Capillary Electrophoresis with Laser Induced Fluorescence - CE-LIF
We have developed a microbeam-coupled Capillary Electrophoresis system with Laser Induced Florescence (CE-LIF). The goal is to allow simultaneous measurement of multiple reactive oxygen and nitrogen species by staining cells with multiple redox-sensitive dyes. As many of these dyes have very similar emission spectra they cannot be separated using imaging techniques but can be separated by CE-LIF.
What is Capillary Electrophoresis?
CE is a separation technique where analytes migrate through a thin glass capillary under high electric field and separated by their electrophoretic mobility differences.
In CE, there are two superimposed flow modalities experienced by the analytes in the sample plug: (1) Electrophoretic flow, responsible for separating the analytes by charge and mobility. (2) Electroosmotic flow, which drives the buffer and analytes (regardless of polarity) toward the outlet of the capillary and the detector. The electroosmotic flow, is driven by the interaction of electrolytic liquid with the capillary walls leading to the formation of a mobile Debye layer of charges and is much stronger than the electrophoretic flow, ensuring that all analytes will reach the detector.
What is Laser Induced Fluorescence?
Laser Induced Fluorescence is consists of using an intense laser as a light source to excite fluorescent molecules. The light emitted by these molecules (at a different color) can then be easily detected and quantified. By using an intense and focused laser the fluorescent molecules can cycle many times as they cross the laser beam, resulting in a strong signal. Coupled to CE, LIF provides highly sensitive detection, particularly for brightly fluorescent molecules. Using LIF, repeatable detection limits on the order of 70 molecules have been achieved.
What do we want to detect?
Within the first few microseconds following a charged particle traversal of a cell, numerous oxygen and nitrogen radicals are formed along the track. In order to detect these transient species (which are not, themselves fluorescent, they can be used to oxidize various chemicals causing those to become fluorescent
The CE-LIF System at RARAF.
Our CE-LIF system is centered on a 70 cm long fused silica capillary, connecting our cell picker to the LIF detection system. As a detector for the light emitted from the fluorophores, we use a spectrometer, which allows identification of different fluorophores based on subtle differences in their emission spectrum.
Simultaneous detection of multiple reactive oxygen species in a fabricated sample
This figure demonstrates separation of a mixed sample of activated dyes, indicating nitrous oxide and hydrogen peroxide – two important reactive species and glutathione – a radical scavenger present in large quantities in the cellular environment.
Detection of Reactive oxygen species and Glutathione (an antioxidant, present in cells) within a single cell, as compared to bulk reagent.