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big, the need is so great, that the people who require it are spending large amounts of money and the large amounts of time that it takes to get that answer.”


Multiplexed fluorescence detection: illumination solutions The MERFISH technique requires the use of multiple colours, which means that selecting the right light source is imperative. A laser source such as Lumecor’s CELESTA laser Light Engine is ideally suited for the task, as Johnson explains: “The light source colour that’s used for this technique is typically an array of five lasers in a box. Because in MERFISH it’s the detecting single molecules present in the cell, it’s a very tiny signal so you have to use a lot of light. The CELESTA produces the amount of light in a very small area that is needed to to actually detect these single molecules, and then the signal that is read out is essentially a barcode that’s used to identify the individual


modules. That barcode can be made up of colours, and different colours are used, again for identification of different individuals in the crowd. So you need to be able to switch quickly between the colours, which is achievable.” Another challenge is that experiments using this technique can take much longer to undertake, due to the incredibly high number of individual identifications. This means that the laser light source has to stay very stable over time for consistent, accurate performance. Johnson elaborates: “If the light source doesn’t remain stable for the longer experiment duration, you could end up in a situation where signals will be crossed, and you won’t be able to tell which target is which. Another big driver is that users want to collect the data quicker, write the same amount of data in less time, so they can get through more experiments in their work. The CELESTA supplies the speed of switching colours, but it also supplies stability


“The light source is the first piece in this long chain of things that must be connected together to retrieve the data. Light is the first step and Lumencor specialises in supplying that light”


over the long timeframe of the experiments.” With the types of microscopy


referred to in this article, there can be a wealth of moving parts, all of which must be correct in order to achieve accurate, repeatable results. There are the microscopes and all of their component parts, cameras, computers, molecular labelling techniques, and of course, the skilled people to carry out the experiments and analyse the data. But before all of this, says Johnson, comes the light.


“The light source is the first


piece in this long chain of things that must be connected together to retrieve the data. Light is the first step and Lumencor specialises in supplying that light. It may only be one part of this long, connected chain of elements that are required for multiplexed fluorescence detection and other techniques, but it is the first part, so, in some ways, you could argue it’s the most important part. If you get the first step wrong, then everything after that is likely to also be wrong, and it can be very difficult from which to recover. “As the starting point of


the whole process, it is very important that it be reliable and consistent. That is why it is our focus.” Find out more in-depth


information about the latest techniques to increase the capabilities of multiplexed fluorescence detection by reading the latest White Paper from Lumencor: Solid-State Illumination for Multiplexed Fluorescence Detection. EO


New White Paper now online


VIEW FOR FREE*


SOLID-STATE ILLUMINATION FOR MULTIPLEXED FLUORESCENCE DETECTION


This White Paper from Lumencor outlines the limitations of multiplexed fluorescence detection based on spectral discrimination, and some of the techniques that have been introduced to expand the number of targets that can be interrogated.


www.electrooptics.com/white-papers


Electro Optics


*Registration required


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