Time-resolved Confocal Fluorescence Microscope with Unique Single Molecule Sensitivity
- Complete confocal system with laser combining unit, inverted microscope body, and multichannel detection unit
- Turn-key diode lasers for multicolor excitation from 375 to 900 nm
- Up to 6 truly parallel detection channels using application-optimized detection with SPADs, PMTs or Hybrid-PMTs
- Time-Correlated-Single Photon Counting (TCSPC) and TTTR mode for investigation of fast dynamics in FCS and FLIM
- Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning
- Two optional exit ports for additional hardware, e.g. spectrographs
- Advanced easy-to-use data acquisition, analysis and visualisation software SymPhoTime 64
- Unique upgrades to 2focus FCS, simultaneous AFM/FLIM, deep UV excitation
- STED add-on for super-resolution imaging available
- NEW: FLIMbee galvo scanner add-on with outstanding flexibility in scanning speed and excellent spatial precision
- Add-on options
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.
Use of the standard piezo scanner is recommended for applications requiring light from the UV (255 to 400 nm) and NIR (1100 to 1400 nm) spectral regions or when pixel sizes smaller than 10 nm are desired.
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.
Detailed specifications are available for download as a PDF document.
Galvo scanner FLIMbee:
|Main optical unit|
All Information given here is reliable to our best knowledge. However, no responsibility is assumed for possible inaccuracies or omissions. Specifications and external appearances are subject to change without notice.
Super-resolution imaging via STED
The MicroTime 200 STED add-on uses the principle of stimulated emission based depletion (STED) to achive spatial resolutions below 50 nm. After exciting fluorophores in the laser focus, a second, donut-shaped focus of a laser with longer wavelength is used to actively de-excite the molecules in the periphery via stimulated emission. In the MicroTime 200 STED, the donut is created using a so-called EASYDOnut phase plate.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.
Combination with optical tweezers
As a joint development, PicoQuant and Ionovation combine time-resolved microscopy and optical tweezers in a single system. Both instruments retain their individual functionalities and can also be used together, thus enabling customers to carry out time-resolved fluorescence microscopy (e.g. fluorescence lifetime imaging) on trapped objects such as cells.
Ionovation’s PicoTweezers system is a compact, robust trapping and force measurement device based on the “optical tweezers” concept which is equipped with real-time video detection. In contrast to common optical tweezers systems, PicoTweezers has no spatial restrictions and is compatible with all sample carriers. The system is well suited for
molecular interaction studies, force spectroscopy, cell trapping, rheology and many
Combination with AFMs
The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single molecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit. For the first time, this development grants access to the effects of precise physical impact on biomaterials like cells and its effects simultaneously measured by fluorescence parameters. The combination of the MicroTime 200 with an AFM has currently been realized for three different AFMs:
The UV upgrade of the MicroTime 200 grants direct access to the native fluorescence of biomolecules originating from naturally occurring chromophoric groups such as tyrosine or tryptophane. The UV upgrade uses a laser emitting at 266 nm along with UV sensitive PMT detectors and quartz optics. The measurement of fluorescence lifetimes and FLIM is feasible with this upgrade. Detection of fluorescence in the visible spectrum is still granted.
- UV upgrade for the MicroTime 200 (Presentation as PDF)
Dual-focus FCS (2fFCS)
In dual-focus FCS (2fFCS) two orthogonally polarized laser diodes, operated in a pulsed interleaved excitation (PIE) scheme are used to generate a robust dual–foci geometry. The robust interfocal distance provides an intrinsic length scale to study diffusion in solution, which allows to overcome various uncertainties and limitations of single-focus FCS that arise because the analysis relies on knowledge of the size and shape of the confocal volume. This enables 2fFCS to dramatically improve the accuracy of measuring absolute diffusion coefficients.
