Microscopy
We have two high-end custom-built inverse microscopes of various configurations and modalities, as well as image analysis workstations and other resources. This allows users to image and analyze a range of samples (multiwell, with flow control, etc.) at various conditions (temperature, humidity, CO2 control).
One of the systems is a widefield (WF) fluorescence microscope with epifluorescence (Epi) and Total internal reflection fluorescence (TIRF) illumination with fluorescence lifetime (FLIM) and Time-Gated Luminescence (TGL) measurement capabilities. Technical specification about the microscope can be found from here.
The second system is a confocal / super-resolution stimulated emission depletion microscope with gated detection (gSTED) with fluorescence lifetime (FLIM) and F(C)CS measurement capabilities. Technical specification about the microscope can be found from here.
Highlights of some available modalities:
Total internal reflection microscopy (TIRF)
TIRF microscopy allows the selective illumination of a very thin optical slice (~60nm to ~300 nm thickness) at the coverslip surface. This is achieved by pointing the excitation laser at a high angle onto the coverslip-sample interface. This leads to the total internal reflection of the excitation laser and only a thin layer directly at the coverslip surface (the so-called "evanescence wave") contains light to excite fluorophores. The advantage is that no fluorophores in the bulk solution will be excited and this leads to a very high signal to background ratio. An additional improvement is the utilization of 360-degree rotation of laser beam at a high angle. This allows to eliminate interference fringes generated in the sample field.
Typical applications of TIRF microscopy include single molecule detection in in vitro experiments; studies related to the basal plasma membrane, such as endo- or exocytosis, membrane translocation of signal molecules.
Fluorescence Lifetime Imaging Microscopy (FLIM)
The monitoring the fluorescence lifetime of a fluorophore (the average time between excitation and photon emission) rather than its intensity („colour“) provides concentration-independent information on local microenvironment. Two main approaches are used for fluorescence lifetime measurements: time domain (TD) using pulsed light sources and frequency domain (FD) using megahertz modulated sources. In our setups TD (particularly Time-Correlated Single Photon counting, TCSPC) measurement is realized in confocal/STED system and FD in WF/TIRF system.
Typical FLIM applications include Fluorescence Resonant Energy Transfer (FRET) analysis of fluorophore interactions, measurement of ions and signalling molecules with biosensors, and analysis of the physical environment of the fluorophore (like plasma membrane viscosity). Additionally, lifetime of auto-fluorescence entities (like NADH/FAD) in cells or tissues could provide useful information.
Time-Gated Luminescence (TGL)
Auto-fluorescence is ubiquitous in nature and some materials fluoresce with great intensity, obscuring or diminishing the visibility of studied fluorescent probes. Time-gated luminescence microscopy effectively eliminates auto-fluorescence and enable high contrast imaging by setting a time delay between the excitation pulse and the luminescence detection and using of long-lifetime molecular probes (e.g. lanthanide complexes).
Typical TGL applications include visualization of biomolecules and metal ions (lanthanides, ARC-LUM protein kinase probes) in cells and image of oxygen consumption.
Stimulated emission depletion microscopy with gated detection (gSTED)
STED is a scanning confocal microscopy based super-resolution technique. In STED imaging, a doughnut-shaped laser beam silences fluorescence around a central excitation spot and thereby diminishes the excitation volume of a confocal laser scanning system to below the conventional diffraction limit. The resolution is improved more than 5 fold (<50 nm in x, and y) by shrinking the confocal volume. Our system is also equipped with gated detection which provides enhanced resolution and better suitability for life cell imaging compared to standard STED.
Typical gSTED applications include imaging with high-resolution biomolecules labelled with red shifted fluorophores (AbberiorSTAR 635P, Atto 647N). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS, FLCS.
