Course detail

Microscopic Imaging Technology

FEKT-FMZTAcad. year: 2016/2017

The course will be a detailed overview of the principle and practice of light microscopy. The emphasis of the course will be on the correct and appropriate use of the light microscope. Course covers optical microscope theory and also advanced optical and imaging techniques.

Language of instruction


Number of ECTS credits


Mode of study

Not applicable.

Learning outcomes of the course unit

The student is able to:
- describe spatial transfer of the electromagnetic wave,
- list and explain meaning of its parameters,
- define the main optical laws,
- apply Fresnel coefficients for specific case,
- describe simple optical system by matrix notation,
- describe the principle of light microscope,
- discuss the function of specific microscopy components,
- revise basic optical abberations and their influence on image quality,
- compare the properties of polarization, dark-field, phase and Nomarsky contrast microscopy techniques,
- describe the fluorescence microscope and its application,
- describe the confocal microscope and its application,
- explain physical principle of two-photon microscopy,
- explain TIRF microscopy,
- discuss, compare and select the optimal light detecion method,
- choose appropriate microscopy technique for specific application.


Student should be able to define basic optical laws and should be able to mathematicaly describe electromagnetic field. The mathematical background from matrix theory is also required.


Not applicable.

Planned learning activities and teaching methods

Techning methods include lectures and practical laboratories. Course is taking advantage of e-learning (Moodle) system. Students have to write 10 assignment during the course.

Assesment methods and criteria linked to learning outcomes

Laboratory work: 0 - 40 points
Final exam: 0 - 60 points
Final exam is focused on testing the knowledge from the light microscopy imaging.

Course curriculum

1. Fundamentals of wave and geometry optic.
2. Fresnel coefficients.
3. Matrix description of the optic systems.
4. Eye as an optical system.
5. Basic microscopy design concept.
6. CCD, CMOS sensors, photomultiplier, Avalanche diode, hybrid detectors its principle and parameters.
7. Dark field microscopy.
8. Phase contrast microscopy.
9. Stereomicroscopy.
10. Nomarsky differential interference contrast microscopy.
11. Hoffman modulation contrast microscopy.
12. Fluorescence microscopy.
13. Laser scanning (confocal) microscopy.
14. 2-photon and multi-photon microscopy. TIRF microscopy.
15. Optical coherent microscopy.
16. Selected fluorescence microscopic techniques.
17. Advanced and superresolution techniques - STED, STORM/PALM, SIM.
18. Basic of electron microscopy.

Work placements

Not applicable.


The main aim of this course is to provide basic orientation in the light microscopy imaging, selected imaging techniques, its principles and its medical and biological applications.

Specification of controlled education, way of implementation and compensation for absences

Labs are obligatory. The properly excused missed laboratory exercise is to be replaced after agreement with the teacher during last week of the semester.

Recommended optional programme components

Not applicable.

Prerequisites and corequisites

Not applicable.

Basic literature

D. B. Murphy Fundamentals of light microscopy and electronic imaging, Wiley-Liss, 2001
P. Mouroulis Visual Instrumentation, McGraw-Hill, 1999
J. Kuběna, Úvod do optiky, MU Brno 1994, skriptum

Recommended reading

Not applicable.

Classification of course in study plans

  • Programme BTBIO-F Master's

    branch F-BTB , 2. year of study, winter semester, compulsory

  • Programme EEKR-CZV lifelong learning

    branch ET-CZV , 1. year of study, winter semester, compulsory

Type of course unit



39 hours, optionally

Teacher / Lecturer


1. Wave and geometry optic - basic optical laws and phenomenon (interference, aberration, diffraction, polarization), optical components. Interaction of light with tissue - absorption, diffraction, attenuation, fluorescence, phosphorescence, autofluorescence.

2. Eye as an optical system, which participate on imaging process. Eye anatomy. Some rules connected to vision process (scotopic/photopic vision, Weber - Fencher law, Stiles - Crawford effect, darkness adaptation)

3. Description of the optical system. Quantitative evaluation of these systems (optical transfer function, modulation transfer function, Strehl ratio, wavefront aberration)

4. Basic microscopy design concept. Description and properties of particular components - holder, eyepiece, lens, condenser, light sources. Examples of microscopes.

5. Analog and digital microscopy. Light detection - CCD and CMOS sensors and their properties (signal-to-noise ratio, spatial resolution, temporal resolution). Videomicroscopy.

6. Upright and inverted microscopy - differences. Dark field microscopy - principle, design, applications. Phase contrast microscopy - physical and mathematic description, design, application.

7. Stereomicroscopy - principle, design, image processing. Nomarsky differential interference contrast (DIC) microscopy, Hoffman modulation contrast (HMC) microscope.

8. Fluorescence microscopy - description of fluorescence, fluorescence dyes, principles, microscope design.

9. Laser scanning microscopy, laser scanning confocal microscopy - principles, spatial resolution. Fluorescence scanning microscopy, 2-photon and mulit-photon microscopy.

10. Optical coherent microscopy and tomography - phenomenon of light interference for tomographic imaging. Systems working in temporal and frequency domain. Applications.

11.Application of light microscopic principles in ophthalmology, dermatology, endoscopy.

12. Basic techniques in microscopic image processing - disparity maps in steremince Energy Transfer (FRET), Stimulated Emission Depletion (STED), holographic microscopy.

12. Preparation of microscopic samples. Live cell imaging - heart cell contractility. Application of light microscopic principles in ophthalmology, dermatology, endoscopy.

13. Basic techniques in microscopic image processing - disparity maps in steremicroscopy, deconvolution, formation of focus image from different focus images sequence.

Laboratory exercise

26 hours, compulsory

Teacher / Lecturer


1. Introductory laboratory - introduction to laboratory equipments, introduction to image acquisition and analysis software NIS - Elements.
2. Basic image operation in Matlab.
3. Stereomicroscopy, influence of illumination, disparity map.
4. Measurement of modulation transfer function using Nikon camera.
5. Image processing from confocal microscope.
6. Measurement of properties of fluorescent dye.
7. Microscopy with immerse objective, basic techniques in NIS-Elements.
8. Dark field and phase contrast microscopy.
9. Microscopy in polarization light, Malus law.
10. Measurement of light wavelength using microscope and interferometric approach.
11. Simulation in geometric optics.
12. Hartmann-Shack abberometry.
13. Free lab.