Microtime 200

Time-resolved Confocal Fluorescence Microscope with Unique Single Molecule Sensitivity


The advances of cutting edge science in many fields depend on single molecule studies. This includes, for example, the quantification of molecular dynamics or molecular properties as well as interaction studies in material and life sciences. Such a wide field of research requires a flexible instrument, which can be adapted to the individual needs. This versatility is given in the MicroTime 200, a time-resolved confocal fluorescence microscope system. This powerful instrument is ready to analyze a multitude of parameters down to the single molecule level using methods such as Fluorescence Lifetime Imaging (FLIM), FLIM/FRET, deep tissue FLIM, PIE, FCS/FCCS, FLCS/FLCCS, dual-focus FCS, anisotropy, burst analysis, simultaneous AFM/FLIM or deep UV detection, to name only the most common. Even high resolution imaging with spatial resolutions below 50 nm is possible with the new MicroTime 200 STED add-on.

Flexible excitation subsystem
The excitation subsystem of the MicroTime 200 consists of a pulsed diode laser driver of the PDL Series and different laser heads with pulses in the picosecond time regime (additional CW mode is available as an option). The available wavelengths range from 375 to 900 nm.The laser power and repetition rate can be flexibly adjusted by the laser drivers of the PDL Series. The multichannel laser driver PDL 828 “Sepia II” even allow to address several lasers in parallel enabling advanced techniques like Pulsed Interleaved Excitation (PIE).The laser heads are integrated in one Laser Combining Unit (LCU) for easier handling, attenuation and coupling into an optical fiber. An alternative incoupling port at the MicroTime 200 can accommodate additional excitation sources such as Ti:Sapphire lasers for multi-photon excitation schemes. Using our dedicated Two-Photon-Excitation unit, the output from external lasers can be easily coupled to the main optical unit of the MicroTime 200.
Choice of scanning technologies
The MicroTime 200 can be equipped with different devices for scanning an image: A galvo scanner for quick image acquisition or piezo devices for maximum flexibility in terms of usable wavelengths. The great versatility of the MicroTime 200 platform is complemented by the FLIMbee galvo scanner which can provide scanning speeds ranging from very slow to fast while maintaining high precision. This high degree of flexibility in speed allows for applications ranging from Phosphorescence Lifetime Imaging (PLIM) to fast fluorescence lifetime measurements using rapidFLIM. Furthermore, with its high precision and sensitivity, the FLIMbee scanner is optimally suited for super-resolution microscopy via STED, enabling imaging down to the single molecule level.
A MicroTime 200 equipped with a FLIMbee scanner is a good choice for Single Molecule Detection (SMD) methods such as spFRET, PIE-FRET, (STED-)FCS, FLCS, FLCCS, dual-focus FCS (2fFCS), and even anisotropy measurements. Additionally, Two-Photon Excitation (TPE) with descanned and non-descanned detection is possible.
The core of the FLIMbee galvo scanner consists of three high precision oscillating mirrors with excellent linearity, repeatability and low drift. The two y-axis galvo mirrors ensure that the laser beam is stationary at the entrance of the objective. This mirror configuration minimizes vignetting of the image field and ensures a constant focal volume over a wide scan range. The FLIMbee scanner provides a minimal pixel size of 10 nm when using a 100x objective.
Detection subsystem with single photon sensitivity
The MicroTime 200 was specially designed for single molecule studies and thus offers unique optics with vastly reduced light absorption. In this confocal microscope, scanning is facilitated through a piezo table optionally combined with a high precision PiFoc element for 3D imaging. The choice of piezo scanning ensures a high repositioning accuracy and stability, which is essential for single molecule studies.The MicroTime 200 can be configured for up to six individual detection channels. Each channel can be equipped with a different detector, chosen from a variety of sensitive detectors. The detectors offer ideal solutions depending on the wavelength to be detected, the signal brightness and the excited state lifetime of the investigated emitters. The choice of detectors include PMA Hybrid detectors, optimized SPADs for efficiency or timing as well as dedicated detectors for experiments in the deep UV.
Timing with picosecond resolution
Time-resolved microscopy requires the registration of not only the photons themselves, but also their position in time and, for imaging, in space. The ideal technique for that purpose is the Time-Tagged Time-Resolved (TTTR) data acquisition developed by PicoQuant, which is an variation of the classical method of Time-Correlated Single Photon Counting (TCSPC). The advantage of TTTR data acquisition mode is that it allows to perform vastly different measurement procedures, like FLIM, FCS or even coincidence correlation (“antibunching”), based on just one fundamental data format. The TTTR format ist supported by all available TCSPC electronics from PicoQuant. Using these high-end integrated devices fluorescence lifetimes down to a few picoseconds or even up to ms for phosphorescence and luminescence studies can be easily resolved.
Intuitive data handling and analysis
Based on the sophisticated data collection and handling, the system software SymPhoTime 64 supports a multitude of methods, such as intensity time trace, burst analysis, lifetime histogramming, Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Correlation Spectroscopy (FLCS), Fluorescence Lifetime Imaging (FLIM), Förster Resonance Energy Transfer (FRET) and anisotropy, to name only a few.SymPhoTime 64 data handling maintains a transparent data structure where all derived data is maintained in one workspace, including a log file to keep track of all measurement and analysis steps.A large number of algorithms for those methods are already integrated in SymPhoTime 64, providing a analysis platform for ready-to-publish data. At the same time, SymPhoTime 64 offers enhanced flexibility for the integration of novel, cutting edge algorithms by the user. A dedicated scripting language interface allows to modify and expand the analysis routines. In addition to data analysis within SymPhoTime 64, data can be exported to standard formats for external analysis.Our interactive user forum as well as our regularly held SymPhoTime training days offer outstanding support for new and advanced users.
Scientific guidance and user training
PicoQuant annually holds the European short course on “Time-resolved Microscopy and Correlation Spectroscopy”. The course is intended for individuals wishing an in-depth introduction to the principles of time-resolved fluorescence microscopy and its applications to the Life Sciences. This 3-day event consists of lectures as well as instrumentation and software hands-on training. For details see the course website.
PicoQuant also hosts a forum that serves as a knowledge exchange platform for users of the company’s systems, components and software packages.