Fluorescence fluctuation spectroscopy (FCS, FCCS, FLCS, PCH)
Fluorescence fluctuation spectroscopy (FFS) exploits fluorescence fluctuations to study various physical and biological systems at the single molecule level. FCS is a method to measure diffusion dynamics and interaction of fluorescent particles in a liquid environment. It is based on the measurement of the autocorrelation of the fluorescence signal from molecules moving through the focal volume of a confocal microscope. The autocorrelation curve calculated by the software gives information on apparent diffusion and concentration of molecules. If the size of the focal volume is calibrated, the diffusion coefficient of the investigated molecules can be accurately calculated. If two types of particles are marked with different fluorophores, one can measure also cross-correlated between the signals (fluorescence cross-correlation spectroscopy, FCCS). If particles move independently there will be no cross-correlation provided that there is no bleed through between fluorescence channels. If particles interact, which implies that they move together, it will create a significant cross-correlation between the signals. Thus the degree of cross-correlation can be an indication of both interaction and the percentage of interacting species. The fusion of TCSPC and FCS, called Fluorescence Lifetime-Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions. FLCS is of particular advantage when using spectrally inseparable fluorophores that differ in their lifetime for FCCS because it offers elimination of spectral crosstalk and background. Photon counting histogram (PHC) is another tool for extracting quantities from fluorescence fluctuation data, it measures photon counts per molecule and the average number of molecules within the observation volume. All those methods can be used for in vitro and in vivo measurements.
Typical use is for determining protein diffusion properties, local concentration, size, binding and clustering.
One of the systems is a widefield (WF) fluorescence microscope with epifluorescence (Epi) and Total internal reflection fluorescence (TIRF) illumination with fluorescence lifetime (FLIM) and Time-Gated Luminescence (TGL) measurement capabilities. Technical specification about the microscope can be found from here.
The second system is a confocal / super-resolution stimulated emission depletion microscope with gated detection (gSTED) with fluorescence lifetime (FLIM) and F(C)CS measurement capabilities. Technical specification about the microscope can be found from here.
Highlights of some available modalities:
Total internal reflection microscopy (TIRF)
TIRF microscopy allows the selective illumination of a very thin optical slice (~60nm to ~300 nm thickness) at the coverslip surface. This is achieved by pointing the excitation laser at a high angle onto the coverslip-sample interface. This leads to the total internal reflection of the excitation laser and only a thin layer directly at the coverslip surface (the so-called "evanescence wave") contains light to excite fluorophores. The advantage is that no fluorophores in the bulk solution will be excited and this leads to a very high signal to background ratio. An additional improvement is the utilization of 360-degree rotation of laser beam at a high angle. This allows to eliminate interference fringes generated in the sample field.
Typical applications of TIRF microscopy include single molecule detection in in vitro experiments; studies related to the basal plasma membrane, such as endo- or exocytosis, membrane translocation of signal molecules.
Fluorescence Lifetime Imaging Microscopy (FLIM)
The monitoring the fluorescence lifetime of a fluorophore (the average time between excitation and photon emission) rather than its intensity („colour“) provides concentration-independent information on local microenvironment. Two main approaches are used for fluorescence lifetime measurements: time domain (TD) using pulsed light sources and frequency domain (FD) using megahertz modulated sources. In our setups TD (particularly Time-Correlated Single Photon counting, TCSPC) measurement is realized in confocal/STED system and FD in WF/TIRF system.
Typical FLIM applications include Fluorescence Resonant Energy Transfer (FRET) analysis of fluorophore interactions, measurement of ions and signalling molecules with biosensors, and analysis of the physical environment of the fluorophore (like plasma membrane viscosity). Additionally, lifetime of auto-fluorescence entities (like NADH/FAD) in cells or tissues could provide useful information.
Time-Gated Luminescence (TGL)
Auto-fluorescence is ubiquitous in nature and some materials fluoresce with great intensity, obscuring or diminishing the visibility of studied fluorescent probes. Time-gated luminescence microscopy effectively eliminates auto-fluorescence and enable high contrast imaging by setting a time delay between the excitation pulse and the luminescence detection and using of long-lifetime molecular probes (e.g. lanthanide complexes).
