/CH 10:15 Coffee break 10:30 12:45 Limitations of the eye. Resolution, contrast, magnification. PJE Lenses, magnifying glasses, compound microscopes. Conjugate planes 12:45 Lunch break 13:30 14:15 Lens defects and their correction PJE 14:15 15:00 Köhler illumination PJE 15:00 Coffee break 15:15 16:30 Practical Köhler illumination Conjugate planes on the optical bench Conjugate planes in the microscope Workbook DIY (1 5, 10, 11, and 14) KQ CH PJE CP 16:30 16:45 Summary of today s work; questions and workbook The objective of the day is that you should be able to understand the geometrical optics of the microscope, know how to set it up, and begin to understand why these steps are necessary.
Tuesday 7 August Time Topic Lecturer 09:00 10:15 Practical Köhler illumination Conjugate planes on the optical bench Conjugate planes in the microscope Workbook DIY (1-5, 10, 11, and 14) KQ CH PJE CP 10:15 Coffee 10:30 11:15 Demonstration Setting up Köhler illumination in transmitted light Depth of field and depth of focus 11:15 13:00 Lecture-demonstration PJE Diffraction, resolution and contrast 13:00 Lunch 13.45 15.45 Practical Diffraction experiments Aperture (p. 15) Resolving power (p. 17) Work Book DIY (p. 4, 7-9) PJE CH KQ CP 15:45 Coffee 16:00 16:45 Practical continued 16:45 17:00 Summary of today s work; questions and workbook The objective of the day is that you should be able to understand how diffraction sets the limits to resolving power and provides the basis for generation of contrast.
Wednesday 8 August Time Topic Lecturer 09:00 09:45 Equations for limit of resolution of optical instruments CH 09:45 Coffee 10:00 11:00 Contrast: Bright field, dark ground, Rheinberg, Phase contrast PJE 11:15 12:00 Practical Dark field patch stop (p. 26) 12:00 13:00 Lunch 13:00 14:30 Practical (continued) Dark field patch stop Rheinberg 14:30 15:00 Coffee (exchange microscopes) 15:00 16:30 Practical Phase contrast (p. 28) 16:30 17:00 Summary of today s work; questions and workbook The objective of the day is that you should be able to understand how the properties of specimens may be exploited in the microscope to give rise to contrast.
Thursday 9 August Time Topic Lecturer 09.00 09.45 The nature and properties of light CH 09.45 10.00 Coffee 10.00 11.00 Polarised light (lecture-demonstration) CH 11.00 11.30 Practical Contrast in the polarised-light microscope; Effects of mounting media 1130 1145 Coffee 1145 1230 Practical Contrast in the polarised-light microscope; Effects of mounting media 1230 1300 Understanding interference colou CH 1300-1345 Lunch 13.45 14.30 Differential interference contrast PJE Polarised light (p. 30 33) DIC (Epi-illumination and transmitted light) (p. 34) Workbook (17-19) 14.30-1445 Coffee 14.45 16.45 Practical Polarised light: examples at lightbox DIC (Epi-illumination and transmitted light) CFIM introduction Workbook (17-19) CH PJE KQ CP 16.15 16.45 Principles of the confocal microscope PJE 16-45 17.00 Summary of today s work; questions and workbook The objective of the day is that you should be able to understand the concept of optical path difference, how polarization colours arise, and how these can be applied to generate contrast in the microscope image.
Friday 10 August Time Topic Lecturer 09.00 09.30 Methods of recording images PJE 09.30 10.30 Principles of digital image recording PJE Optical considerations in fitting a camera to a microscope 10.30 10.45 Coffee 10.45 11.30 Stereomicroscopes PJE 11.30 12.00 Cleaning and maintenance PJE 12.00 12.45 Lunch 12.45 14.15 Principles of electron microscopy PJE/CH 14.10 14.30 Coffee 14.30 16.30 Practical Transmission electron microscopy Scanning electron microscopy Image recording; fitting the camera Methods of stereoscopic viewing RL KQ PJE CH 16.15 17.00 Summary of today s work and questions Objectives of the day is that you should know the principles; see you in a week.
