Microtime 200 STED

Time-resolved Confocal Fluorescence Microscope with Super-resolution Capability


In the recent years, super-resolution microscopy has gained more and more attention. It has now evolved beyond the stage of development and permits to investigate biological systems that were formerly obscured by the diffraction limit of light. One of the most popular techniques for super-resolution imaging is Stimulated Emission Depletion (STED)microscopy. STED is usually performed with confocal microscopes and is therefore ideally suited to be added to the MicroTime 200. The integration of STED into the system has been driven towards highest robustness and ease-of-use. The system permits to perform STED microscopy without lengthy alignment preparations while still having the choice to modify the system and use the full capability of the open microscopy platform MicroTime 200.


The MicroTime 200 STD is a time-resolved confocal microscope with single molecule sensitivity and super-resolution capabilities. Hence, numrous applications are possble with this instrument, including:

  • Stimulated Emission Depletion Microscopy (STED) / gated STED
  • Single Molecule Spectroscopy / Detection
  • Time-Resolved Fluorescence
  • Fluorescence Lifetime Imaging (FLIM)
  • Phosphorescence Lifetime Imaging (PLIM)
  • Fluorescence Correlation Spectroscopy (FCS)
  • Fluorescence Lifetime Correlation Spectroscopy (FLCS)
  • Foerster Resonance Energy Transfer (FRET)
  • Dual-focus Fluorescence Correlation Spectroscopy (2fFCS)
  • Pulsed Interleaved Excitation (PIE)
  • Fluorescence Anisotropy (Polarization)
  • Pattern Matching Analysis
  • Time-Resolved Photoluminescence (TRPL)
  • TRPL Imaging
  • Antibunching
Optical resolution
  • below 50 nm (STED)
  • below 300 nm (confocal)
Excitation system
  • Picosecond diode lasers (375 nm – 900 nm) with repetition rates up to 80 MHz inside a compact Laser Combining Unit
  • Single or multichannel laser driver
  • Optional: excitation down to 266 nm*
  • Optional: external laser (e.g., Titanium:Sapphire laser)

For STED

  • 640 nm (excitation), 765 nm (STED laser)
  • Dual species STED with 640 nm and 595 nm excitation
  • Dual species STED with 640 nm and 660 nm excitation
Microscope
  • Inverted microscope IX 73 or IX 83 from Olympus
  • Specially designed right side port for confocal microscope
  • 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 range
  • Standard sample holder for 20 mm x 20 mm cover slips
  • Optional: epifluorescence illumination
  • Optional: cryostat for low temperature measurements
  • Optional: combination with Atomic Force Microscope (AFM)
Objectives
  • Air 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)
Scanning Piezo:

  • Computer controlled
  • 2-dimensional piezo objective scanning with 80 µm x 80 µm scan range at nominal 1 nm positioning accuracy
  • PIFOC for 3-dimensional imaging, 80 µm range at nominal 1 nm positioning accuracy
  • Optional: sample scanning
  • Optional: large area scanning table with cm 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 set-up in a compact housing with up to four parallel detection channels
  • Specialized high-end major dichroics with enhanced stability
  • All optical elements easily accessible, adjustable, and exchangeable
  • CCD camera for beam diagnostics and photodiode for relative power measurements
  • Variable beam-splitting units and exit ports to connect external devices
Detectors
  • Single Photon Avalanche Diodes
  • Hybrid-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 six channels
Software
  • SymPhoTime 64

Single Molecule Imaging

Detecting the emission of single molecules is important in biochemistry, drug development, and fundamental research. Single molecule sensitive systems aim at minimizing the number of optical elements to maximize light throughput, which is why piezo scanning systems were commonly used. With the FLIMbee add-on, the MicroTime 200 retains its outstanding single molecule sensitivity, but now with a much higher scanning speed. The images shown in this example were obtained from single ATTO 655 molecules bound to a glass coverslip, imaged under STED and confocal conditions with polarization- and time-resolved data acquisition. By using Pulsed Interleaved Excitation (PIE), STED and confocal data can be acquired quasi simultaneously, making the analysis of blinking and bleaching of single molecules straightforward.

FLIM-STED

Super-resolution STED imaging is now a well established tool in molecular biology. Single molecules, cells, and tissue samples can be investigated using a wide variety of parameters, like emission color, polarisation, and fluorescence lifetime. With the MicroTime 200 STED, super-resolution FLIM images can be easily recorded while the high scan speed of the FLIMbee helps reducing phototoxicity and bleaching during STED imaging. The example shows an image of the microtubule network in an adherent cell. The higher resolution of STED allows studying the interaction within the network in much more detail.

Simultaneous Measurement of Topological and Fluorescence Parameters

In this example, both fluorescence and topological information of dye-labeled beads were recorded simultaneously from the same sample area. Fluorescence parameters like emission intensity and lifetime shown in the top figure were obtained with STED super-resolution (<50 nm) on a MicroTime 200 STED. A JPK NanoWizard 3 atomic force microscope (AFM) was interfaced to the time-resolved fluorescence microscope, which allowed recording a height map from the same sample are (as shown in the lower picture). The easy-to-use combination of fluorescence lifetime imaging (FLIM) with topological information from AFM offers exciting avenues for investigating the structure-fluorescence interactions in e.g., biomaterials.

Multicolor Nanorulers for Super-resolution Microscopy

Nanorulers labeled with two different dyes are available to evaluate the resolution of diffraction-limit-breaking microscopes. This example shows GATTAquant STED 140RYR nanorulers, labeled with two red (ends) and a yellow dye (middle part). The distance between a red and yellow marker is 70 nm, while two red dyes on the same nanoruler are separated by a distance of 140 nm.

PIE-STED Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) is a well established method for studying the diffusion behavior of molecules in solution or membranes. Under confocal conditions, the minimal size of the FCS observation volume is restricted by the diffraction limit. However, this limit can be circumvented by employing Stimulated Emission Depletion (STED) and the observation volume can be shrunk in a gradual manner by increasing the STED laser intensity. At low STED laser powers, a small increase in intensity leads to a rapid shrinking of the lateral observation area diameter. Going from 0 to 50 mW reduces the spot diameter from 250 to less than 100 nm, depending on the fluorophore used. At high STED powers, however, increases in intensity will result in slower shrinking until a diameter below 50 nm can be reached. Shrinking the detection volume helps in overcoming averaging issues in long transit paths and also enables determining the type of hindered diffusion behavior of fluorophores in lipid membranes. Furthermore, using a fully Pulsed Interleaved Excitation (PIE) illumination scheme for excitation and STED lasers allows collecting FCS data under both confocal and STED conditions quasi-simultaneously. PIE-STED-FCS allows for a straightforward check whether the STED laser has an influence on the investigated diffusion dynamics.

FLIM-STED pattern matching for dual species separation

Two fluorescent species can be distinguished by pulsed interleaved excititation (PIE) with two different diode lasers at 640 and 660 nm. Fluorescence is detected in two spectral bands. Fluorescence pattern matching was used to identify the contributions of each fluorophore. The example shows part of a U2OS cell labeled for tubulin with Abberior STAR 635p (green) and giantin with Atto647N (red) utilizing a single STED laser wavelength.

Evaluating resolution with DNA origami

Specifically labeled DNA origami is now commonly used to evaluate the resolution of diffraction-limit-breaking microscopes. Here, origami with two rows of 11 dye molecules with a distance of 71 nm was used. Image size: 4 µm x 4µm.

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