Typical TGL applications include visualization of biomolecules and metal ions (lanthanides, ARC-LUM protein kinase probes) in cells and image of oxygen consumption.
Stimulated emission depletion microscopy with gated detection (gSTED)
STED is a scanning confocal microscopy based super-resolution technique. In STED imaging, a doughnut-shaped laser beam silences fluorescence around a central excitation spot and thereby diminishes the excitation volume of a confocal laser scanning system to below the conventional diffraction limit. The resolution is improved more than 5 fold (<50 nm in x, and y) by shrinking the confocal volume. Our system is also equipped with gated detection which provides enhanced resolution and better suitability for life cell imaging compared to standard STED.
Typical gSTED applications include imaging with high-resolution biomolecules labelled with red shifted fluorophores (AbberiorSTAR 635P, Atto 647N). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS, FLCS.
Fluorescence fluctuation spectroscopy (FCS, FCCS, FLCS, PCH)
Fluorescence fluctuation spectroscopy (FFS) exploits fluorescence fluctuations to study various physical and biological systems at the single molecule level. FCS is a method to measure diffusion dynamics and interaction of fluorescent particles in a liquid environment. It is based on the measurement of the autocorrelation of the fluorescence signal from molecules moving through the focal volume of a confocal microscope. The autocorrelation curve calculated by the software gives information on apparent diffusion and concentration of molecules. If the size of the focal volume is calibrated, the diffusion coefficient of the investigated molecules can be accurately calculated. If two types of particles are marked with different fluorophores, one can measure also cross-correlated between the signals (fluorescence cross-correlation spectroscopy, FCCS). If particles move independently there will be no cross-correlation provided that there is no bleed through between fluorescence channels. If particles interact, which implies that they move together, it will create a significant cross-correlation between the signals. Thus the degree of cross-correlation can be an indication of both interaction and the percentage of interacting species. The fusion of TCSPC and FCS, called Fluorescence Lifetime-Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions. FLCS is of particular advantage when using spectrally inseparable fluorophores that differ in their lifetime for FCCS because it offers elimination of spectral crosstalk and background. Photon counting histogram (PHC) is another tool for extracting quantities from fluorescence fluctuation data, it measures photon counts per molecule and the average number of molecules within the observation volume. All those methods can be used for in vitro and in vivo measurements.
Typical use is for determining protein diffusion properties, local concentration, size, binding and clustering.
Highlights of some available modalities:
Total internal reflection microscopy (TIRF)
TIRF microscopy allows the selective illumination of a very thin optical slice (~60nm to ~300 nm thickness) at the coverslip surface. This is achieved by pointing the excitation laser at a high angle onto the coverslip-sample interface. This leads to the total internal reflection of the excitation laser and only a thin layer directly at the coverslip surface (the so-called "evanescence wave") contains light to excite fluorophores. The advantage is that no fluorophores in the bulk solution will be excited and this leads to a very high signal to background ratio. An additional improvement is the utilization of 360-degree rotation of laser beam at a high angle. This allows to eliminate interference fringes generated in the sample field.
Typical applications of TIRF microscopy include single molecule detection in in vitro experiments; studies related to the basal plasma membrane, such as endo- or exocytosis, membrane translocation of signal molecules.
Typical applications of TIRF microscopy include single molecule detection in in vitro experiments; studies related to the basal plasma membrane, such as endo- or exocytosis, membrane translocation of signal molecules.
Fluorescence Lifetime Imaging Microscopy (FLIM)
The monitoring the fluorescence lifetime of a fluorophore (the average time between excitation and photon emission) rather than its intensity („colour“) provides concentration-independent information on local microenvironment. Two main approaches are used for fluorescence lifetime measurements: time domain (TD) using pulsed light sources and frequency domain (FD) using megahertz modulated sources. In our setups TD (particularly Time-Correlated Single Photon counting, TCSPC) measurement is realized in confocal/STED system and FD in WF/TIRF system.
