Confocal Imaging Techniques
1. Scan modes:
xy, xz, xyz, xyt, xzt, xyzt, xyl, xzl, xyzl, xyzlt
a. 2D recordings:
xy acquires image from single optical plane.
xz collects "real time" axial images.
xt allows fast repeated detection of a single line within the optical plane.
b. 3D recordings:
xyt performs repeated recording of an optical plane over time.
xyz scans several sections at different optimized positions of the optical axis.
xyl or xzl, so called lambda scans, allow the acquisition of emission spectra in the axial or lateral axis.
c. 4D recordings:
xyzt allows the repeated acquisition of complete volumes, so called 3D-time lapse.
xylt, xzl t is used for acquisition of of combined lambda and time series.
2. Regions of interest scan (ROI Scan)
The illumination can be controlled separately for any number of objects within a field of view without illuminated the rest of the field. Besides the background, individual regions of the specimen can be excited with different wavelengths and intensities. This function is especially suited to techniques which require intentional photo-bleaching.
Spot bleaching can also be achieved by 'parking' the laser over a spot to bleach the fluorochrome over the smallest possible area. The size of the induced damage depends on the laser intensity and duration of radiation.
3. Sequential and simultaneous image acquisition.
Although simultaneous excitation with image acquisition for double/triple staining is by far more desirable, more often than not there is bleed through from one channel to next. In some cases fine tuning of the laser power/gain settings can over come this problem but when all else falls the only remedy is Sequential scanning. In this mode each separate fluorophore is optimised for laser power, gain and offset and these settings are saved to perform the sequential scan. Because each fluorophore is collected separately there is no risk of obtaining a false positive signal in any channel. Up to four fluorescence channels can be acquired sequentially, whilst five channels can be acquired simultaneously, one being the transmitted light channel.
4. High-end performance of intra- and inter-cell dynamics using complex time lapse.
A specialised time lapse module enables the user to combine different scan modes, whereby external triggering, detection ranges, scan speeds, different image formats and zoom can be integrated into one process. In this bespoke image acquisition modules can be developed to observe live cell processes.
5. Bleaching application:
a. FRAP ~ Fluorescence Recovery After Photo Bleaching.
FRAP is an analytical imaging method that is used to assess a number of biological processes within and between live cells. The three main steps involved in FRAP are; acquisition of image is done before photo bleaching, followed by photo bleaching of an area of interest with a high laser power. The final stage is re-imaging over time under the original laser settings to monitor the recovery of fluorescence. The results are then analysed to obtain information about dynamic behaviour of macromolecules in the cytoplasm or within membrane systems such as Golgi and plasma membranes. The ROI Scan function is well suited to perform FRAP.
b. FRET ~ FLuorescence-Resonance Energy Transfer.
Spatially adjacent fluorescent molecules can exchange energy. A donor fluorophore in an excited state transfers its energy radiation-free (without fluorescence) directly to an absorbing acceptor fluorophore which, in turn, may fluoresce at its own emission wavelength. Using FRET, it is possible to show biological or chemical processes in which the spatial distance between donors and acceptors is less than 10nm. Using appropriately fluorescent tagged macromolecules it is possible to observe, for example, conformational changes of protein-protein interaction, homo- and hetero-dimerisation of receptors etc.
6. FLIM ~ Fluorescence Lifetime Imaging Microscopy.
FLIM is a technique in which the average fluorescence lifetime of a chromophore is measured at every spatially resolvable element of a microscope image. It is the lifetime of the fluorophore signal, rather than its intensity, that is used to create the image in FLIM. The nanosecond excited-state lifetime is independent of probe, concentration or light path length, but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biomedical reactions to be followed at each microscopically resolvable location within the cell. (Philippe I. H. Bastiaens & Anthony Squire)
7. FLIP ~ Fluorescence Loss In Photobleaching.
A technique in fluorescence microscopy which can be used to examine the movement or diffusion of molecules inside cells or membranes. The region of interest is repeatedly bleached using a confocal microscope. The decrease of fluorescent intensity from around the ROI is measured over time, from which can be derived a measure of 2D or 3D motility of the fluorescent molecules.
8. Kaede ~ Photoconversion.
Kaede is a protein that originates from a stony coral. When irradiated by UV light, it undergoes an irreversible photoconversion from green to red fluorescence. After photoconversion the Kaede protein emits a bright and stable red fluorescence, which can last for months without anaerobic conditions. Because of its ability to change colour it is a valuable cell marker. Some of its most useful applications include cell tracking, protein labelling, optical markers for gene expression and visualisation of neurons.
9. Leica Confocal Software: LCS
In addition to the user interface to control all microscope image acquisition functions, Leica software incorporates a number of image processing and quantitative modules to enable the user to further analyse their images. These modules include:
LCS 3D: Visualisation of of volumetric data: Creating projections from any desired viewing angle and creating animated sequences of rotating objects.
LCS Multicolour: Colocalisation analysis of multicoloured samples. Using the function the user can prove the existence of colocalised fluorochromes based on the relationship between high correlation of intensity values and spatially colocalised voxels. The scattergram produced to calculate colocalisation can also be used to detect spectral crosstalk (bleed-through) of signal from one channel to another (see below). The multicolour function is also useful for determining the results of FRET experiments where the colour of donor and acceptor fluorophore's can be separated.
LCS Dye Finder: Is used to correct for spectral crosstalk. Two principle methods are used to achieve this; Adaptive Dye Separation and Spectral Dye Separation.
LCS Physiology: Measurements and analysis of physiological processes and advanced time critical events.
LCS Macro: Visual Basic ® compatible development environment.
LCS Materials: Roughness analysis according to DIN/ISO values.
In addition to these modules there are also a limited set of image analysis algorithms to perform basic image analysis on users data sets. These are particularly useful for despeckling noisy background of edge enhancing for example.