Characteristics

The major aim of the course is to introduce the powerful tools of advanced fluorescence microscopy to potential users in cell and developmental biology. The course is designed to maximize a hands-on experience, and participants are encouraged to bring their own samples. During the course, each technique will be introduced theoretically, followed by an extensive practical part. After data acquisition, the results will be processed and/or analyzed to demonstrate the complete procedure starting from the image acquisition to data presentation and interpretation.

The course is intended as a practical hands-on exercise with 4 participants per a workgroup.

The course requires basic to intermediate knowledge of fluorescence microscopy, at least at the level of the course "Microscopy Methods in Biomedicine".

The applications will be accepted based on the first come first served basis but also taking into account the evaluation of scientific relevance explained in the motivation letter. Preference will be given to applicants in early stage of their career.

We look forward to seeing you in Prague!

The course will be taught in English.





Venue


Institute of Molecular Genetics of the AS CR, v.v.i., Vídeňská 1083, Prague 4, Czech Republic







Scientific supervisors





Content of the course

 
SIM

Structured illumination microscopy (SIM) is a widefield microscopy technique. It is one of several superresolution methods available in light microscopy. In principle, the technique using the patterned light increases resolution by exploiting interference patterns – moiré patterns. In fact, a grid pattern is generated and superimposed on the specimen. The pattern is projected on the specimen in three angles and five translations. The final set of images is processed by mathematical algorithm based on Fourier transformation. The resolution of final reconstructed image is approximately two times better than the Abbe limit for particular wavelength and could be around 100 nm.

We will use OMX 3D-SIM microscope, image reconstructions will becomputed in Softworx software package and in SIMToolbox Matlab programme.

STED

Stimulated emission depletion (STED) is a superresolution confocal microscopy technique using two laser lines – one for an excitation and the second for an emission depletion. The lasers are co-aligned to form a donut shape when the inner spot is the excitation laser and the outer ring is the depletion laser (STED laser).

The function of STED laser is to interrupt the normal emission process after the excitation when the photon is released. In the STED, the excited electron is forced to relax into a higher vibration state – due to the higher vibration state the energy of the emitted photon is lower and shifted to the red spectra.

The resolution of STED microscopy is around 70 nm and lower depending on experiment conditions.
We have Leica TCS SP8 STED 3X, 660nm cw STED laser and pulse wide light laser in combination with HyD hybrid detector that allows time-gating. For STED deconvolution we routinely use Huygens professional, SVI.

FRAP

Fluorescence recovery after photobleaching (FRAP) is a well-established photo-kinetic method. Together with iFRAP, FLIP or photo-activation it is used to study dynamics of molecules within living cells. The aim of these methods is perturbation of the steady-state fluorescence distribution in a selected region of the specimen and observation and then analysis of fluorescence recovery towards the steady state.
The analysis of the FRAP experiment allows to calculate half-time of the recovery and percentage of mobile/immobile fractions of the studied component. Then it is possible to calculate the diffusion coefficients and binding constants.

The FRAP experiments will be done on DeltaVision Core microscope equipped with 405 and 488 nm lasers for photo-manipulation; enclosed in climate chamber for work at 37°C and regulated CO2 atmosphere. For the FRAP analysis the FiJi ImageJ, Matlab software and MS Excel will be used.

FLIM-FRET

Förster resonance energy transfer (FRET) enables to study molecular interactions such as binding or protein clustering or can be used for microenvironment sensing through FRET based sensors.  The method is based on non-radiative energy transfer between two fluorophores (named donor and acceptor) that are maximally few nanometers apart, appropriately oriented and for which donor emission spectra and acceptor absorption spectra overlap. The occurrence of FRET and its efficiency can be determined by monitoring: 1) relative changes in donor and acceptor fluorescence intensity (sensitized emission) and/or 2) shortening of donor fluorescence lifetime.  The later approach is considered more robust against possible artefacts. Excited state lifetimes are acquired by FLIM technique (fluorescence lifetime imaging microscopy), in which the fluorescence decay is recorded in every pixel.  

FLIM-FRET will be measured on laser scanning confocal microscope Leica TCS SP8 WLL SMD-FLIM (located in IMCF at BIOCEV) using internal spectral HyD detectors and WLL pulsed laser, and analyzed by SymphoTime64 and LabVIEW software.

 


Faculty




Aleš Benda
Michaela Blažíková
Martin Čapek




          








The course is organized by the Microscopy Centre - Core Facility for Light Microscopy & Core Facility for Electron Microscopy, which are supported from the program for large research infrastructures of the Ministry of Education, Youth and Sports within the project “National Infrastructure for Biological and Medical Imaging (Czech-BioImaging – LM2015062)“ and from the National Programme for Sustainability I of Ministry of Education, Youth and Sports ("Biomodels for health" - LO1419).