Description

Molecular Imaging: What Happens Inside a Cell?

How much Calcium, Sodium, Magnesium is there? And where is it? What is the pH and how is it distributed over the cell? Are two proteins connected to each other? If so, how many of them are connected in pairs and how many are unconnected? Are these parameters changing over the area of the cell? What is the voltage over the cell membrane? These are questions addressed by molecular imaging. There are 'molecular probes' for almost any conceivable cell parameter. These probes are molecules that change their configuration in dependence of their molecular environment. These changes cause changes in the quantum yield and in the fluorescence lifetime. Another probing mechanism is FRET (Förster Resonance Energy transfer). FRET is sensitive to the distance between a donor and an acceptor. Changes in protein binding and protein configuration change the efficiency of FRET, which, in turn, causes changes in the fluorescence lifetime of the donor. Measure the lifetime changes by FLIM, and you know what happens in the cell, and where exactly it happens. Measuring cell parameters by fluorescence lifetime is more reliable than measuring them by fluorescence intesity: In contrast to the intensity the fluorescence lifetime does not depend on the concentration of the probe, the laser intensity, filter characteristics, and other instrumental parameters.

Molecular imaging by FLIM requires accurate mapping of the fluorescence lifetime in cells and tissues. The FLIM technique should be highly sensitive, deliver absolute values for the fluorescence lifetime at a minimum of recorded photons, and reliably resolve multi-exponential decay profiles. Moreover, a sufficient number of excitation wavelengths should be available to provide flexibility in the choice of probes, and beamsplitters and filters should allow the user to select any reasonable detection wavelength. This exactly is the application the bh Molecular FLIM System has been designed for.

Basic Features:

• Fully motorized sample stage
• 4D Z-stack FLIM Video rate recording ready – Express FLIM^(*)
• Seamless analysis integration including Phasor Plot +, image segmentation by lifetime, FCS analysis
• Area and line scanning modes, as well as true point measurements for correlation measurements.
• Fast laser scanning unit for optimal image acquisition
• Basic detection filter kit, chosen for your experiment

^(*) Depends on choice of time-tagging unit

Options:

• Choice of laser wavelengths
• Choice of fast and efficient Becker & Hickl hybrid single photon detectors
• Incubator with optional environment control for live cell work.

Specifications

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Molecular Imaging by FLIM

Principle

When a molecule is in the excited state it can return to the ground state by emitting a photon, by internally converting the energy into heat, or by exchanging energy with another molecule. Consequently, the time the molecule stays in the excited state - or the fluorescence lifetime - depends on the molecular environment of a fluorophore. Accurate measurement of the fluorescence lifetimes or, more exactly, the fluorescence decay functions in the pixels of a FLIM image can therefore be used to obtain reliable information on biological systems. An inherent advantage of FLIM in this respect is that the fluorescence lifetime, within reasonable limits, does not depend of the concentration of the fluorophore, the laser power, the detector gain, or other experimental or instrumental details. Measurement of molecular parameters by FLIM is therefore more reliable than by intensity measurement.

Requirements to a Molecular FLIM Technique

Molecular Imaging experiments have, of course, to be performed in live cells or live tissue. This implies that the photon rates available from the sample are very limited. High sensitivity and high photon efficiency, i.e. maximum signal-to-noise ratio for a limited number of detected photons, are therefore important. Other requirements are high time resolution, high timing stability, and capability to record and resolve multi-exponential decay profiles. Helpful are also optical sectioning capability, absence of lateral crosstalk, and the capability to record in several wavelength intervals simultaneously. Exactly these requirements are inherent features of the bh TCSPC FLIM technique and the bh TCSPC FLIM systems.

Molecular Parameters Measured by FLIM

In most instances molecular imaging is performed by incubating or transfecting the samples with a fluorophore or several fluorophores that show the desired dependence of the molecular parameter of interest. There is a wide variety of such 'molecular probes', please see W. Becker (ed.), Advanced TCSPC Applications, Springer 2015.

Molecular parameters that can be favourably measured by FLIM are pH, concentrations of Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, Hg⁺⁺, Cl^-, Glucose, membrane potential, local viscosity, local temperature, and a wide variety of other cell parameters for which molecular probes are available.

FRET Imaging

Förster Resonance Energy Transfer, or FRET, is an interaction of two molecules in which the emission band of one molecule overlaps the absorption band of the other. In this case the energy from the first molecule, the donor, can transfer into the second one, the acceptor. FRET can result in an extremely efficient quenching of the donor fluorescence and, consequently, in a considerable decrease of the donor lifetime.

The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance. Therefore it is noticeable only at distances shorter than 10 nm. FRET is used as a tool to investigate protein-protein interaction and protein conformation. In the first case, different proteins are labelled with the donor and the acceptor, and FRET is used as an indicator of the binding between these proteins. In the second case, donor and acceptor are attached to the same protein. The intensity of FRET is then an indicator of the protein conformation. Correct FRET measurement requires double-exponential decay analysis and thus cannot be performed with FLIM systems which do not record the full decay curves, please see 'A Common Mistake in Lifetime-Based FRET Measurement'. Double-exponential FTET measurement is not only free of external calibration, it also delivers the classic FRET efficiency, the FRET efficiency of the interacting donor, the fraction of interacting donor, and the donor-acceptor distance from a single FLIM measurement. Please see 'Double-Exponential FLIM-FRET Approach is Free of Calibration'.