The microscope system present within the group is a Nikon Eclipse Ti, inverted microscope base unit with additional components for extended imaging capabilities. The microscope imaging modes available with the system are: brightfield/DIC, fluorescence, confocal, total internal reflection fluorescence (TIRF) and Förster resonance energy transfer (FRET). The microscope is coupled with an Andor Neo sCMOS camera which allows for high speed time lapse imaging (100 fps) and a confocal detector unit (PMT). For the varying applications the microscope system also contains different objectives 20X and 40X standard objectives and a 60X oil immersion objective.
Brightfield/DIC – Standard white light illumination microscopy is a technique that is easily implemented on samples and used in this system for micropillar measurements. In the imaging of transparent samples, such as cells, differential interference contrast (DIC) is introduced into the imaging system. In DIC, polarised light is separated into two mutually coherent parts polarised at 90ᵒ to one another. These pass through the sample and are recombined, variations in the optical path length (sample thickness, refractive index) lead to contrast from constructive or destructive interference.
Fluorescence – Fluorescence imaging of samples is possible in this system through illumination with a mercury lamp. Fluorescence imaging allows specific regions of cells to be imaged and thus differentiated through immunofluorescent staining. Higher resolution can be achieved with fluorescence compared to brightfield due to high signal-to-noise of fluorophores and site specific staining leading to a less pronounced background signal.
Confocal – The microscope system has two colour confocal capabilities to image samples stained with Green or Red fluorescent markers allowing for higher resolution fluorescence imaging to be achieved. In confocal imaging, a laser is focused onto a diffraction limited volume at a specific focal plane on the sample of interest and scanned across the sample. The fluorescent emission from the sample is passed through a pinhole which blocks out of focus light which arises from emission outside of the focal plane of interest thus improving on resolution from other fluorescence imaging techniques. Confocal imaging can be used to construct a 3D image of the sample by altering the focal plane that is imaged at specific intervals throughout the sample and reconstructing this in the imaging software.
TIRF – Total Internal Reflection Fluorescence (TIRF) microscopy is a high resolution imaging technique which can be used with the microscope system to image the sample surface to high precision. An excitation beam travelling at high incidence is used to illuminate the sample which induces a thin electromagnetic field (evanescent field) in a 200 nm region at the coverslip/cell boundary. Emission from fluorophores in this thin optical section allows high resolution to be achieved from high signal-to-noise due to minimal out of focus light contributing to images.
FRET – Förster resonance energy transfer (FRET) microscopy is a technique that can be used in the microscope system to investigate molecular interactions and act as a method to investigate length scales between proteins. FRET is a mechanism describing energy transfer between two fluorophores where a donor in its excited state may transfer energy via nonradiative dipole coupling. The efficiency of the transfer is inversely proportional to the sixth power of the separation distance between the donor and acceptor. Measurement of the FRET efficiency either through the intensity of the acceptor emission or through a ratio of donor and acceptor emission can indirectly reveal the separation distances between specific proteins on length scales that are below the diffraction limit.