Adding a spectrograph (SR-163 from Andor) along with a single molecule sensitive CCD camera to the MicroTime 200 opens up new prospects in the investigations of single molecules. The spectrograph is attached via a multimode fibre to an exit port of the main optical unit of the MicroTime 200. Inside the main optical unit, a suitable optical element is used to direct a defined part of the emitted light towards the exit port. An easy exchange of the optical element (100% mirror, 50/50 beamsplitter, ...) allows for the customization of the experimental conditions. Depending on the context of the experiment, a defined part of the fluorescence is utilized to acquire the spectral information. Intensity fluctuations, fluorescence decay properties and spectral data of a single molecule can be obtained simultaneously. Both, the observation of spectral changes as a function of time, and the comparison of spectra from different molecules are possible with this combination.
The main optical unit of the MicroTime 200 can be equipped with an additional set of opto-mechanical and electrical components for coupling of an external laser. This includes bringing the laser beam to the correct height, expanding the beam for focused excitation, and providing a synchronization signal that can be processed by the TCSPC electronics. The optics will be pre-aligned by PicoQuant, and re-alignment can be performed by the user, should it be necessary. Inside the TPE unit, the laser beam is fully enclosed to ensure the user's safety.
Applications of two-photon excitation include deep-tissue imaging and time-resolved photo-luminescence in materials science.
Unilamellar vesicles can be produced in different sizes ranging from giant (GUVs), to large (LUVs) and even down to small (SUVs). They are a very powerful model for investigating, e.g., the formation of microdomains or the organization of proteins in membranes. The flexibility in the composition of a vesicle membrane allows introducing specifically labeled lipids, thus increasing their importance in biophysical studies using highly sensitive lifetime measurement techniques. Both the fast processes occurring within the vesicle membranes and their high mobility require very fast lifetime imaging to prevent information loss. Thanks to the FLIMbee scanner add-on and rapidFLIM approach, multiple FLIM images per second can be acquired, making it possible to accurately observe and analyze both unilamellar vesicles as well as processes occurring in their membranes. The series of FLIM images of Giant Unilamellar Vesicles (GUVs) shown above reveals information on vesicle movement and the fluorescence lifetime of dyes embedded in the membranes. The vesicle membranes contain the dyes rhodamine and NBD. Slight variations in fluorescence lifetime can be observed since the dye ratio is not constant over the surface. The image sequence was acquired with a frame rate of 6 images per second.
- GUVs with NBD and Rhodamin labeled lipids (no phase separation): DOPC + 0.5 mol % Palmitoyl-C6-NBD-PC + 0.5 mol % N-Rhd-DOPE
- NBD fused to Palmitoyl-C6-NBD-PC (phosphatidyl choline)
- Rhodamine fused to N-Rhd-DOPE (Di-oleyl-phosphatidyl-ethanolamin)
- MicroTime 200
- Galvo scanner: FLIMbee
- TCSPC unit: TimeHarp 260 NANO
- Excitation: 485 nm, 40 MHz with LDH-D-C-485
- Long pass filter: 488 nm
- 75 x 75 µm, 300 x 300 pixel, 1 µs/pixel
- 300 frames with 5.6 fps
- Data analysis: SymPhoTime 64
GUV preparation by Ivan Haralampiev, Lab of Molecular Biophysics, Humboldt Universität zu Berlin
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.
- MicroTime 200
- Galvo scanner: FLIMbee
- TCSPC unit: TimeHarp 260 PICO
- Excitation: 640 nm with LDH-D-C-640
- Data analysis: SymPhoTime 64
Monitoring Chloride Concentration in Insect Organs
Example for exploiting the fluorophore lifetime dependence on the local environment
Cockroach glands are a well-established model system for studying epithelial ion transport. Salivary glands of the american cockroach were stained with the Cl--sensitive dye MQAE. Thus after calibration, Cl--concentration can be determined as well as the response time to various stimuli like different buffer concentrations wherein the salivary glands are embedded. Recording FLIM images with physiological Cl--concentration and reduced Cl--concentration allows the mapping of Cl--concentration to measured fluorescence lifetime and thereby enabling the mapping of the Cl--concentration throughout the salivary system. The two FLIM images show the fluorescence lifetime in the salivary gland at physiological (left) and low (right) Cl- concentrations. The central salivary reservoir displays a significant change in chloride concentration (blue → red). The lifetime difference in the surrounding salivary glands is much less pronounced (green). This can also be seen in the fluorescence lifetime distribution histogram.
