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pE-4000 by CoolLED

14 March 2020 Tags: microscopy light sources fluorescence microscopy

When working with Dr. Leslie Vanderpant from Digital Pixel to get the environmental chamber and the new camera installed he also talked about switching from the metal halide light source that came with our microscope system to a more modern LED light source, an idea that I had in the back of my mind for some time. There are a number of rather obvious advantages, for example the fact that no bulb warm-up time is required, bulb changes are obsolete and that, once fully adjusted, a very even illumination of the entire field of vision can be achieved, which then essentially does not have to be adjusted again. But a LED slight source such as the pE-4000 from CoolLED also can interact directly with the camera, thereby minimising exposure times, as illumination and picture taking can be synchronised. This aspect in particular made this light source very attractive, as we do a lot of time-lapse work.

Again we received some finanical help from Brunel University, which enabled us to equip our Nikon Ti-U system with a pE-4000 by CoolLED, which was bought from and installed by Digital Pixel. All things considered the installation went rather smoothly. However, one issue did arise: our existing version of the Nikon NIS Elements Br software, 4.3, was supposed to fully support the pE-4000, but it did not. It allowed basic functionality, and of course the pE-4000 can be operated manually via the existing buttons, but this scenario is not ideal for more complex illuminations with multiple wave lengths.

The issue was in the end solved directly via Nikon, as they kindly agreed to provide a free minor upgrade to version 4.5. This allowed full integration of the light source.

Overall the light source is a big improvement over the old metal halide system. One of the most helpful parts is the fact that the intensity can be smoothly regulated from 1 to 100%. The various fluorescent proteins all react differently to illumination. For some of them a longer exposure time at a reduced intensity yields much-improved time-lapse experiments. The use of neutral density filters with the conventional metal halide system is of course possible and works fine in principle, but the intensity regulation allows for a much more systematic analysis of the right conditions, which I found tremendously helpful.

Responsiveness is overall slightly faster than with the old metal halide system, allowing for quicker exposures with the right duration, which does make a difference over the duration of a time-lapse, so all these points are clear improvements. And this is the case even without the direct interaction between camera and light source. A discussion with CoolLED revealed that such an interaction is mostly beneficial if whole clusters of images are taken in a very short time frame, a requirement that we simply do not have at the moment. Interactivity would be even faster, but the system would be considerably more expensive and the implementation technically more challenging. In retrospect I still believe this was the right decision to make, as the responsiveness of the system is very good and better than the old illumination system. A mild downside of the switch from metal halide to the pE-4000 is that a switch between some wavelength groupings will require a motorised filter change. These are short, admittedly, but longer than with metal halide, which does not have the same requirement. However, for most of the combinations that we use this restriction does not apply. Nevertheless, in order to switch between experiments with different fluorophores we found that the adjustment of for example our regular brightfield exposure needs to be adjusted, a step not necessary with the old system. It might be that this is down to the interaction with Nikon's NIS Elements Br software, but nevertheless it requires the occasional fiddle with the settings.

Overall the change to the LED system is another step towards a modern and very powerful system, even though it only uses conventional wide-field and relatively long exposure times. In fact, following a conversation with Nikon, it is one of the best-equipped Ti-U systems they have seen in some time. Any additional major upgrades would very likely require an upgrade of the body of the microscope to allow aspects such X-Y-motorisation of the stage etc. All things considered I would definitely recommend an upgrade like this, but it might be a good idea, depending on the specific user applications, to try a unit by doing actual experimental work if at all possible.