In our last post we showed it was possible to encapsulate plant protoplasts. The next step is to couple encapsulation to analysis of fluorescence. This is important as it will allow us to measure the abundance of a given fluorescent protein if we want to test the strength of a promoter or the output of a genetic circuit. Additionally, the technique could be used to measure the amount of a particular chemical produced by cells in each droplet (such as ethanol) if we wanted to test novel metabolic pathways.
To do this we are using a laser setup in Chris Abell's lab shown in Figure 1.
 |
| Figure 1: Laser setup for analysis of protoplast fluorescence. Courtesy of Dr. Sara Abalde-Cela. Photomultiplier tube (PMT). |
We use the laser beam to illuminate each droplet at a particular wavelength as it passes through the microfluidic device. The choice of wavelength is dependent on the molecule you wish to detect and the experimental conditions (such as pH), (a great online tool with detailed information about the parameters for fluorescent proteins can be found here).
Photons from the laser beam are absorbed by chemicals and re-emitted in a process known fluorescence, which can then be read by a detector. Fluorescence is often found in nature (Figure 2), and use of a protein from jelly fish (known as green fluorescent protein 'GFP') for visualization of biological processes has become so vital to biological research it was awarded the Nobel Prize for Chemistry in 2008.
 |
| Amazing Fish. By Sparks, J. S.; Schelly, R. C.; Smith, W. L.; Davis, M. P.; Tchernov, D.; Pieribone, V. A.; Gruber, D. F. - Sparks, J. S.; Schelly, R. C.; Smith, W. L.; Davis, M. P.; Tchernov, D.; Pieribone, V. A.; Gruber, D. F. (2014). "The Covert World of Fish Biofluorescence: A Phylogenetically Widespread and Phenotypically Variable Phenomenon". PLoS ONE 9: e83259. DOI:10.1371/journal.pone.0083259., CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=30540872 |
For our first test run we used the principle of chlorophyll autofluorescence (Figure 3). Chlorophyll is one of the molecules that photosynthetic organisms use to absorb sunlight, and when an excess amount of light is absorbed, chlorophyll molecules fluoresce.
 |
| Figure 3: Absorption spectra of chlorophyll a and b, the two main photosynthetic pigments in higher plants. http://www.austincc.edu/biocr/1406/labm/ex7/prelab_7_4.htm |
This gives us a nice proxy for presence or protoplasts within a droplet, and using fluorescence intensity we were were determine which droplets were empty, contained intact protoplasts or ruptured cells (Figure 4).
 |
| Figure 4: Measurement of chlorophyll fluorescence as droplets pass through the laser set up (Time/s). |
This was very exciting as it means we can now expand this approach to analyse fluorescence at a wide range of wavelengths, providing the basis for our test. Further, the fluorescence signal intensity can potentially be used to sort droplets.
"@jimhaseloff: A fully encapsulated story from PLOS blogger Steven Burgess (https://t.co/oL8TfjnrC2) pic.twitter.com/H3Ilw1dtHN" @sjb015— Susana Sauret-Gueto (@SusanaSaGu) 28 July 2016
We presented our project at the OpenPlant Forum in July. It was encouraging to see the excitement the work generated and we had a number of good discussions with people about improving the quality of our protoplasts.
Following on from this we will be teaming up with Dr. Oleg Raitskin at the Earlham Institute to couple fluorescence analysis to protoplast transformation.
