Microscopy and instrumental methods in biology

General description

This course overviews modern measurements and imaging techniques used in biophysics, molecular and cell biology. Its main goal is to teach students about the capabilities and limitations of each of the most popular techniques, and explain how to select the simplest and the most effective approach to answer a particular experimental question. Practical works will include a complete workflow of processing real experimental datasets from fluorescence and CD experiments, planning chromatography experiments, advanced work with fluorescence microscopy images using ImageJ and Pyton

Details of the curriculum

Registration is open and will be possible until April 23. (closed)

1. Light.

Light absorption. Color. Spectrophotometers. Absorption-based methods. Dipole moment of molecules and absorption wavelength. (Shvadchak V.).


2. Fluorescence

Principles of fluorescence. Jablonsky diagram. Fluorescence quantum yield. Fluorophores. Tryptophan and other natural fluorophores. GFP. Brightness. Solvatochromism. (Shvadchak V.).

3. Advanced fluorescence methods

Protein labeling. Cys-, Lys- reactive dyes. Intercalating dyes. FRET and its application to study protein interactions. Polarized light. Fluorescence anisotropy. (Shvadchak V.).

4. CD and IR

CD spectroscopy to determine protein structure. IR spectroscopy.  (Shvadchak V.).

5. Methods to determine the size of molecules

Electrophoresis of proteins and oligonucleotides. DLS. FCS. FCCS. (Shvadchak V.).

6. Chromatography

HPLC principle, preparative and analytical applications. Types of columns. Ion exchange chromatography. Size-exclusion. (Shvadchak V.).

MIM-L6_(HPLC).pdf (1.76 MB)
7. Mass-spectrometry

Mass-spectrometry. LC-MS. ESI, MALDI and other ionization methods. Types of mass detectors. Fragmentation. LC-MS in proteomics. (Shvadchak V.).

8. Transmission and fluorescence microscopy

Transmission microscopy, phase contrast. Fluorescence microscopy. Principal schemes of microscopes. Lasers. Filters. Dichroic mirrors. Channels. Digital image collection. Image resolution, micrones and pixels. Confocal microscopy. Z-slices. (Kovalchuk  Yu.).

9. Applied fluorescence microscopy

Membrane trackers, staining of nuclei. ImageJ/Fiji. Colocalization. TIRF. (Kovalchuk  Yu.).

10. Advanced fluorescence microscopy

FRET and detection of interactions in microscopy. Fluorescence lifetime and FLIM. Diffraction limit. Superresulution, STORM. PALM. Application to image actin fibrils. (Shvadchak V.)

11. Fluorophores for microscopy

Fluorescent proteins. Small molecule dyes. Channel crosstalk and selection of fluorophores. Photodegradation during measurements. FRAP. Light intensity and damage to cells. Caged molecules and controllable photorelease. Photoswithchable molecules. (Shvadchak V.)

12. How to build or customize a microscope?

(Khoroshyy P.)

13. How to build or customize a microscope? (1)

(Khoroshyy P.)

14. How to build or customize a microscope? (2)

(Khoroshyy P.)

15. Electron microscopy

Principle. Resolution. Modes. Sample preparation. (Bondarenko  N.)

16. CryoEM

(Bondarenko N.)

17. Atomic Force Microscopy

Principle and scheme of microscopes. XY and Z resolution. Sample preparation. Scanning speed and sample damage. Application for protein unfolding. (Shvadchak V.)

18. NMR and ESR

Spin. 13C and 15N protein labeling. NMR for protein structure analysis. Solid state NMR. ESR and free radicals. (Shvadchak V).

19.  X-ray

Protein crystallization. SAXS. (Shvadchak V.). 

1. Light absorption and concentration

(Shvadchak V.)

2. Real data processing: Protein-membrane binding followed by Trp fluorescence.

Kd determination. Origin software for non-linear fitting.

Kd-9.zip (38.13 KB)
Kd-8.zip (86.66 KB)
3. Fluorescence anisotropy. FRET, stopped flow

(Shvadchak V.)

4. CD, FCS, DLS, Electrophoresis

(Shvadchak V.)

CD_protein-membrane (84.58 KB)
5. Ion exchange chromatography: construction of gradient. HPLC analysis

(Shvadchak V.)

6. LC-MS

(Shvadchak V.)

7. ImageJ and image processing

(Shvadchak V.)

Fiji ImageJ - безкоштовна програма для обробки файлів даних з мікроскопів (завантаження Fiji).
Підручник по Image J

8. Fluorophores for microscopy.

(Shvadchak V.)

9. Python

 (Khoroshyy P.)

10. EM and CryoEM

(Bondarenko N.)

11. How to select method to solve the problem?
Додаткові матеріали та рекомендована література
Підручники по абсорбції та флуоресцкнції
Візуалізація структури біомолекул

Сторінка завантаження програми VMD https://www.ks.uiuc.edu/Development/Download/download.cgi?PackageName=VMD

Cайт де можна скачати файли структур молекул з координатами атомів https://www.rcsb.org/  (в форматі PDB).

