Instrumentation of Department of Biophysical Chemistry

A: Major Equipment - Advanced light Microscopy and Spectroscopy

1) PicoQuant MicroTime 200, confocal time-resolved microscope with diffraction limited spatial resolution, single molecule sensitivity and sub-nanosecond time resolution

  • Four detection channels based on single photon avalanche (SPAD) detectors
  • Multicolor excitation modalities (pulsed-interlieved excitation, pulse burst excitation)
  • Available excitation wavelenghts: 375, 405, 440, 470, 532, 640 nm
  • 3D objective scanning using XY piezo stage with 10nm moving precision and objective Z-positioner
  • Wide field fluorescence observation using mercury lamp and CCD camera

2) Home built confocal time-resolved microscope with diffraction limited spatial resolution, single molecule sensitivity and sub-nanosecond time resolution

  • Two detection channels based on single photon avalanche (SPAD) detectors
  • Multicolor excitation modalities (pulsed-interlieved excitation, pulse burst excitation)
  • Available excitation wavelenghts: 375, 405, 440, 470, 532, 640 nm
  • 3D objective scanning using XYZ piezo stage with 10nm moving precision

3) Olympus FluoView1000 MPE, confocal laser scanning microscope with multi-photon as well as single photon excitation capability

  • Three built-in PMT detectors
  • Two channel, non-descanned detection unit based on PMT detectors with sub-nanosecond TCSPC (FLIM) capability
  • Two channel confocal detection unit based on single photon avalanche (SPAD) detectors with sub-nanosecond TCSPC (FLIM) and FCS capability
  • Transmitted light detector
  • Wide field fluorescence observation using mercury lamp and color CCD camera
  • Coherent Chameleon, tunable Ti Sapphire laser for multiphoton excitation
  • Available conventional excitation wavelenghts: 470, 488, 532, 640 nm

4) Home built single molecule sensitive fluorescence microscope with TIRF objective based on Olympus IX71 frame and Andor DU897_BV EMCCD camera

  • wide-field epifluorescence time-lapse imaging with TIRF capability
  • image splitter for dual color (or dual plane or astigmatic) observation
  • single molecule localisation microscopy (e.g. PALM and SOFI) capability
  • TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling) capability
  • Coherent Sapphire (488 and 560nm) an Coherent Cube (405 and 640nm) CW excitation sources
  • Combined micro (XY) and piezo-nano (XYZ) positioner from MadCityLabs

5) Home built TIRF microscope targeted at camera based Imaging FCS and Single Particle Tracking based on Olympus IX81 frame and Andor iXon Ultra 897 BV EMCCD camera

  • TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling) and Single Particle Tracking (SPT) capability
  • Coherent Sapphire 488nm and Lasos 640nm CW excitation sources
  • image splitter for dual color (or dual plane or astigmatic) observation

6) IBH model 5000U Time Resolved Spectrofluorimeter

  • based on Time-Correlated Single-Photon Counting (TCSPC)
  • Hamamatsu MCP detector
  • broad selection of laser diode and pulsed LED excitation sources
  • excitation and emission polarization accessories
  • temperature controlled sample holder
  • intensity decay, anisotropy decay, TRES measurement capabilities

B: Available Methods

1) Advanced modalities of Fluorescence Correlation Spectroscopy (FCS)

  • Fluorescence Cross-Correlation Spectroscopy (FCCS)
  • Fluorescence Lifetime Correlation Spectroscopy (FLCS)
  • z-scan FCS (invented in our laboratory)
  • Raster Image Correlation Spectroscopy (RICS)
  • Nanosecond FCS and Total correlation (FCS from zero lag-time)

2) FLIM (Fluorescence Lifetime Imaging Microscopy

  • based on Time Correlated Signle Photon Counting (TCSPC)

3) Total Internal Reflection Fluorescence (TIRF) microscopy

  • Thinning Out Clusters while Conserving Stoichiometry of Labeling (TOCCSL)
  • Single Particle Tracking

4) Super-resolution techinques

  • PALM and SOFI super-resolution microscopy with high-end data processing including generation of density maps and cluster analysis

5) Number&Brightness and 6) Antibunching analysis of confocal microscopy data

6) Fluorescence anisotropy

  •  both steady-state and time-resolved, in microscopy as well as in cuvette format

7) Time Resolved Emission Spectra (TRES)

  • sub-nanosecond dynamic Stokes shift, measurements in solvents, proteins, DNA and biomembranes

C: Auxiliary equipment

1) Fluorescence steady state spectrophotometers

  • Jobin Yvon SPEX Fluorolog 3
  • Edinburgh Instruments FS5

2) Circular Dichroism (CD) spectrophotometer

  • Jasco J-1500 with custom made sample holders enabling measurements of orientation CD

3) UV-VIS absorption spectrometer

  • Schimadzu UV2600, including integrating sphere

4) System for preparation of model phospholipid bilayers

  • including unilamellar vesicles (SUVs, LUVs, GUVs), giant plasma membrane vesicles (GPMVs) and supported phospholipid bilayers (SPBs)
  • Avestin

5) Langmuir troughs

  • Microtrough XS setup (Kibron) enabling monolayer preparation and its visualization on microscope (with temperature control)

6) Osmometer

  • Marcel OS 3000

7) Facilities for mammalian cell culture

  • Including laminar flowbox and 2 CO2 incubators
  • Verification of cells (viability, mycoplasma infection, stress, ...)
  • Cell transfection capability using electroporation (NEON system, Invitrogen) and lipofection
  • Small-scale autoclave for sterilisation of tools and solutions

Department of Biophysical Chemistry

Head
doc. RNDr. Šachl Radek Ph.D.
Deputy Head
doc. Ing. Petráková Vladimíra Ph.D.