Molecular interactions between 1-10nm can be detected in cell biology by using Time-resolved Förster Resonance Energy Transfer detected by Fluorescence lifetime imaging microscopy (FLIM). Cell Biophysics laboratory (7W 3.11) has two FLIM setups one in the time domain and another in the frequency domain. Expertise and training are provided by the specialised engineer in Cell Biophysics Laboratory. These set-ups are part of the Centre for Therapeutic Innovation (CTI-Bath).
Our high-throughput automated multiple frequency domain FLIM is a lifetime imaging microscope from Lambert Instruments modified to set up an automated multiple frequency high-throughput lifetime imaging microscope. It is an inverted Nikon TI wide-field microscope. The diode modulated laser is used for 488nm excitation. This microscope is also combined with a TIRF (total internal reflection). This set up is ideal for automatic imaging in fixed tissue for determining molecular interactions in in less than 10s. Its main advantage compared to the Two-photon FLIM is its automation and speed of image acquisition.
Relevant References:
Image caption: determination of interactive states of immune check point regulators, programme death ligand1 and programme death receptor (PDL1-PD1) on fixed lung carcinoma. The red regions have high interactive states (measured molecular distance is 5.5nm) and blue regions the ligand and receptor do not interact. Interactions are determined by High-throughput automated multiple frequency domain Fluorescence imaging microscope using amplified coincidence-Förster resonance energy transfer. (Courtesy of Cell Biophysics Laboratory- Centre for Therapeutic Innovation 2023).