Difference between revisions of "Team:IIT Delhi/Photobleaching"
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Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.<br/><br> | Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.<br/><br> | ||
Revision as of 20:30, 1 November 2017
Photobleaching
Characterization of Rate of Photobleaching of Wild Type GFP (BBa_E0040)
Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.
Here, we characterized the photobleaching effect in wildtype GFP (E0040), which was the reporter in our oscillator using fluorescent microscopy with the etaluma Lumascope 500 microscope. Cells expressing GFP under the PhlF repressible promoter (BBa_K2525016) in the absence of PhlF, so that it constitutively expressed GFP. Cells were loaded in microfluidic chambers and droplet encapsulation was performed to capture a small number of cells. This droplet was continuously exposed to light corresponding to the excitation wavelength of GFP (~485 nm) and the emission was captured continuously as well. ImageJ was used to analyze the images, to obtain the rate of photobleaching as shown in Fig 1. Where we have fitted an exponential curve to the total intensity over time. It is known that photobleaching has a first order decay. We obtain a photobleaching rate of 0.002 per second (7.2 per hour).
Total Intensity in the encapsulated droplet over time. Since photobleaching is known to be a first order process, we have fitted an exponential curve to the data. The high R-squared value implies a good fit of the exponential model to the data. The rate of photobleaching turns out to be 0.002 per second (7.2 per hour)
A timelapse gif of the image is as shown below. Photobleaching over time can be clearly seen as time progresses (notice the time stamp).
Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.
Here, we characterized the photobleaching effect in wildtype GFP (E0040), which was the reporter in our oscillator using fluorescent microscopy with the etaluma Lumascope 500 microscope. Cells expressing GFP under the PhlF repressible promoter (BBa_K2525016) in the absence of PhlF, so that it constitutively expressed GFP. Cells were loaded in microfluidic chambers and droplet encapsulation was performed to capture a small number of cells. This droplet was continuously exposed to light corresponding to the excitation wavelength of GFP (~485 nm) and the emission was captured continuously as well. ImageJ was used to analyze the images, to obtain the rate of photobleaching as shown in Fig 1. Where we have fitted an exponential curve to the total intensity over time. It is known that photobleaching has a first order decay. We obtain a photobleaching rate of 0.002 per second (7.2 per hour).
Total Intensity in the encapsulated droplet over time. Since photobleaching is known to be a first order process, we have fitted an exponential curve to the data. The high R-squared value implies a good fit of the exponential model to the data. The rate of photobleaching turns out to be 0.002 per second (7.2 per hour)
A timelapse gif of the image is as shown below. Photobleaching over time can be clearly seen as time progresses (notice the time stamp).
