Course detail

Microscopic Imaging Technology

FEKT-MPA-MZTAcad. year: 2024/2025

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

English

Number of ECTS credits

5

Mode of study

Not applicable.

Guarantor

doc. Ing. Radim Kolář, Ph.D.

Department

Department of Biomedical Engineering (UBMI)

Entry knowledge

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.

Rules for evaluation and completion of the course

Laboratory work: 0 - 40 points
Final exam: 0 - 60 points
Final exam is focused on testing the knowledge from the light microscopy imaging.
Labs are obligatory. The properly excused missed laboratory exercise is to be replaced after agreement with the teacher during last week of the semester.

Aims

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.
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.

Study aids

Not applicable.

Prerequisites and corequisites

Not applicable.

Basic literature

D. B. Murphy Fundamentals of light microscopy and electronic imaging, Wiley-Liss, 2001
J. Kuběna, Úvod do optiky, MU Brno 1994, skriptum
Murphy,D.B.: Fundamentals of light microscopy and electronic imaging, Wiley, 2011 (CS)
P. Mouroulis Visual Instrumentation, McGraw-Hill, 1999
Sharma, K.K.: Optics: Principles and Applications, Academic Press, 2006 (CS)

Recommended reading

Not applicable.

Classification of course in study plans

  • Programme MPA-BTB Master's 2 year of study, winter semester, compulsory

Type of course unit

 

Lecture

39 hod., optionally

Teacher / Lecturer

doc. Ing. Radim Kolář, Ph.D.

Syllabus

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 hod., compulsory

Teacher / Lecturer

doc. Ing. Radim Kolář, Ph.D.

Syllabus

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.