Leica SP5 spectral scanning confocal microscope

The success of fluorescence microscopy is reliant on using a fluorophore that attaches to the specific site of interest within the material being studied. When there are two or more sites of interest it is not always possible to choose fluorophores that emit light at wavelengths that are significantly different so as to be readily distinguished and quantified. The problem of distinguishing fluorophores that emit light at similar wavelengths can be overcome by the use of a confocal microscope with the ability to tune the detection of emitted light to specific wavelengths or a specific range of wavelengths. The Leica SP5 is an advanced scanned-laser confocal microscope that has five, high-sensitivity detectors for which the range of detected wavelengths can be accurately defined. This allows the emissions from fluorophores to be readily distinguished.

The Leica SP5 confocal microscope at Adelaide Microscopy has seven, laser-generated, excitation wavelengths: 405 nm (pulsed), 458 nm, 478 nm, 488 nm, 496 nm, 514 nm, 561 nm and 633 nm. The intensity of all excitation lines can be independently adjusted. The Leica SP5 offers a number of advanced features such as a continuously variable confocal aperture and adjustable zoom. The movement of the microscope stage is computer controlled and has attachments that allow features including sample heating and control of the sample environmental. Additionally the Leica SP5 can be used in transmitted light mode to obtain high quality phase and DIC images.

The software supplied with the Leica SP5 can be used to process z-stacks to produce projection images and quantify co-localisation of fluorophores. When combined with the 3D analysis software at Adelaide Microscopy (AMIRA and analySIS) it becomes possible to visualise and quantify the full structure of a sample.

The high-sensitivity and flexibility of the Leica SP5 allows specialised techniques such as fluorescence recovery after photo-bleaching (FRAP) and fluorescence resonance energy transfer (FRET) to be utilised. For FRAP the sample is imaged in its initial state and then a region of sample is irradiated at a high intensity causing the fluorophore in the irradiated region to photo-bleach and thereby no longer fluoresce. The fluorescent light emission from the irradiated region is quantified subsequent to intense irradiation and any increase in light emission with time indicates a diffusion of photo-bleached fluorophore out of the irradiated region and a diffusion of unbleached fluorophore into the irradiated region. This technique can therefore be used to study effects such as diffusion through cell membranes and the diffusion of lipids and proteins within cells.

FRET uses two fluorophores; a donor fluorophore and an acceptor. When a donor fluorophore is excited, rather than emitting fluoresced light, it can pass energy to the acceptor fluorophore in a non-radiative process (e.g. dipole-dipole interactions) over relatively large distances (approximately 10nm). Importantly, the acceptor fluorophore must not be fluoresced by the radiation used to excite the donor and the acceptor fluorophore must emit light at a wavelength distinct from the wavelength of the light emitted by the donor. A commonly used donor-acceptor pair is CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein). If the acceptor is attached to one structure of interest (e.g. a specific protein) and the donor is attached to another structure of interest (e.g. a second specific protein or DNA) then the interactions between the structures (i.e. the spatial proximity of the structures) can be quantified by measuring the relative amounts of fluorescence from the donor and acceptor fluorophores. Clearly, the interactions can be studied and quantified over time and in response to various external factors such as temperature and chemical environment.

The pulsed 405nm laser on the Leica SP5 at Adelaide Microscopy allows the system to be used for fluorescence lifetime imaging (FLIM). When a fluorophore is excited by light, the emission of fluorescent radiation is not instantaneous but rather the fluorophore remains in a transition (excited) state for a short length of time (of the order of a few tens of nano-seconds). Thus after excitation by a pulse of radiation the emitted intensity from a sample containing a fluorophore will decay exponentially over time. The lifetime of this transition state is dependent on both the fluorophore and the environment surrounding the fluorophore. One application of FLIM is to distinguish between fluorophores that emit at very similar wavelengths but have different fluorescent lifetimes. Similarly, FLIM can be used to remove the auto-fluorescence component from a fluorescent signal. As fluorescence lifetime is dependant on the environment surrounding the fluorophore, by fitting the intensity decay after irradiation to exponentials it is possible to determine if the fluorophore exists in a single environment or a number of different environments.