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
Price: Single room standard DKK 1225,-
Booking: Please contact Hotel Kong Arthur, Mr. Jesper Holmberg,
Hotel CABINN Scandinavia
Address: Vodroffsvej 55, 1900 Frederiksberg
Website
Price: Single room standard/double DKK 535,- /DKK 665,-
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 KQ
Conjugate planes on the optical bench CH
Conjugate planes in the microscope PJE Workbook DIY (1 – 5, 10, 11, and 14) 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 KQ
Conjugate planes on the optical bench CH Conjugate planes in the microscope PJE Workbook DIY (1-5, 10, 11, and 14) 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 PJE
Aperture (p. 15) CH
Resolving power (p. 17) KQ
Work Book DIY (p. 4, 7 - 9) 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 CH DIC (Epi-illumination and transmitted light) PJE
CFIM introduction KQ
Workbook (17 - 19) 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 RL Scanning electron microscopy KQ Image recording; fitting the camera PJE Methods of stereoscopic viewing CH 16.15 – 17.00 Summary of today’s work and questions
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
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
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
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
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
