Team:Tsinghua/Measurement
Measurement
Overview
This year, PhASE explored the measurement of protein distribution in a cell. To reflect the spatial distribution, we used fluorescence intensity profile to quantify signal density along a straight line. To reflect the distribution change towards time, we utilized the intensity ratio of phase to cytosol to quantify the relative amount of proteins in different area in each time unit.
Microplate Reader
(1) Fluorescence intensity profile
Tagging target proteins with fluorescence proteins, we can firstly quantify the relative density of them by recording fluorescence intensity. Then, in order to determine the relationship between fluorescence intensity and subcellular location, we defined an origin from which distance could be calculated. Next, we measured the fluorescence intensity on a particular orientation derived from the origin to reflect distribution of protein along this dimension in an intuitive way. This measurement method is called intensity profile and could be carried out easily using NIS viewer. We successfully characterize the recruitment of mCherry to phase using this method. We propose that intensity profile could be utilized to study the redistribution of proteins, DNA, RNA and other visible molecules in cells. (Figure 1, 3, 4)
Microplate Reader
(2) Intensity ratio of phase to cytosol
To show Cry2 distribution in certain region during stimulation, we tagged Cry2 with mCherry and measure mCherry intensity in certain areas. These areas are called “ROIs”. ROI locates in phase and cytosol is measured their intensity to show the distribution of Cry2 in these area and the normalized intensity is intensity of phase/cytosol. The change is normalized intensity shows the “movements” of Cry2. When perform time measurements, which measures the change of ROIs intensity with time, ROIs drew in phase and cytosol is set as standard ROIs. While a background ROI is also need to remove the affect of background and a reference ROI is to ensure the intensity of the whole zoom didn’t change much during stimulation.(Figure 2)
Figure 1 Intensity profile of phASE#1 before and after stimulation. The intensity of mCherry is measured, which is fused with Cry2. The measurement shows Cry2 distribution. Figure 1A Measurement of mCherry intensity profile before light stimulation. Figure 1B Measurement of mCherry intensity profile after light stimulation. Figure 1C Result of intensity profile before light stimulation. Figure 1D Result of intensity profile after light stimulation.
Figure 2 Measurement of mCherry intensity in phase and cytosol during light stimulation, which indicates the distribution of Cry2 in certain ROI regions. Figure 2A ROIs measured during stimulation(Red for phase, purple for cytosol, yellow for reference and blue for background). Figure 2B Result of ROI intensity measurements. Normalized intensity is intensity of phase/cytosol.
Figure 3 Intensity profile of phASE#2 before and after stimulation. The intensity of mCherry is measured, which is fused with Cry2. The measurement shows Cry2 distribution. Figure 3A Measurement of mCherry intensity profile before light stimulation. Figure 3B Measurement of mCherry intensity profile after light stimulation. Figure 3C Result of intensity profile before light stimulation. Figure 3D Result of intensity profile after light stimulation.
Figure 4 Intensity profile of phASE#1, stimulated by FITC and incubate in dark for 1 h. The reversible system then stimulated by 488nm light. Figure 4 is the reversible system before and after stimulation by 488 nm. The intensity of mCherry is measured, which is fused with Cry2. The measurement shows Cry2 distribution. Figure 4A Measurement of mCherry intensity profile before light stimulation. Figure 4B Measurement of mCherry intensity profile after light stimulation. Figure 4C Result of intensity profile before light stimulation. Figure 4D Result of intensity profile after light stimulation.
Confocal
We performed data analysis using Excel, NIS-Elements AR Analysis, and Prism8.
Confocal
Fluorescence recovery after photobleaching (FRAP)
The emission intensity of each channel (mostly 488nm excitation channel) was measured before and after photobleaching. Apart from the bleaching area, the intensity of the background and a reference area (separate from the bleached cell) was also measured for calibration. A plot of normalized intensity (the ratio of intensity at t s to intensity at 0 s) versus time was made afterwards. The videos and screenshots of the photobleaching were also exported for demonstration.
Confocal
Light induction
The ratio of intensity within phase to cytosol versus time was measured and plotted, in order to quantitively demonstrate the distribution change of mCherry fluorescence signal. The videos and screenshots of the light induction were also exported for demonstration.