Confocal and Fluorescence Microscopy Monday 13 August Time Topic Lecturer Location 09.00 09.15 Welcome & introductions KQ 15.2.18 09.15 10.15 Lecture Atoms, light and matter AE 15.2.18 10.15 Coffee 10.30 11.30 Lecture Fluorescence and fluorophores AE 15.2.18 11.30 13.30 Interactive lecture Computers and software AE & JC 15.2.18 (Includes 30 45 minutes free time for lunch) 13.30 14.30 Lecture Fluorescence microscopy: an overview AE 15.2.18 14.30 15.15 Interactive lecture The fluorescence microscope AE 15.2.17b 15.15 Coffee 15.30 16.40 Lecture Signals, noise and detectors AE 15.2.18 16.40 17.00 Lecture Fluorescence microscopy: an overview (cont.) AE 15.2.18
Tuesday 14 August Time Topic Lecturer Location 09.00 10.00 Lecture Confocal and wide-field fluorescence microscopy AE 15.2.18 10.00 Coffee 10.15 11.15 Lecture CCD cameras and detecting fluorescence 15.2.18 11.15 12.15 Lecture Confocal and wide-field fluorescence microscopy (cont.) AE 15.2.18 Practicals in 5 groups of 4 people 1) Zeiss LSM 710 (integration time & pixel density) AE CFIM 2) Zeiss LSM 700 (Collect 3D data / discuss sampling) JC CFIM 3) Zeiss LSM 780 (use spectral collection) CP CFIM 4) Zeiss ( real time )(Compare and contrast) THB CFIM 5) CCD cameras (Andor) LH CFIM 12.15 13.00 Practical 1 CFIM 13.00 Lunch 13.45 15.15 Practicals 2 & 3 CFIM 15.15 Coffee CFIM 15.30 17.00 Practicals 4 & 5 CFIM
Wednesday 15 August Time Lecturer Lecturer Location 09.00-09.45 Lecture Fluorescence Recovery After Photobleaching (FRAP) DZ 15.2.18 09.45 10.45 Lecture JC 15.2.18 3D Reconstruction 10.45 Coffee 11.00 12.00 Lecture Fluorescent Resonance Energy Transfer (FRET) DZ 15.2.18 12.00 13.00 Lecture 3D Reconstruction JC 15.2.18 13.00 Lunch 13.45 15.45 Practicals in 5 groups 1) Zeiss LSM 710 Checking the confocal microscope AE CFIM 2) 3D reconstruction JC CFIM 3) Zeiss LSM 780 FRAP, FRET & FCS DZ CFIM 4) Zeiss LSM 700 collecting confocal data CP CFIM 5) Fluorescence, alignment of the Hg arc THB/BBJ/KQ 15.2.17b 15.45 Coffee 16.00 17.00 Lecture Beyond the diffraction limit JC 15.2.18
Thursday 16 August Time Lecturer Location 9.00 11.00 Practicals in groups (continued) CFIM 11.00 Coffee CFIM 11.15 13.15 Practicals in groups (continued) CFIM 13.15 Lunch 14.00 16.00 Practicals in groups (continued) CFIM 16.00 Coffee 16.15 17.00 Lecture Digital fluorescence micrographs and their presentation 15.2.18
Friday 17 August Time Lecturer Lecturer Location 09.00 10.00 Lecture Quantification of fluorescence AE 15.2.18 10.00 Coffee CFIM 10.15 12.15 Practicals in groups (continued) CFIM 12.15 13.00 Interactive lecture Deconvolution and image restoration JC 15.2.18 13.00 Lunch 13.45 14.45 Interactive lecture Deconvolution and image restoration (cont.) JC 15.2.18 14.45 15.30 Lecture Fluorescence Localization After Photobleaching (FLAP) DZ 15.2.18 15.30 Coffee 15.45 16.45 Lecture Immunofluorescence and affinity fluorescent staining AE 15.2.18 16.45 17.00 Evaluation of course 15.2.18