The combination between MicroTime 200 and the BioScope Catalyst AFM from Bruker has been realized. Such a combination allows investigation, prediction and control of photophysical processes on the nm-scale, high-resolution imaging or investigation of inter- and intramolecular distances.

The MicroTime 200 time-resolved fluorescence microscope system is a powerful instrument capable of Fluorescence Lifetime Imaging (FLIM) with single molecule detection sensitivity. It contains the complete optics and electronics for recording virtually all aspects of the fluorescence dynamics of microscopic samples or femtoliter volumes. The instrument gains its exceptional sensitivity and flexibility in combination with unprecedented ease-of-use from a unique fusion of miniaturized and highly sophisticated state-of-the-art technologies. For the first time, these technologies enable to run an instrument of comparable complexity and power to be operated in routine work, yet without having to spend more time on instrument maintenance than on original scientific content. The underlying key technologies are the proven Picosecond Diode Lasers and the Time-Correlated Single Photon Counting electronics developed by PicoQuant, complemented by state-of-the-art piezo-scanning technology and optics from industry leaders.

All optics needed to achieve confocal excitation, detection and beam / focus diagnostics are installed together with the detectors in the self-contained main optical unit. The coupling to the inverted microscope body is achieved through the infinity beam port of the IX 71 microscope body. The MicroTime 200 can be equipped with two scanner configurations: Object or objective scanning. Using object scanning, the sample holder is designed either to accommodate 20 × 20 mm² microscope cover slips or microscope slides. Objective scanning allows free access to the sample, e.g. for live cell investigations or applications using a cryostat. Up to four detectors can be mounted into the optical unit. Each detector channel has its dedicated filter holder and a mechanical shutter.

The excitation subsystem consists of a pulsed diode laser driver, laser head(s) and optical components to attenuate and couple the laser's output into a polarization maintaining single mode fibre. The Laser Coupling Unit (LCU) can host up to 5 laser heads. With a special multichannel laser driver, Pulsed Interleaved Excitation (PIE) can be performed.

For data acquisition the outstanding Time-Correlated Single Photon Counting (TCSPC) units HydraHarp 400 or PicoHarp 300 are used. These highly integrated devices provide several measurement modes. One especially powerful mode is of pivotal importance for the realisation of the MicroTime system: in Time-Tagged Time-Resolved (TTTR) measurement mode each photon is recorded individually. Each photon record contains the picosecond timing of the photon relative to the laser pulse and a coarser nanosecond timing with respect to the start of the experiment. This combination allows to perform vastly different measurement tasks based on one fundamental data format, yet without any sacrifice of information available from every detected photon. It also allows to handle all measurement data in a standardized and yet very flexible way. Due to the independent measurement channels of the Time-Correlated Single Photon Counting units, it is also possible to store the absolute arrival time of each detected photon with picosecond resolution. This permits to calculate correlations from picoseconds to seconds or coincidence correlations ("antibunching").

Based on this clean concept of data handling, the operating software "SymPhoTime" of the MicroTime 200 was designed with almost unlimited flexibility for integration of virtually all algorithms and methods for the analysis of fluorescence dynamics users may require. Based on the powerful TTTR data collection, users can perform an unlimited number of analysis steps without losing track of interdependence and origin of their measurement and analysis data. Derived results can be obtained through a vast set of analysis tools, such as intensity time trace, burst analysis, lifetime histogramming, Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Imaging (FLIM) and Förster Resonance Energy Transfer (FRET), to name only a few. All derived data is maintained in the workspace, including a log file keeping track of all measurement and analysis steps. Image data can be processed further or exported to standard formats. A newly developed scripting language interface allows to modify and expand the analysis routines according to the individual needs towards e.g. static anisotropy, multi-parameter burst analysis or FLIM-FRET analysis.

• MFP-3D-BIO from Asylum Research (see technical note)
• BioScope Catalyst from Bruker (see technical note)