State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single
molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as
well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it
is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence
resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics.
The main quantities such performance standard must address are:
• Spatial resolution
• Sensitivity
• Fluorescence lifetime
To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the
corresponding standards have to be ready made, easy to handle and to store. The measurement procedures
necessary should be available on as many different setups as possible and the procedures involved in their
deployment should be as easy as possible.
To this end, we developed two performance standards to accomplish the mentioned goals:
• Resolution reference
• Combined molecular brightness and fluorescence lifetime reference
The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single
molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a
liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are
sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to
ascertain durability and road capabilities.
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 performed quantitative tests in order to compare the practical limits of FCS and FLCS. Unlike
conventional FCS, FLCS yields precise and correct concentration values from as low as picomolar
to micromolar concentrations. We discuss some of the inherent technical limitations of FCS and
demonstrate that they are easily overcome by FLCS employing the simplest confocal detection
scheme.
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.
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.
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.
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