Emphasis of research is set on the investigation of cell biology and protein dynamics by optical and scanning-probe techniques. Next to standard equipment available at the Institute of Physics or in service facilities at the Faculty of Sciences, specialized instrumentation for single-molecule microscopy is available. We frequently host visitors in our laboratory for joined research projects, or for initial proof-of-principle experiments which might lead to a joined proposal. If you have interest in a pilot experiment you should contact Thomas Schmidt.
Optical Fluorescence Microscopy Scanning Probe Microscopy Standard Lab Equipment
Optical Fluorescence Microscopy
Fluorescence microscope
Inverted fluorescence microscope (Zeiss, Axiovert 40) with halogen (50W) and Hg-lamp (25W) illumination. The microscope can be equipped with objectives ranging between 10x,NA0.3 to 100x, NA1.4. Standard filtersets (GFP, YFP, FM, etc) are installed. The images are acquired onto a high-sensitivity monochrome CCD camera (Watec 902H). The setup is controlled by LabView through a NI PCI-1405 card (National Instruments).
Specs:
Reference: AxioVert40
Spinning-disk confocal setup
Inverted microscope (Zeiss, Axiovert 200) with halogen- and Hg-lamp illumination. The microscope is equipped with a motorized xy-stage (Märzh$auml;ser) and a piezo-controlled objective positioner (Physik Instrumente, PiFoc) allowing for 50nm lateral and 2nm axial sample positioning. The sample stage is temperature controlled (5-50oC) by means of a water bath. The microscope is equipped with objectives ranging between 10x,NA0.3 to 100x, NA1.45. Fluorescence excitation is alternatively achieved by an Ar-laser (Coherent, Innova300, 5W all lines), fiber-coupled into the microscope (20mW) and a set of DPSS and diode lasers. The single-line power at the objective <20mW and the wavelength and excitation timing is controlled by an acousto-optical filter (AA-OptoElectronic, AOTFnC-VIS). The signals are detected on a back-illuminated electron-multiplying CCD camera (Photometrics, Cascade, 512×512 pixel, QE>0.92, noise<7cnt/pxl). The setup is controlled by LabView (National Instruments) and WinView (Photometrics). Analysis is performed offline using our Matlab package.
Specs:
Reference:
2 single-molecule setups, microscope based
Inverted microscopes (Zeiss, Axiovert 100TV) with halogen-lamp illumination. The sample stage is temperature controlled (5-50oC) by means of a water bath. The microscopes are equipped with objectives ranging between 10x, NA0.3 to 100x, NA1.45. Fluorescence excitation is achived by an Ar/Kr-laser (Spectra Physics, 2060-10, 3W all lines), fiber-coupled into the microscope. The single-line power at the objective is < 20mW. The wavelength and excitation timing is controlled by an acousto-optical filter (AA-OptoElectronic, AOTFnC-VIS). Signals are detected on a N2-cooled, thinned back-illuminated CCD camera (Photometrics, SpectraMax 1340×400 pixel, QE>0.92, noise<3cnt/pxl). By means of our tailor-made dichroic wedges (Chroma, ‘Schmidt wedge’) and a Wollaston prism in the detection path, 4 images (4 x 100×100, i.e. 2 colors & 2 polarizations) can be acquired simultaneously. The setup is controlled by LabView (National Instruments) and WinView (Photometrics) software. Analysis is performed offline using our Matlab package.
Specs: single-molecule (eYFP) sensitivity with S/B>30 at 50 Hz on live cells; positional accuracy <40nm in xy
Reference: Single-Molecule Microscopy; Watching Single-Molecule Diffusion; Multidimensional Single-Molecule Imaging
Single-molecule setup for 3D-tracking, microscope based
Inverted microscope (Zeiss, Axiovert 100TV) with halogen-lamp illumination. The microscope is equipped with a 3D piezo scanner (Physik Instrumente, P-733.2DD & P-721.CDQ) allowing for nanometric position control of the sample. The sample stage is temperature controlled (5-50oC) by means of a water bath. The microscope is equipped with objectives ranging between 10x,NA0.3 to 100x, NA1.45. Fluorescence excitation is alternatively achieved by an Ar-laser (Coherent, Innova300, 5W all lines), fiber-coupled into the microscope (20mW) and a diode-laser at 635nm and output power of 30mW. The single-line power at the objective <20mW and the wavelength and excitation timing is controlled by an acousto-optical filter (AA-OptoElectronic, AOTFnC-VIS). The signals are detected on a back-illuminated electron-multiplying CCD camera (Photometrics, Cascade, 512×512 pixel, QE>0.92, noise<7cnt/pxl). By means of dichroic wedges (Chroma, ‘Schmidt wedge’), 2 images can be acquired simultaneously. The insertion of controlled astigmatism into the detection beampath the 3-dimensional position of objects are determined at nanometer accuracy. The setup is controlled by LabView (National Instruments) and WinView (Photometrics). Analysis is performed offline using our Matlab package.
