Scanning FCS (sFCS) is a great tool for studying slowly diffusing species as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for simultaneous measurement at multiple locations. The shorter residence times also lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. Here, we show results from sFCS measurements on supported lipid bilayers (SLBs) where fluorescence lifetime information is used to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET).
To overcome limitations of indirect immunofluorescence, a new method is presented to employ the ostensible disadvantage of cross-labeling secondary antibodies by separation of the fluorescence signals via spectral FLIM-FRET. The undesirable cross-labeling among secondary antibodies leads to the generation of new characteristic FRET emission spectra including a change in the donor lifetime. We used a spectrally resolved FLIM detection system with pulse interleaved excitation. The combined spectral FLIM-FRET and pattern-matching analysis forms an excellent tool for use in indirect immunofluorescence that overcomes the undesirable effect of secondary antibody cross-labeling by assigning separate color channels to cross-labeled fluorescent antibodies.
In this research work, we present a novel indirect immunolabeling method for labeling up to three different antigens using just two primary and fluorophore tagged secondary antibodies. We propose a viable solution to overcome the limitations imposed by limited variability in primary and secondary antibody type by leveraging cross-labeling phenomenon. Cross-labeling among two fluorophore conjugated secondary antibodies leads to FRET effects, resulting in changed spectral and fluorescence lifetime properties of donor molecule. To detect and quantify these changes in photophysical properties of immunolabeled species, we developed an eight-channel spectrally resolved fluorescence lifetime imaging (sFLIM) system. We demonstrate the capabilities of our approach in case of multi-targeted immunostaining in A549 cells. Efficient excitation of samples is achieved using two pulsed laser of wavelengths 485 nm and 561 nm operating in alternating/interleaved manner. Acquired multi-dimensional sFLIM data was pre-processed and analysed using state-of-the-art pattern-matching algorithm1 which takes into account the information of fluorescence emission spectra as well as lifetime. The sFLIM detection system together with pattern-matching analysis enables separation of cross-linked labels from single labeled species.
We present here a novel time-domain diffuse optical detection chain consisting of a large area Silicon PhotoMultipliers (SiPM) coupled to a high count-rate timing electronics (TimeHarp 260 PICO) to achieve sustainable count-rates up to 10 Mcps without significant distortions to the distribution of time-of-flight (DTOF). Thanks to the large area of the detector (9 mm2) and the possibility to directly place it in contact with the sample (thus achieving a numerical aperture close to unity), the photon collection efficiency of the proposed detection chain is almost two orders of magnitude higher than traditional fiber-mounted PMT-based systems. This allows the detection also of the few late photons coming from deeper layers at short acquisition times, thus improving the robustness of the detection of localized inhomogeneities. We then demonstrate that, despite the high dark count rate of the detector, it is possible to reliably extract the optical properties of calibrated phantoms, with proper linearity and accuracy. We also explore the capability of the new detection chain for detecting brain activations. This work opens up the possibility of ultimate performance in terms of high signal and photon throughput, with compact, low cost, relatively simple front-end electronics detector coupled to innovative timing electronics, with exciting opportunities to expand it to tomographic applications.
Using Time-Correlated Single Photon Counting (TCSPC) for the purpose of fluorescence lifetime measurements is often limited in speed due to pile-up and dead-time artifacts. This is particularly critical in fast imaging applications. With modern instrumentation this limitation can be lifted by reducing the dead-time of the TCSPC electronics to the absolute minimum imposed by the speed of the electrical detector signals. Another, complementing approach to speedy image acquisition is parallelization by means of simultaneous, time tagged readout of many detector channels. This of course puts high demands on the data throughput of the TCSPC system. Here we present a new integrated design, providing up to 8 independent input channels, an extremely short dead-time, very high time tagging throughput over USB 3.0, and a timing resolution of 80 ps. Apart from design features and benchmark results of the instrument as such, we show application results from spectrally resolved (sFLIM) and high speed confocal fluorescence lifetime imaging (rapidFLIM). We put special focus on life science applications, paving the way to monitor sub-second dynamics in live cell imaging, including lifetime based Förster Resonant Energy Transfer (FLIM-FRET) imaging. We furthermore show how the inevitable pulse-pile-up occurring in the detector signals at high photon flux can be corrected for and how this data acquisition scheme excels in terms of photon collection efficiency in comparison to other approaches.
Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential for imaging dynamic processes in life science. The rapidFLIM approach dramatically reduces acquisition times through a combination of fast beam scanning, hybrid photomultiplier detectors, which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high spatial resolution offered in confocal microscopy. Depending on image size, rapidFLIM enables following dynamic processes like protein interactions, chemical reactions or highly mobile species in live cell imaging with a rate of several frames per second.
The separation of overlapping fluorescence emissions in biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains challenging, especially when strong tissue autofluorescence (AF) overshadows specifically labeled structures. Combining the spectral approach with fluorescence lifetime measurements based on a simultaneous acquisition of both spectral and lifetime parameters could significantly improve the separation quality between multiple labels and tissue AF. We demonstrate this approach in highly autofluorescent human lung tissue, where the fluorescence signals from specific stainings are sometimes weaker than tissue AF. We use dual color Pulsed Interlevaed Excitation (PIE) in conjunction with a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and analyze the data by applying a unique pattern matching technique.
Fluorescence Correlation Spectroscopy (FCS) is an important technique for understanding molecular dynamics and motion on timescales ranging from nanoseconds to seconds. The high concentrations found in some biological systems reduce the significance of FCS, as too many fluorophores are present within a standard confocal volume. Combining FCS with Stimulated Emission Depletion (STED) is one technique to overcome this problem by reducing the observation volume in the sample, reducing the number of molecules within this volume. This technique also allows the observation volume to be tuned to access additional information, such as the parameters which characterize hindered diffusion.
In addition, by utilizing galvoscanners, scanning FCS can increase the number of transits recorded and reduce the residence time of each molecule, increasing the statistics and reducing the effects of photobleaching respectively. This technique is also useful for increasing the number of transits observed for slowly diffusing species at low concentrations, such as observed in membranes.
Measuring multiple species simultaneously saves time and allows interactions between molecules to be investigated, which may not be clear from multiple experiments investigating single species. By exploiting the lifetime information available from a microscope equipped with Time Correlated Single Photon Counting (TCSPC) hardware, pattern matching can be used to separate similar fluorophores allowing up to three superresolved species to be resolved using a single STED laser. This pattern matching analysis can be combined with STED-FCS and scanning FCS to investigate complex diffusion in membranes.
KEYWORDS: Ultrafast phenomena, Data acquisition, Sensors, Photons, Fluorescence lifetime imaging, Signal processing, Single photon, Fluorescence resonance energy transfer, Image processing, Time resolved spectroscopy
Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key systems are available either as stand-alone units or as upgrades for confocal laser scanning microscopes (CLSM). Data acquisition on such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors.
Up to now, TCSPC data acquisition is considered a somewhat slow process as a large number of photons per pixel is required for reliable data analysis, making it difficult to use FLIM for following fast FRET processes, such as signal transduction pathways in cells or fast moving sub-cellular structures. We present here a novel and elegant solution to tackle this challenge.
Our approach, named rapidFLIM, exploits recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher detection count rates. Thanks to these improved components, it is possible to achieve much better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical resolution. FLIM imaging can now be performed with up to several frames per second making it possible to study fast processes such as protein interactions involved in endosome trafficking.
Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.
The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM)
allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and
spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel
and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect
to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and
degree of binding.
Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to
condense the inherent information to answer the scientific questions in a straightforward way. Different analysis
techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination
of environmental parameters and distance measurements via FLIM FRET.
In order to reach highest sensitivity in single photon detection, different detector types are investigated and
developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration
of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels.
High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the
USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.
Photon coincidence analysis is nowadays a widely used technique to study fluorescence intensity fluctuations, taking
place on a timescale from seconds down to picoseconds. Photon bursts in the microsecond regime are e.g. used to study
diffusion properties via Fluorescence Correlation Spectroscopy (FCS). Photon bunching in the microsecond regime
allows to study fast conformational changes as well as internal photophysics like singulett-triplet transitions. Interphoton
delay times in the ns regime carry information about the fluorescence lifetime and can also be used to characterise
molecular rotation. Down in the picosecond regime, photon antibunching is used to quantify a small number of emitters
and especially to proof the existence of a single emitting dye molecule.