- Excitation wavelength 780 nm (TPE), SpectraPhysics MaiTai
- Detection: 420 nm -460 nm
- Analysis: SymPhoTime
Courtesy of Carsten Hille, Carsten Dosche, Potsdam University
Multistep FRET analysis of a single photonic wire
Multistep FRET in a photonic wire was detected by Time-correlated Single Photon Counting (TCSPC). To generate a photonic wire, rigid DNA was stained with the five dyes RHG, TMR, ATTO590, LCR and ATTO680, spaced each by approx. 3.4 nm. The immobilized multichromophoric molecules were excited at 470 nm and, consequently, undergo step by step unidirectional FRET. The emission was detected in four spectrally separated channels (Fig. B-C). The photons can easily be contributed to the four spectrally different subpopulations which enables to calculate the FRET efficiency step by step. For example, the photonic wire marked with 4 in Fig. A undergoes FRET up to the last Atto680 dye but also shows some leakage for all single FRET steps. In contrast, a second photonic wire (marked with 1 in Fig. A) shows FRET only up the second dye.
After spectral splitting, the macroscopic time was used to monitor temporal intensity fluctuations (see relative fluorescence intensity). Due to the non trivial emission patterns of the involved dyes (spectral bleedthrogh of each dye into several detection channels) it is diffucult to determine the number of actually emitting dyes just from the intensity based information. However, every dye shows one dominant lifetime in a first approximation. Thus, by taking into account also the microscopic time seen as changes of the TCSPC histogram (fluorescence decay in the figure), it is possible to extract the number of involved emitters from the fluorescence lifetime decay. In this way, the simultaneous acquisition of fluorescence intensity and lifetime data allows to explain the complicated FRET mechanism in this multichromophoric system. Especially, nonfluorescing relaxation pathways can be investigated, because the lifetime senses this as a quenching of the radiative channels.
In collaboration with M. Heilemann, P. Tinnefeld, M. Sauer, University of Bielefeld, Germany
Quasi-multichannel FCS with one detector
FLCS can be used as quasi-multichannel detection scheme even if only one laser wavelength and one detector is used. This can be understood as a lifetime analogy of a multicolor FCS measurement, with several inherent advantages.
It separates the autocorrelation curves (ACF) not by discriminating spectrally but based on their fluorescence decay characteristics. FLCS separates different signal components (e.g. emitters) quantitatively, because it uses all photons. It is possible to separate even several contributions, provided that their decay patterns are sufficiently different. In general, no assumptions on the functional form of the decay patterns are involved.
Owing to the separation principle based on TCSPC decay behaviour, distinct ACFs of two compounds can be obtained even if they have equal diffusion times. FLCS works equally well for emitters with completely overlapping fluorescence spectra.
No assumptions are made on the resulting ACFs. One can continue the analysis with the usual fitting of standard FCS models.
Once the intensity contributions are separated, even cross-correlation is straightforward. Note that the signal containing the various contributions is recorded by a single detector, hence the separately calculated ACFs correspond to the same detection volume, which can be interpreted as two (or more) perfectly overlapped excitation/detection volumes.
Here, as a proof of principle experiment the individual autocorrelation curves of a 50:50 mixture of Atto655 and Cy5 are retrieved from a single detector measurement. In total four different components contribute to the signal:
- Fluorescence from Cy5
- Fluorescence from Atto655
- Scattering of the excitation light
- Afterpulsing, dark counts, residual room light
The TCSPC decays of all four contributions are recorded in a control experiment. Fig. (a) displays the correlation curves and their respective fits of the pure Cy5 and ATTO-655 stock solutions. They are used to extract the filter curves necessary to separately calculate the individual FCS curves of the four contributions to the signal of the mixture measurement (only the fluorescence contributions are shown here).Fig. (b) shows the correlation curve of a 1:1 mixture of the two stock solutions of Cy5 and ATTO-655 together with a fit of a single component diffusion model including a triplet term. Note that this model, although false, is able to reproduce the correlation curve of the mixture. The decay curve decomposition and FLCS filters for a 1:1 Cy5 and ATTO-655 mixture is shown in Fig. (c) containing scaled patterns of the four signal components (ATTO-655: red, Cy5: blue, light scattering: green, background plus afterpulsing: grey) and the TCSPC histogram of the measurement (black). The scaling factors of the normalized patterns represent their respective contributions to the measured TCSPC curve (black line): 48.5% Cy5, 48.5% ATTO-655, 0.8% Scattering, 2.2% background and afterpulsing. Filter curves for these signal components are displayed in (d). The residuals indicate that the four chosen patterns are able to reconstruct the measured histogram of (c). By using the filter curves depicted in (d), FLCS is able to separate correlation curves of the same mixture (Fig. f). Apart from the doubled correlation amplitudes due to the halved specific concentrations FLCS yields almost identical correlation curves as for the pure stock solutions shown in (a).