Приставка STED для MicroTime 200:

Excitation System
  • Picosecond diode lasers with adjustable output power and repetition rates up to 80 MHz inside a compact fibre coupling unit
  • Wavelengths between 375 and 900 nm
  • Single or multi-channel laser driver
  • Optional: external laser (e.g. Titanium:Sapphire laser)
Microscope
  • Inverted microscope IX 73 or IX 83 from Olympus
  • Left side port and back port still accessible (for e.g. widefield imaging or TIRF)
  • Transmission illumination unit included
  • Special manual sample positioning stage with 25 mm × 25 mm range
  • Standard sample holder for 20 mm × 20 mm cover slips
  • Optional: Epifluorescence illumination
  • Optional: Cryostat for low temperature measurements
  • Optional: Combination with Atomic Force Microscope (AFM)
Objectives
  • Air spaced objectives with 20x and 40x magnification (standard)
  • Various high end objectives available (Oil / water immersion, air spaced, IR/UV-enhanced, TIRF or long working distance objectives upon request
Scanning Piezo:

  • 2-dimensional piezo scanning table with 80 µm x 80 μm imaging range at nominal 1 nm positioning accuracy
  • PIFOC for 3-dimensional imaging, 80 μm range at nominal 1 nm positioning accuracy
  • Objective scanning with 80 µm x 80 μm imaging range at nominal 1 nm positioning accuracy
  • Optional: large area scanning table with centimetre scan range

Galvo scanner FLIMbee:

  • Image size ranging from 10 × 10 to 2048 × 2048 pixel
  • Maximum field-of-view: 250 × 250 µm (60x objective)
  • Up to 2.6 kHz line frequency (bi-directional scanning), 5.2 FPS @ 512 × 512 pixel
  • Optional z-axis control, e.g., for z-stacks (piezo-based, up to 100 µm)
  • Pixel dwell times from 0.5 µs up to 1 s
Main optical unit
  • Confocal detection setup in a compact housing with up to six detection channels
  • Variable beam splitting units
  • Exit ports to connect external devices
  • Prealigned major dichroic mirror holder for easy change of optics
  • CCD camera and photodiode for beam diagnostics
  • All optical elements easily accessible and adjustable
Detectors
  • Single Photon Avalanche Diodes
  • Hybrid-Photomultiplier Tubes
  • Photomultiplier Tubes
Data acquisition
  • Based on the method of Time-Correlated Single Photon Counting (TCSPC) in the unique Time-Tagged Time Resolved (TTTR) measurement mode
  • Simultaneous data acquisition of up to four channels
Software
  • SymPhoTime 64

Введение в микроскопию единичных молекул:

Silver-coated nanoporous gold skeletons for fluorescence amplification

Lee M.-J., Yang W.-G., Kim J.H., Hwang K., Chae W.-S. Microporous and Mesoporous Materials, Vol.237, p.60-64 (2017)

Reference to: MicroTime 200, SymPhoTime Related to: FLIM


Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

Andersen T.G., Nintemann S.J., Marek M., Halkier B.A., Schulz A., Burow M. Scientific Reports, Vol.006, 27766 (2016)

Reference to: MicroTime 200, LSM Upgrade Kit, SymPhoTime Related to: FLIM, FRET


Influence of plasmonic array geometry on energy transfer from a quantum well to a quantum dot layer

Higgins L.J., Morocico C.A., Karanikolas V.D., Bell A.P., Gough J.J., Murphy G.P., Parbrook P.J., Bradley A.L. Nanoscale, Vol.008, p.18170-18179 (2016)

Reference to: MicroTime 200 Related to: FRET, TRPL


Temperature-dependent luminescent decay properties of CdTe quantum dot monolayers: impact of concentration on carrier trapping

Murphy G.P., Zhang X., Bradley A.L. The Journal of Physical Chemistry C, Vol.120, p. 26490–26497 (2016)

Reference to: MicroTime 200 Related to: FLIM, TRPL


Ag colloids and arrays for plasmonic non-radiative energy transfer from quantum dots to a quantum well

Murphy G.P., Gough J.J., Higgins L.J., Karanikolas V.D., Wilson K.M., Garcia Coindreau J.A., Zubialevich V.Z., Parbrook P.J., Bradley A.L. Optics (2016)

Reference to: MicroTime 200 Related to: FLIM, TRPL


Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer

Grudzinski W., Sagan J., Welc R., Luchowski R., Gruszecki W.I. Scientific Reports, Vol.006, 32780 (2016)

Reference to: MicroTime 200, FluoTime 300 Related to: FLIM, Anisotropy


Spatial inhomogeneity in spectra and exciton dynamics in porphyrin micro-rods and micro-brushes: Confocal microscopy

Chattoraj S., Bhattacharyya K. Journal of Chemical Sciences, Vol.128, p.1717-1724 (2016)

Reference to: MicroTime 200 Related to: FLIM


Exploring the HYDRAtion method for loading siRNA on liposomes: the interplay between stability and biological activity in human undiluted ascites fluid

Dakwar G.R., Braeckmans K., Ceelen W., De Smedt S.C., Remaut K. Drug Delivery and Translational Research, Vol.007, p.241-251 (2016)

Reference to: LSM Upgrade Kit, SymPhoTime Related to: FCS


Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis

Zhang X., Poniewierski A., Jelińska A., Zagożdżon A., Wisniewska A., Hou S., Hołyst R. Soft Matter, Vol.012, p.8186-8194 (2016)

Reference to: PicoHarp 300, LSM Upgrade Kit, SymPhoTime Related to: FCS


Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon S.V., Tabarin T., Yamamoto Y., Ma Y., Nicovich P.R., Bridgeman J.S., Cohnen A., Benzing C., Gao Y., Crowther M.D., Tungatt K., Dolton G., Sewell A.K., Price D.A., Acuto O., Parton R.G., Gooding J.J., Rossy J,. Rossjohn J., Gaus K. PNAS, Vol.1113, p.5454-5463 (2016)

Reference to: MicroTime 200 Related to: FCS