Typical FLIM applications include Fluorescence Resonant Energy Transfer (FRET) analysis of fluorophore interactions, measurement of ions and signalling molecules with biosensors, and analysis of the physical environment of the fluorophore (like plasma membrane viscosity). Additionally, lifetime of auto-fluorescence entities (like NADH/FAD) in cells or tissues could provide useful information.
Typical FLIM applications include Fluorescence Resonant Energy Transfer (FRET) analysis of fluorophore interactions, measurement of ions and signalling molecules with biosensors, and analysis of the physical environment of the fluorophore (like plasma membrane viscosity). Additionally, lifetime of auto-fluorescence entities (like NADH/FAD) in cells or tissues could provide useful information.
Time-Gated Luminescence (TGL)
Auto-fluorescence is ubiquitous in nature and some materials fluoresce with great intensity, obscuring or diminishing the visibility of studied fluorescent probes. Time-gated luminescence microscopy effectively eliminates auto-fluorescence and enable high contrast imaging by setting a time delay between the excitation pulse and the luminescence detection and using of long-lifetime molecular probes (e.g. lanthanide complexes).
Typical TGL applications include visualization of biomolecules and metal ions (lanthanides, ARC-LUM protein kinase probes) in cells and image of oxygen consumption.
Typical TGL applications include visualization of biomolecules and metal ions (lanthanides, ARC-LUM protein kinase probes) in cells and image of oxygen consumption.
Stimulated emission depletion microscopy with gated detection (gSTED)
STED is a scanning confocal microscopy based super-resolution technique. In STED imaging, a doughnut-shaped laser beam silences fluorescence around a central excitation spot and thereby diminishes the excitation volume of a confocal laser scanning system to below the conventional diffraction limit. The resolution is improved more than 5 fold (<50 nm in x, and y) by shrinking the confocal volume. Our system is also equipped with gated detection which provides enhanced resolution and better suitability for life cell imaging compared to standard STED.
Typical gSTED applications include imaging with high-resolution biomolecules labelled with red shifted fluorophores (AbberiorSTAR 635P, Atto 647N). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS, FLCS.
Typical gSTED applications include imaging with high-resolution biomolecules labelled with red shifted fluorophores (AbberiorSTAR 635P, Atto 647N). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS, FLCS.
Fluorescence fluctuation spectroscopy (FCS, FCCS, FLCS, PCH)
Fluorescence fluctuation spectroscopy (FFS) exploits fluorescence fluctuations to study various physical and biological systems at the single molecule level. FCS is a method to measure diffusion dynamics and interaction of fluorescent particles in a liquid environment. It is based on the measurement of the autocorrelation of the fluorescence signal from molecules moving through the focal volume of a confocal microscope. The autocorrelation curve calculated by the software gives information on apparent diffusion and concentration of molecules. If the size of the focal volume is calibrated, the diffusion coefficient of the investigated molecules can be accurately calculated. If two types of particles are marked with different fluorophores, one can measure also cross-correlated between the signals (fluorescence cross-correlation spectroscopy, FCCS). If particles move independently there will be no cross-correlation provided that there is no bleed through between fluorescence channels. If particles interact, which implies that they move together, it will create a significant cross-correlation between the signals. Thus the degree of cross-correlation can be an indication of both interaction and the percentage of interacting species. The fusion of TCSPC and FCS, called Fluorescence Lifetime-Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions. FLCS is of particular advantage when using spectrally inseparable fluorophores that differ in their lifetime for FCCS because it offers elimination of spectral crosstalk and background. Photon counting histogram (PHC) is another tool for extracting quantities from fluorescence fluctuation data, it measures photon counts per molecule and the average number of molecules within the observation volume. All those methods can be used for in vitro and in vivo measurements.