The MT 200 picoquant system provides single molecule sensitivity and high temporal resolution equipped with variable repletion rate from 1kHz to 80MHz and confocal volume is about 1 fL (femtoliter) or less, such that, at 1 (nM) nanomolar concentration of a diffusing sample can be easily monitored, hence, combined with outstanding capabilities and ease-of-use.

Principal of measurements based on fluorescence intensity fluctuation of probe under confocal volume with ability to detect and follow single molecules dynamics, hence widely applicable for depth information underlying heterogeneity of the systems.

It allows numerous measurement includes T2 (fast dynamics in ns time scale such as single molecule emitter) and T3 (Time tagged time resolved) modes with single excitation and dual excitation established as a versatile tool for current research and analysis.

Sample preparation:

Only immobilized/viscous sample for FLIM imaging: sample should be prepared on standard glass slide and sandwiched with cover slip on one side. One sample only mount on centre of glass slide.

Applications

1. Single molecule FCS and FERT dynamics:

FCS measures translation diffusion dynamics captured from translational diffusion in and out of the confocal volume. It is very relevant to get information about aggregation, protein folding unfolding or conformational dynamics of molecule; dynamics of enzyme ligands binding or complex formation between molecules. Single molecule FRET dynamics using single excitation is a relative method to demonstrate information of donor-acceptor interaction. The analyses are based on control where only donor present and then donor in presence of acceptor. Single excitation FRET infers how donor molecule dynamics vary in absence and presence of acceptor molecule.

2. PIE FCCS, FLCCS, FRET dynamics:

interaction between two non-identical molecules labelled with two different fluorophores excited both simultaneously to measure actual dynamics information either intermolecular or intermolecular (two fluorophores labelled at different poles or position of a molecule). Additionally cross correlation is a method to identify Co-localization between two molecules. It infers about strong overlap or superimposed interaction and complexation between two fluorophores tagged molecules.

3. FLCS/FLCCS: Fluorescence life time correlation or Cross correlation

The fusion of Time-Correlated Single Photon Counting and Fluorescence Correlation Spectroscopy, called Fluorescence Lifetime Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions. FLCS is of particular advantage when using spectrally inseperable fluorophores that differ in their lifetime. Fluorescence lifetime Cross-Correlation Spectroscopy (FLCCS) offers elimination of spectral cross talk and background. It also offers a way around detector after pulsing artifacts.

4. FLIM/FLIM-FRET/PIE FLIM-FRET

Fluorescence Lifetime Imaging (FLIM) produces an image based on the differences in the excited state decay rate from a fluorescent sample. Thus, FLIM is a fluorescence imaging technique where the contrast is based on the lifetime of individual fluorophores rather than their emission spectra. The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon. As the fluorescence lifetime does not depend on concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity it is more robust than intensity based methods. At the same time, the fluorescence lifetime depends on a wealth of environmental parameters such as pH, ion or oxygen concentration, molecular binding or the proximity of energy acceptors making it the technique of choice for functional imaging of many kinds.