The standard fluorescence widefield microscope described in our last article has one major disadvantage: it collects not only the desired image information from the focal plane, but it also records a large amount of out-of-focus light, leading to a blurred image. In the 1950’s, system developers started thinking about how to get rid of the out-of-focus light and eliminate this unwanted information. The major confocal microscopes currently used in labs and imaging facilities are described in this article.
Confocal Laser Scanning Microscopes – ‘CLSM’ is a technology that uses a laser beam which is focussed by the objective lens into a tiny spot in the focal plane and this is used to illuminate (or ‘excite’) fluorescent marker molecules in the sample. A high-precision mirror unit allows accurate steering (scanning) of the illumination spot across the sample in order to record the light emitted from all points in an image. Photomultiplier tubes (PMTs) at the end of the light path are the detectors used to amplify the signal and to quantify the photon flux emitted (the ‘emission’) from the fluorescent molecules. The most crucial component in a CLSM is the adjustable pinhole in front of the detectors in the back-focal image plane, which blocks out a large proportion of the unwanted out-of-focus light. Therefore, the pinhole of the CLSM generates the ‘confocality’ of the system- most of the image information comes from the same focal plane which significantly improves the axial resolution of the microscope.
Modern CLSMs have been developed as imaging platforms with added advanced functionality. Additional functions include spectroscopic light separation, spectral foot-printing and un-mixing, a powerful method for ratiometric FRET measurements and the removal of autofluorescence. This makes them very flexible and versatile microscopes, in particular if complex mixtures of fluorophores with partially overlapping emission spectra need to be imaged.
Imaging in three dimensions
This technology has the advantage in that it delivers excellent 2-D and 3-D images. The technical lay-out also allows zooming into the sample and thus achieving the accurate setting of the sampling rate for a large range of objective lenses, which is unique to this technology. Sophisticated spectral separation of the emitted light on these systems facilitates the use of up to seven fluorophores in the same sample. The use of scanning mirrors also provides the option to define regions of interest to only scan or manipulate these parts of the sample. This is of particular advantage for special applications that require photobleaching of certain areas e. g. for fluorescence recovery after photobleaching (FRAP). Line scanning is suitable for imaging fast events, making up for the relatively slow 2-D acquisition.
The major disadvantages of point scanning systems are the relatively slow image acquisition and, depending on the laser power setting, the relatively large amount of energy which is put into the sample- this may become a problem in live specimen imaging.
To overcome the problem of the relatively slow scanning process, manufacturers have started to include a second, much faster resonant scanner in addition to the conventional scanner to provide so-called ‘tandem scanners’. This additional resonant scanner ‘swings’ at high speed and thus allows much faster image acquisition of up to 12,000 lines per second. The disadvantage of the resonant scanner becomes obvious; as the pixel dwell time decreases, so does the sensitivity of the system, which can be in part compensated by PMTs with higher sensitivity. However, with weak fluorescent labels, as often encountered when using encoded fluorescent proteins, images can become dim and noisy.
Spinning disk confocal microscope- As for the point scanning system, this technology uses pinholes to exclude out-of-focus light and laser beams for fluorophore excitation. But unlike steering a focussed laser beam across the sample point by point, the spinning disk confocal simultaneously generates multiple beams using a rotating disk which holds a distinct pattern of small lenses and these focus the laser beams into the sample plane. The returning light, emitted from the fluorophores, has to pass through a second, coupled rotating disk that holds a similar array of pinholes to eliminate a large proportion of the out-of-focus light. The rotation of the two disks with the specific arrangement of lenses and pinholes generates a sweeping pattern across the sample, which generates high rates of 2-D or 3-D image acquisition. The emitted light is reflected by a dichroic mirror onto a highly sensitive camera. In multi-channel systems, the emitted light is spectrally split into defined channels, each recorded by a separate camera.
These systems are particularly suited for fast 4-D image acquisition (up to approx. 30 fps) limited mainly by the read-out-speed of the camera.
A disadvantage of these systems is the lack of flexibility- the instruments are usually only optimised for the use of one or two lenses, so great attention should be paid to the configuration of the microscope available to you.
Structured illumination confocal- This is a cost-efficient and user-friendly alternative to a point scanning system for standard applications. A dark grid is projected into the focal plane and blocks out fluorophore excitation in the part of the focal plane covered by the grid. The grid is then shifted by a third of a phase to acquire a second image and this is repeated a third time to get the signal from the last third of the image. The resulting three images are used to subtract the out-of-focus information from the final, calculated confocal image. This very user-friendly, time-saving technology provides 2-D and 3-D images with improved axial resolution, sufficient for most standard applications.
The basic systems which we have looked at are the ‘workhorses’ of light microscopy. These instruments are available in most research institutes and should be accessible wherever you work. If this is not the case, you can check on the BioImaging UK website. This has the all the details of external imaging facilities, the equipment they have and contact information.
We will cover even more advanced light microscopes in future articles, so stay tuned to this Channel!