Principles of Microscopy and Confocal and Fluorescence Microscopy

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1 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 ,- must be paid by bank transfer to CFIM, Copenhagen University Bank information: Reg. no Account no It is very important that you remember to write your name and CFIM project number # 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: Price: Single room standard DKK 1225,- Booking: Please contact Hotel Kong Arthur, Mr. Jesper Holmberg, [email protected] Hotel CABINN Scandinavia Address: Vodroffsvej 55, 1900 Frederiksberg Website: Price: Single room standard/double DKK 535,- /DKK 665,- Booking: Please contact CFIM, Ms. Ragnhild Mostert, [email protected]

2 Hotel Wake up Address: Carsten Niebuhrs Gade 11, 1577 Copenhagen V Website: 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.

3 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 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.

4 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.

5 Thursday 9 August Time Topic Lecturer The nature and properties of light CH Coffee Polarised light (lecture-demonstration) CH Practical Contrast in the polarised-light microscope; Effects of mounting media Coffee Practical Contrast in the polarised-light microscope; Effects of mounting media Understanding interference colou CH Lunch Differential interference contrast PJE Polarised light (p ) DIC (Epi-illumination and transmitted light) (p. 34) Workbook (17-19) Coffee Practical Polarised light: examples at lightbox DIC (Epi-illumination and transmitted light) CFIM introduction Workbook (17-19) CH PJE KQ CP Principles of the confocal microscope PJE 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.

6 Friday 10 August Time Topic Lecturer Methods of recording images PJE Principles of digital image recording PJE Optical considerations in fitting a camera to a microscope Coffee Stereomicroscopes PJE Cleaning and maintenance PJE Lunch Principles of electron microscopy PJE/CH Coffee Practical Transmission electron microscopy Scanning electron microscopy Image recording; fitting the camera Methods of stereoscopic viewing RL KQ PJE CH Summary of today s work and questions Objectives of the day is that you should know the principles; see you in a week.

7 Confocal and Fluorescence Microscopy Monday 13 August Time Topic Lecturer Location Welcome & introductions KQ Lecture Atoms, light and matter AE Coffee Lecture Fluorescence and fluorophores AE Interactive lecture Computers and software AE & JC (Includes minutes free time for lunch) Lecture Fluorescence microscopy: an overview AE Interactive lecture The fluorescence microscope AE b Coffee Lecture Signals, noise and detectors AE Lecture Fluorescence microscopy: an overview (cont.) AE

8 Tuesday 14 August Time Topic Lecturer Location Lecture Confocal and wide-field fluorescence microscopy AE Coffee Lecture CCD cameras and detecting fluorescence Lecture Confocal and wide-field fluorescence microscopy (cont.) AE 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 Practical 1 CFIM Lunch Practicals 2 & 3 CFIM Coffee CFIM Practicals 4 & 5 CFIM

9 Wednesday 15 August Time Lecturer Lecturer Location Lecture Fluorescence Recovery After Photobleaching (FRAP) DZ Lecture JC D Reconstruction Coffee Lecture Fluorescent Resonance Energy Transfer (FRET) DZ Lecture 3D Reconstruction JC Lunch 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 b Coffee Lecture Beyond the diffraction limit JC

10 Thursday 16 August Time Lecturer Location Practicals in groups (continued) CFIM Coffee CFIM Practicals in groups (continued) CFIM Lunch Practicals in groups (continued) CFIM Coffee Lecture Digital fluorescence micrographs and their presentation

11 Friday 17 August Time Lecturer Lecturer Location Lecture Quantification of fluorescence AE Coffee CFIM Practicals in groups (continued) CFIM Interactive lecture Deconvolution and image restoration JC Lunch Interactive lecture Deconvolution and image restoration (cont.) JC Lecture Fluorescence Localization After Photobleaching (FLAP) DZ Coffee Lecture Immunofluorescence and affinity fluorescent staining AE Evaluation of course