Seeing is believing II - Advanced Microscopy for Everyone - MB100P02

• Title: Seeing is believing II - Advanced Microscopy for Everyone
• Czech title: Vidět znamená chápat a věřit II - Pokročilá mikroskopie pro každého
• Guaranteed by: Biology Section (31-101)
• Faculty: Faculty of Science
• Actual: from 2023 to 2023
• Semester: summer
• E-Credits: 4
• Examination process: summer s.:
• Hours per week, examination: summer s.:2/1, C+Ex [HT]
• Capacity: 20
• Min. number of students: unlimited
• 4EU+: no
• Virtual mobility / capacity: no
• State of the course: taught
• Language: English
• Explanation: Observing and quantifying cellular processes at molecular level
• Note: enabled for web enrollment; the course is taught as cyclical
• Guarantor: Mgr. Aleš Benda, Ph.D.; Mgr. Marek Cebecauer, Ph.D.
• Teacher(s): Mgr. Aleš Benda, Ph.D.; Mgr. Marek Cebecauer, Ph.D.; Mgr. Markéta Dalecká; Ing. Dalibor Pánek, Ph.D.; Ing. Petra Prokšová, Ph.D.

Annotation

Last update: Mgr. Aleš Benda, Ph.D. (11.01.2024)

Rapid development of microscopy techniques and labelling probes enabled observation of cellular physiology at the molecular level and often in living specimens. In Seeing is believing I. course, we focused on basic light and electron microscopy techniques, which enable everyday analysis of biological specimens. In this second part (Seeing is believing II.), we explore application of more advanced imaging methods, including super-resolution microscopy and spectroscopy-based tools. Importantly, non-standard modes of some basic microscopy techniques and the use of specialised probes to monitor cellular processes and analyse local environment is discussed in this course (see Syllabus for more details).

The practical part: hands-on demonstration of selected applications, including sample preparation, data acquisition, data analysis and data management.

Literature

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

Supporting literature (not required for the exam):

Pawley et al. Handbook of Biological Confocal Microscopy, 3^rd Edition. Springer Science; 2006. ISBN-13: 978-0387259215

Peng Xi. Optical Nanoscopy and Novel Microscopy Techniques. CRC Press; 2014. ISBN 9781466586291

Diaspro et al. Nanoscopy and Multidimensional Optical Fluorescence Microscopy. Chapman and Hall/CRC; 2010. ISBN 9781420078862

http://www.olympusmicro.com

http://www.microscopyu.com

http://micro.magnet.fsu.edu

Requirements to the exam

Last update: Mgr. Aleš Benda, Ph.D. (15.09.2020)

A student needs to participate at minimum 60% of the lectures. For the exam, the student selects 2 papers (from 25-30 available). The exam is in the form of discussion on the technology used and its suitability to generate exciting observation(s) presented in the selected papers.

Active participation at the practical part.

The exam can be in Czech or Slovak in case the student does not feel comfortable with an exam in English.

Syllabus

Last update: Mgr. Aleš Benda, Ph.D. (11.01.2024)

Seeing is believing II. – Advanced Microscopy for Everyone.
1. Structural details observable using standard fluorescence microscopy are insufficient. Is there a
way how to “see below the resolution limit”? Yes, and even a little resolution improvement can do

miracles. Cellular structures and processes visualised with ‘widefield’ superresolution microscopies:
structured illumination microscopy (SIM), lattice light sheet microscopy (LLM). AireScan.

2. What if “a little resolution improvement” is not enough? In case a broad field is required, there is
a confocal-based superresolution microscopy: Stimulated emission depletion (STED) microscopy.
Alternatively, we can increase the size of our sample. How? Let’s discuss principles of expansion
microscopy (ExM) and see deserved details with standard microscopes.

3. Still more’s wanted! Can I see individual biomolecules forming a fine three-dimensional structure
inside a cell? Single molecule fluorescence, single molecule localisation microscopy (SMLM; PALM,
dSTORM, dTRABI), interferometric scattering (iSCAT) microscopy. Photoconversion, photoactivation
and photoswitches.

4. Can we follow molecular interactions directly in living cells? What about monitoring cellular
environment? Főrster (fluorescence) resonance energy transfer (FRET), anisotropy, fluorescence
lifetime and ratiometric imaging. Imaging Flow Cytometry.

5. Static snapshots are boring. Can I track individual molecules inside a cell? Dynamics of molecules
in the cytosol, on fibres (the cytoskeleton) and in membranes. Fluorescence recovery after
photobleaching (FRAP + variants), fluorescence (cross-)correlation spectroscopy (F(C)CS) and single
particle tracking.

6. Structural details visualised by electron microscopes are amazing. How can I get molecular
specificity with these methods? Specific labelling for the EM (immuno-gold, APEX, APEX-gold).
CryoFM. Correlated light and electron microscopy (CLEM).

7. A bull is a powerful animal. However, can a cell exert a force? Does tumour environment differ
from normal tissues? Measuring mechanical forces in cells. Tissue stiffness. Tricks and tips. Atomic
force microscopy, optical tweezers, nanopillars, Brillouin microscopy, …

8. Can we image specific atoms, ions, bonds, structures? Chemical specificity by Raman Spectroscopy, CARS, EDS/EDX (EM mode), pH/ion imaging/voltage
imaging.

9. How to get my sample ready to win ‘Imaging contest’? It must be bright, colourful and artifact
free? Advanced labelling methods: 30 colour microscopy (antibody exchange), DNA-PAINT for
labelling without antibodies and ligands, biorthogonal (direct) labelling of molecules, …

Entry requirements

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

The lectures will be taught in English.

Prerequisite:

Biologie buňky (Biology of the cell) - MB150P31(E)

Recommended:

Fluorescenční mikroskopie v buněčné biologii (Fluorescence microscopy of cells) - MB151P96

Landmarks/Milestones of Cell Biology - MBCPLUS002

Registration requirements

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

The course is designed for Master and PhD students.