Specs: single-molecule (eYFP) sensitivity with S/B>20 at 100 Hz on live cells; positional accuracy <40nm in xy and <60nm in z
Reference: 3D Single-Molecule Tracking
One-photon and two-photon confocal lifetime setup, microscope based
Inverted microscope (Zeiss, Axiovert100TV) with halogen-lamp illumination. The microscope is equipped with a 3D piezo scanner (Physik Instrumente, P-733.2DD & P-721.CDQ) allowing for nanometric position control of the sample. The microscope is built as a sample-scanning instrument. Fluorescence excitation is achived by a pulsed Ti:Saphire laser (Spectra; Millenia X, 10W, 532nm; Tsunami, 2W, 700-900nm, 76MHz, 70fs). After split into 2 wavelength or polarization channels the signals are detected on 2 photon-counting self-quenched avalanche photodiodes (PerkinElmer, SPCM-AQR-14, noise <100Hz). Photons are counted by a multichannel scaler card (Becker&Hickl, PMS-200) or by a 4-channel time-correlated single-photon counting module (Picoquant, TimeHarp-200). The instrument response function has a width <120ps. The setup is controlled by LabView. Analysis is performed offline using our Matlab suite.
Specs: sample-scanning 2-photon confocal, 2-channel detection, excitation wavelength 700-900nm, IRF <120ps
Reference: .
Scanning-Probe Microscopy
2 atomic force multimode microscopes
Commercial atomic force microscopes (Digital Instruments, NanoScope III). The microscopes are vibration (Halcyonics, Micro40) and acoustical isolated. Both are equipped with liquid cell, break-out box and phase module. One instrument is optimized for low noise applications allowing to resolve the structure of individual biomolecules. The second has an optical access and has the option for electric readout in a combined AFM/STM mode. All usual SPM-modes (contact, tapping, force-volume, …) are available. Various scanners allow for scan areas from 15um to 150um.
Specs: Digital Instruments NanoScope III with fluid cell.
Reference: (Bio-) Molecular resolution
Atomic force microscope, optical microscope based
Commercial atomic force microscopes (Digital Instruments, BioScope) mounted on an inverted optical microscope (Zeiss, Axiovert100TV). The microscope is acoustical isolated. The samples can be scanned using either the internal DI scanner or by a 2D piezo table (Physik Instrumente, P-733.2DD) which serves as a sample-mounting stage. A home-built analog module allows that both modes are controlled via the original DI-software. The confocal is equipped with a photon-counting self-quenched avalanche photodiodes (PerkinElmer, SPCM-AQR-14, noise <100Hz). The instrument is equipped with liquid cell, break-out box and phase module supporting all usual SPM-modes (contact, tapping, force-volume, …). This unique instrument allows to simultaneously obtain fluorescence and heights/mechanical images, as well allows for following cellular changes after mechanical stimulation or following mechanical changes of cells on optical release of a process.
Specs: Digital Instruments BioScope on top of a confocal microscope.
Reference: Combined AFM/optical microscopy BioScope
Other Instrumentation
Fluorescence spectrometer
Commercial fluorescence / fluorescence excitation spectrometer (PerkinElmer, LS55). Standard 1cm cuvettes. T-controlled system.
Specs: excitation: 400-900nm, emission: 400-900nm
Reference: LS55
Gel-imager
Commercial gel documentation system (UVP, GelDocIt). Gel imaging & analysis system using CCD detection.
Specs: epi- and transillumination, CCD detection, EtBr filters
Reference: GelDocIt
Langmuir trough
Commercial Langmuir trough (KSV, Minitrough) with T-control and dipper. The instrument is placed inside a glass box.
Specs: 365x75mm area, 2 barriers, sample lift
Reference: KSV Minitrough
UV/VIS absorption spectrometer
Commercial 2-beam absorption spectrometer (Shimazu, UV-1650PC). Standard 1cm cuvettes. T-controlled system.
Specs: 190-1100nm, dual beam
Reference: Shimazu UV-1650PC
