You can choose up to 12 channels, but we explain the factors to consider when choosing the number of channels.
It can be confusing when it comes to deciding how many channels an OEM microscope light source requires, especially when it is possible to choose anywhere between two and 12 channels. In this blog, we aim to make this process clearer by explaining some of the factors to consider.
Channels vs wavelengths
Firstly, it is important to understand that the terms ‘channel’ and ‘wavelength’ cannot always be used interchangeably. Some light sources feature multiple LEDs (and therefore wavelengths) for a given channel. In fact, in the case of ‘white light sources’, the whole light source features a single channel. Imaging multiple fluorophores on the same channel can be slow, limiting temporal resolution and increasing imaging times. In the case of the Amora, each LED instead has its own channel, and any combination has the potential to be switched on, pulsed or controlled as required at speeds faster than 7 µs.
Unlike traditional lamps, LEDs have discrete spectra which offers benefits in terms of superior signal to noise ratio. Adding more LED channels to a light source increases the number of wavelengths and therefore the number of compatible fluorophores. However, careful attention must be paid to exactly which fluorophores will be required, their absorption spectra and compatible filter sets.
This is because even if the peak of an LED does not match the peak absorption of a fluorophore, it may still be compatible. Not all LEDs are the same spectral width. For example, an Amora with three channels can provide coverage for the most popular fluorophores, with LEDs at UV, blue and green-yellow-red (GYR) wavelengths. The GYR is broad spectrum, and using the example of TRITC and Cy5 as shown in Figure 1A, can achieve a good spectral separation when using excitation filters.
On the other end of the proverbial spectrum is a 12 channel Illumination System. With 12 channels, this means the choice of fluorophore is very unlikely to be limited by the light source. In the same example of TRITC and Cy5, the LED peaks are tailored to the absorption spectra of popular fluorophores (Figure 1B). This provides superior signal to noise ratio, and enables sophisticated control since each LED can be selected and irradiance modulated individually. For light sources with 12 channels, the optimal fluorophore set can be chosen and signal to noise is maximised whichever assay is running (as long as the correct optical filters are in place).
Figure 1: The importance of matching LED spectra and absorption profiles of fluorophores. A: It is possible for a single broad-spectrum GYR LED channel such as that in the CoolLED pE-300 Series or Amora to excite TRITC and Cy5 (LED irradiance is normalised relative to channel of highest irradiance). B: Exciting TRITC and Cy5 with two narrower LEDs at 550 nm and 635 nm enhances signal to noise and enables extra control. Excitation filters based on Chroma 89022 multi-band set; FPbase used to visualise data: Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature Methods. 16, 277–278. doi: 10.1038/s41592-019-0352-8
Future proofing your light source
LEDs have extremely long lifetimes, and so it also makes sense to consider which applications the imaging system could be used for in the coming years. Moreover, new fluorophores are being developed all the time. In particular, there is a shift into the longer regions of the spectrum, since the lower energy of the near-IR and IR regions reduces the risk of phototoxicity and photobleaching, which improves data accuracy during live cell analysis. Taking this into account, we recommend opting for the maximum number of channels your system has the space for.
The number of channels is just one feature that can be tailored to suit individual requirements – take a look at the other options available with Our Technology.