- Excitation: 640 nm
- Detection: 655 nm -725nm
- Analysis: SymPhoTime
Courtesy: S. Rüttinger, Physikalisch-Technische Bundesanstalt Berlin, Germany
Nitrogen Vacancy (NV) Centers in Diamond
Luminescent single-nanometer-sized diamond-like carbon particles have received considerable attention in biophysics, material science, nano-medicine, and photonics. Partly this is related to their extraordinary material properties, such as chemical inertness, biocompatibility, and hardness, and also partly due to their exceptional photo stability. All of these applications desire the availability of sub-10 nm particles that show bright and stable photoluminescence. However, it was questionable if very small crystals in the range below 10 nm would still be photostable. To demostrate that a FLIM image was taken that proved the stability since no distortions in the individual crystals due to photobleaching could be seen. The anitbunching trace also shows that indeed there is only a single fluorescent emitter in the nanocrystal investigated.
- Excitation: 532 nm
- Emission: 600-800 nm
- image size: 13 x 13 µm (512 x 512 pixels)
- image: time per pixel: 4.00 ms, total image: 45 min
- antibunching point measurement: 120 s
Sample courtesy of Fedor Jelezko and Jörg Wrachtrup, University of Stuttgart, Germany
The MicroTime 200 is a time-resolved confocal microscope with single molecule sensitivity. Hence, numerous applications are possible with this instrument, including:
- 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
The following documents are available for download:
- Brochure about the MicroTime 200
- Specifications of the MicroTime 200
- MicroTime 200 - UV Upgrade
- Brochure about the FLIMbee galvo scanner
- Poster: Quick Reference for confocal time-resolved microscopy (FLIM, FRET, FCS)
- Practical Manual for Fluorescence Microscopy Techniques
Presentations (as PDF files)
Related technical and application notes:
- Technical note: Spectrograph Add-on for the MicroTime 200
- Technical note: Combination of MicroTime 200 with Asylum AFM
- Technical note: Combination of MicroTime 200 with Bruker AFM
- Technical note: Combination of MicroTime 200 with JPK AFM
- Technical note: Sample temperature controller for the MicroTime 200
- Technical note: Time-Correlated Single Photon Counting (TCSPC)
- Technical note: Phosphorescence Lifetime Imaging Microscopy (PLIM) Measurements. Practical Aspects
- Application note: Quantitative Fluorescence Correlation Spectroscopy
- Application note: Time-gated Fluorescence Correlation Spectoscopy
- Application note: Absolute diffusion coefficients
- Application note: Two-Focus Fluorescence Correlation Spectroscopy (2fFCS)
- Application note: Fluorescence Lifetime Correlation Spectroscopy (FLCS)
- Application note: Fluorescence Lifetime Imaging (FLIM) in Confocal Microscopy
- Application note: Imaging of molecular distances using FLIM-FRET
- Application note: FRET analysis with Pulsed-Interleaved Excitation (PIE)
- Application note: Two-photon excitation using the MicroTime 200
- Technical note: FLIM: Scanning Speed vs. Excitation Rate
Latest 10 publications referencing MicroTime 200, SymPhoTime
The following list is an extract of 10 recent publications from our bibliography that either bear reference or are releated to this product in some way. Do you miss your publication? If yes, we will be happy to include it in our bibliography. Please send an e-mail to email@example.com containing the appropriate citation. Thank you very much in advance for your kind co-operation.