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Precise details of the optical adapter system will vary according to the type of light source and microscope (or cuvette holder in use), but the general principles remain the same. We should not need to say this, but all the lenses in the optical adapter system are silica, and the light guide also has good UV transmission (better than 80% down to 300nm).
The optical adapter always attaches directly to the light source, and connection to the microscope is normally via a liquid light guide (always so for the cuvette holder). As an alternative, it is possible to omit the light guide and instead connect the optical adapter directly to the microscope; this option requires an alternative rear plate on the optical adapter, to which the lens coupling for the microscope's epifluorescence input port is attached. Although the direct optical connection allows more light to reach the microscope, in practice the flexible connection provided by the light guide is much more convenient, and the amount of light reaching the specimen is still high enough to require further attenuation in many cases. This is particularly true when our light source is used, as the ellipsoidal reflector design collects substantially more light than those using a condenser lens.
Our lamphouse is supplied with four removable legs, which holds it, and the optical adapter, at the correct height for the rotor. Correct horizontal alignment is also important, although the design of the system should automatically ensure this. However, if you do wish to check it, you can use an oscilloscope to observe the output from the input amplifier module when a suitable optical signal is present (see testing and trouble-shooting section). The output from the input amplifiers consists of the integrated output for each filter in turn, and is reset to zero between filter signals. Each integral should be sigmoid in shape, i.e. following each reset there should be a flat region at zero output, which subsequently rises and then levels off again to a plateau. Horizontal misalignment of the rotor may cause the integrals to be reset too soon or too late, so either the plateau will not be reached or the signal will start rising again before being reset. If either condition occurs, the signals from individual filters will not be separated properly by the system electronics.
The iris diaphragm in the optical adapter should typically be set to about 10mm diameter. Openings much larger than 10mm are not recommended when the rotor is in use, as they may allow some light to reach the light guide via more than one of the filters in the rotor. This possibility can be checked by viewing the output signal from the input amplifier, as described above, to verify that the optical output signal reaches a stable plateau before it is reset. Use of a much smaller aperture than 10mm will significantly reduce the optical efficiency of the system, but of course this may be desirable under many circumstances.
An additional way of reducing the amount of light reaching the specimen is to fit one or more neutral-density filters in the slots at the front of the optical adapter. Filters, which reduce the light uniformly through the near ultraviolet, must be of quartz or silica rather than normal optical glass, but a much more economical, and robust, alternative is to use metal gauze (supplied free with our light source).
Xenon bulbs produce a significant infrared output, which together with the large amounts of optical energy gives a significant overall heating effect. This is no problem when the rotor is spinning, but if the full output from the light source is sent through the rotor when it is stopped at one filter position for a significant period of time (tens of seconds or longer), the filter may overheat and be damaged. A heat filter can help significantly in this respect, but such filters can also reduce the amount of UV, particularly at wavelengths around 340nm or shorter, so it may not be appropriate to use one under all circumstances. If a heat filter is not used, we recommend use of at least two of our neutral-density gauze filters, which will reduce the overall energy levels by about 75%. These may well be required in any case, in order to avoid excessive photobleaching. For the record, just about any optical system can bleach indo1 in a few minutes, but ours is powerful enough to do the same to fura2 when optimally aligned.
Alternatively, if single-excitation-wavelength measurements are being performed on a regular basis, then the rotor (or just one or more of its filters) can be removed, and a 25mm diameter filter can be installed in the carrier provided. The carrier is located at a point in the optical adapter where the diameter of the light beam is correspondingly larger than at the rotor, where 12.5mm filters are used to obtain a more compact and hence faster filter-changing system. Since the light beam is larger here, its energy density is correspondingly lower, so a higher overall intensity can be sustained in this case. Nevertheless, it is important to realise that the optical filters do not last indefinitely even when used within their recommended operating conditions, although our experience suggests that they should give several years of normal use. However, because of the high overall energy levels achievable with our system, neither our filter suppliers nor we can guarantee this part of the system, so we do please ask you to be careful with them. In particular, all the filters have a preferred orientation, which is with the more reflective surface facing the light source. In this orientation, the majority of the light not transmitted through the filter is reflected rather than absorbed, thereby reducing the energy dissipated within the filter.
The optical adapter also has provision for an electrically-operated shutter, which will have already been fitted if the two items have been ordered together. It is driven by the switcher module, and defaults to the closed state if power is no longer applied to it for any reason. The shutter facility is very useful, since it is inadvisable to keep switching the light source on and off. This shortens the bulb life, and the high voltages required to strike the arc can interfere with other equipment. Whatever light source you use, we strongly recommended that it is switched on BEFORE any other equipment, particularly computers or sensitive amplifiers. Failure to observe this precaution with some light source designs (we name no names here!) can and has damaged such equipment, so do please be careful. Our own light source has been designed specifically to minimise the risk of such damage, and although we appear to have been completely successful in this respect, it nevertheless remains good practice to observe this precaution.
Optimum alignment of the optical system is the single most important aspect of setting up, and it is worth spending time on this process. While we do not recommend random twiddling of the various components, a systematic experimental approach to the alignment will probably give better results than a purely theoretical one would do, although the theoretical settings certainly form the best starting point for any experimentation. There is particular scope for experimentation with regard to the uniformity of the fluorescence illumination of the specimen; for example, by sacrificing uniformity of illumination it may well be possible to increase the intensity of the illumination in the centre of the field (but of course this increases the risk of damaging the specimen by excessive illumination). However, with a powerful light source such as ours, there is unlikely to be any great advantage in exploring such possibilities.
Note: Please observe full safety precautions when setting up or adjusting the light source.