All of these methods can be carried out with the single molecule sensitive confocal fluorescence microscope MicroTime
200 and are based on time-correlated single photon counting (TCSPC). We developed a generalized approach to store the
individual photon arrival time information with ps accuracy on a timescale up to hours which allows to study all
mentioned phenomena in a single measurement (Full Correlation Analysis). Using the new HydraHarp 400 TCSPC unit
we can now acquire photon information in 4 completely independent detection channels. This paper present the
straightforward experimental concept as well as typical results and recent application examples.
We report about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on
TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as
Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS).
FLCS is a fundamental improvement of standard FCS overcoming many of its inherent limitations. The basic idea of
FLCS is a weighting of the detected photons based on the additional picosecond timing information (TCSPC start-stop
time) when using pulsed laser excitation. 2fFCS goes even further, combining Pulsed Interleaved Excitation
(PIE) with a time-gated FCS analysis. The basic implementation of 2fFCS uses two synchronized but interleaved
pulsed lasers of the same wavelength but of different polarisation to generate two close by excitation foci in a pre-determined
distance acting as a submicron ruler. In this case it it no longer necessary to have prior knowledge about
the size and shape of the confocal volume. Maintaining the information about the photon´s origin, the dual focus
data allows a precise calculation of absolute diffusion coefficients.
Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local
environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal
different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard
laser scanning confocal microscopes towards a time-resolved system, which is based on picosecond pulsed lasers,
fast detectors and sophisticated single photon counting electronics.
We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to
detect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET efficiencies
can be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow to
measure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneously
monitored using two spectrally separated detectors and a router.
A very special feature of the upgrade kit is that it uses an unrestricted data acquisition approach. With this approach,
not only Fluorescence Lifetime Imaging Microscopy (FLIM) with single molecule sensitivity is realized, but the
provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy
(FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Lifetime
Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove
the influence of detector afterpulsing, which is classically done by cross correlation between two detectors.
Single molecule detection methods, in particular those based on fluorescent labels offer the possibility to gain not only
qualitative but also quantitative insight into the function of complex biological systems. Fluorescence Correlation
Spectroscopy (FCS) is one of the favourite techniques to determine concentrations and diffusion constants as well as
molecular brightness in the pico- to nano-Molar concentration range, with broad applications in Biology and Chemistry.
Although FCS in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity
to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results
and restricts FCS to relative measurements mainly. The determination of the confocal volume in situ is difficult because
it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed
in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume
and to obtain the confocal parameters by FCS-curve fitting, a FCS dilution series and confocal bead scanning. The
results are compared in the view of quantitative FCS measurement and analysis.
In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule
microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and
offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In
contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which
allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full
experimental information. This format allows us not only to easily study dependencies between various fluorescence
parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to
improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated
for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a
simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the
arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example,
cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional
multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the
respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take
advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify
and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a
piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing
performance can be significantly increased by using new SPAD detectors which enable, in conjunction with new TCSPC
electronics, an overall IRF width of less than 120 ps maintaining single molecule sensitivity.
Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Förster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.
We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS 1, 2. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).
We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This procedure offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 to 800 nm for excitation which are coupled to the microscope via a single mode fiber. The corresponding emission is guided to a fibre coupled photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC system, via the unique Time-Tagged Time-Resolved (TTTR) data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently create the full fluorescence lifetime image (FLIM). The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows for example to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for advanced Forster Resonance Energy Transfer (FRET) analysis. In this paper, we present some selected results acquired with standard laser scanning microscopes upgraded towards temporal resolution.
The need for cost reduction and flexibility in semiconductor production will result in a wider application of computer based automation systems. With the setup of a new and advanced CMOS semiconductor line in the Fraunhofer Institute for Silicon Technology [ISIT, Itzehoe (D)] a new line information system (LIS) was introduced based on an advanced model for the underlying data structure. This data model was implemented into an ORACLE-RDBMS. A cellworks based system (JOSIS) was used for the integration of the production equipment, communication and automated database bookings and information retrievals. During the ramp up of the production line this new system is used for the fab control. The data model and the cellworks based system integration is explained. This system enables an on-line overview of the work in progress in the fab, lot order history and equipment status and history. Based on this figures improved production and cost monitoring and optimization is possible. First examples of the information gained by this system are presented. The modular set-up of the LIS system will allow easy data exchange with additional software tools like scheduler, different fab control systems like PROMIS and accounting systems like SAP. Modifications necessary for the integration of PROMIS are described.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.