19 лекцій
Practical classes
11 семінарів
2 місяці
Date Time Subject
1 May, Mon 16:00-18:00 Lecture 1. Light (VS)
1 May, Mon 18:00-20:00 Seminar 1, group 1. Light absorption and concentration (VS)
3 May, Wed 16:00-18:00 Seminar 1, group 2. Light absorption and concentration (VS)
3 May, Wed 18:00-20:00 Seminar 1, group 3. Light absorption and concentration (VS)
4 May, Thu 16:00-18:00 Lecture 2. Fluorescence (VS)
4 May, Thu 18:00-20:00 Seminar 2, group 1. Real data processing: Protein-membrane binding followed by Trp fluorescence (VS)
6 May, Sat 10:00-12:00 Seminar 2, group 2. Real data processing: Protein-membrane binding followed by Trp fluorescence (VS)
6 May, Sat 12:00-14:00 Seminar 2, group 3. Real data processing: Protein-membrane binding followed by Trp fluorescence (VS)
8 May, Mon 16:00-18:00 Lecture 3. Advanced fluorescence methods (VS)
8 May, Mon 18:00-20:00 Seminar 3, group 1. Fluorescence anisotropy. FRET, stopped flow (VS)
10 May, Wed 16:00-18:00 Лекція 4. CD and IR (VS)
10 May, Wed 18:00-20:00 Seminar 3, group 2. Fluorescence anisotropy. FRET, stopped flow (VS)
11 May, Thu 16:00-18:00 Лекція 5. Methods to determine the size of molecules (VS)
11 May, Thu 18:00-20:00 Seminar 3, group 3. Fluorescence anisotropy. FRET, stopped flow (VS)
13 May, Sat 10:00-12:00 Seminar 4, group 1. CD, FCS, DLS, Electrophoresis (VS)
13 May, Sat 12:00-14:00 Seminar 4, group 2. CD, FCS, DLS, Electrophoresis (VS)
13 May, Sat 14:00-16:00 Seminar 4, group 3. CD, FCS, DLS, Electrophoresis (VS)
15 May, Mon 16:00-18:00 Lecture 6. Chromatography (VS)
15 May, Mon 18:00-20:00 Seminar 5, group 1. Ion exchange chromatography: construction of gradient. HPLC analysis (VS)
17 May, Wed 16:00-18:00 Seminar 5, group 2. Ion exchange chromatography: construction of gradient. HPLC analysis  (VS)
17 May, Wed 18:00-20:00 Seminar 5, group 3. Ion exchange chromatography: construction of gradient. HPLC analysis (VS)
18 May, Thu 16:00-20:00 Lecture 7. Mass-spectrometry (VS)
18 May, Thu 18:00-20:00 Seminar 6, group 1. LC-MS (VS)
22 May, Mon 16:00-18:00 Seminar 6, group 2. LC-MS  (VS)
22 May, Mon 18:00-20:00 Seminar 6, group 3. LC-MS (VS)
24 May, Wed 16:00-18:00 Lecture 8. Transmission and fluorescence microscopy (YK)
25 May, Thu 16:00-18:00 Lecture 9. Applied fluorescence microscopy (YK)
29 May, Mon 16:00-18:00 Seminar 7, group 1. ImageJ and image processing (VS)
29 May, Mon 18:00-20:00 Seminar 7, group 2. ImageJ and image processing (VS)
31 May, Wed 16:00-18:00 Lecture 10. Advanced fluorescence microscopy (VS)
31 May, Wed 18:00-20:00 Seminar 7, group 3. ImageJ and image processing (VS)
5 June, Mon 16:00-18:00 Lecture 11. Fluorophores for microscopy (VS)
5 June, Mon 18:00-20:00 Seminar 8, group 1. Fluorophores for microscopy (VS)
7 June, Wed 16:00-18:00 Seminar 8, group 2. Fluorophores for microscopy (VS)
7 June, Wed 18:00-20:00 Seminar 8, group 3. Fluorophores for microscopy (VS)
12 June, Mon 16:00-18:00 Lecture 12. How to build or customize a microscope? (PK)
12 June, Mon  18:00-20:00 Seminar 9, group 1. Python (PK) 
14 June, Wed 16:00-18:00 Lecture 13. Python for image processing (1)  (PK) 
14 June, Wed 18:00-20:00 Seminar 9, group 2. Python (PK) 
15 June, Thu 16:00-18:00 Lecture 14. Python for image processing (2) (PK) 
15 June, Thu 18:00-20:00 Seminar 9, group 3. Python (PK) 
19 June, Mon 16:00-18:00 Lecture 15. Electron microscopy (NB)
21 June, Wed 16:00-18:00 Lecture 16. CryoEM  (NB)
21 June, Wed 18:00-20:00 Seminar 10, group 1. EM and CryoEM (NB)
17 June, Thu 16:00-18:00 Seminar 10, group 2. EM and CryoEM (NB)
17 June, Thu 18:00-20:00 Seminar 10, group 3. EM and CryoEM (NB)
26 June, Mon 16:00-18:00 Lecture 17. Atomic Force Microscopy (VS)
26 June, Mon 18:00-20:00 Lecture 18. NMR and ESR (VS)
28 June, Wed 16:00-18:00 Lecture 19. X-ray (VS)
28 June, Wed 18:00-20:00 Seminar 11, group 1. How to select method to solve the problem? (VS)
29 June, Thu 16:00-18:00 Seminar 11, group 2. How to select method to solve the problem? (VS)
29 June, Thu 18:00-20:00 Seminar 11, group 3. How to select method to solve the problem? (VS)
3 July, Mon 16:00-18:00 Test

Pathologist and Research Scientist with PhD.

Researcher at the Institute of Organic and Biochemistry in Prague

Employee of the Precarpathian University (Ivano-Frankivsk) and the Institute of Organic and Bioorganic Chemistry (Prague). Doctorate degree in Life sciences (PhD) was obtained from Strasbourg University in 2009 for research work on the development of solvatochromic fluorescent labels for studies of protein interactions.

Senior research scientist Department of Neurophysiology, University of Tübingen