Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells

Ao Yin; Shixian Zhai; Han Sun; Zhi Liu; Zhengfei Zhuang; Tongsheng Chen

doi:10.3788/CJL202148.2107001

Chinese Journal of Lasers, Volume. 48, Issue 21, 2107001(2021)

Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells

Ao Yin^1,2, Shixian Zhai^1,2, Han Sun^1,2, Zhi Liu^1,2, Zhengfei Zhuang^1,2,3、**, and Tongsheng Chen^1,2,3、*

¹Key Laboratory of Laser Life Science, Ministry of Education, College of Biophotonics, South China Normal University, Guangzhou, Guangdong 510631, China

²Guangdong Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, Guangdong 510631, China

³SCNU Qingyuan Institute of Science and Technology Innovation Co., Ltd., Qingyuan, Guangdong 511517, China

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Figures & Tables(8)

Fig. 1. Excitation-emission spectral fingerprints of CFP, YFP, and CFP-YFP sensitization. (a) Left images are fluorescence images of CFP at 436-nm laser extation and YFP at 470-nm laser extation under different emission filters, right image is normalized emission spectrum of CFP and YFP; (b) top images are fluorescence images of CFP captured by 510-nm emission channels under laser excitation at 436 nm and 470 nm wavelengths and YFP captured by 530-nm emission channels under laser excitation at 436 nm and 470 nm wavelengths, bottom image is normalized excitation spectrum of CFP and YFP; (c) spectral fingerprints of S_D, S_A, and S_S, scale bar is 10 μm

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Fig. 2. Spectral crosstalk coefficient measurement. (a) I_DD(DA), I_DA(DA), and I_AA(DA) typical fluorescence images of CFP; (b) I_DD(DA), I_DA(DA), and I_AA(DA) typical fluorescence images of YFP; (c) statistical histogram of spectral crosstalk coefficient of at least 30 cells expressing CFP and YFP

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Fig. 3. Predetermining the F_C/I_DD(DA) ranges of three kinds of cells and measuring G and γ factors. (a) Fluorescence images of C4Y cells and corresponding histogram of F_C/I_DD(DA) ranges; (b) fluorescence images of C40Y cells and corresponding histogram of F_C/I_DD(DA) ranges; (c) fluorescence images of C80Y cells and corresponding histogram of F_C/I_DD(DA) ranges; (d) fluorescence images of cells expressing C4Y, C40Y, and C80Y and corresponding histogram of F_C/I_DD(DA) ranges; (e) calibration line obtained from cells expressing C4Y, C40Y, and C80Y cultured in one dish and implementation of E-FRET method for measuring the E values of cells

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Fig. 4. Measuring system parameters using C4Y cells. (a) Representative excitation-emission spectral fluorescence images of C4Y cells; (b) corresponding pixel-to-pixel pseudo-color map and column statistical map of f_sc; (c) corresponding pixel-to-pixel pseudo-color map and column statistical map of r_K; (d) statistical f_sc and r_K values of 100 living cells

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Fig. 5. Quantitative FRET measurements for cells expressing C4Y, C10Y, C40Y, and C80Y under different R_SN. (a) Fluorescence images of cells expressing C4Y, C10Y, C40Y, and C80Y; (b) corresponding pixel-to-pixel E pcolor images and R_C pcolor images; (c) statistical E and R_C values of cells under R_SN≤3; (d) statistical E and R_Cvalues of cells under R_SN>3

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Table 1. E and R_C values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

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Table 1. E and R_C values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

Plasmid	Cell	R_SN	E		R_C		Reported Evalue(E^ref)^[28]
Plasmid	Cell	R_SN	mExEm-spFRET	E-FRET	mExEm-spFRET	E-FRET	Reported Evalue(E^ref)^[28]
C4Y	1	6.86	0.298	0.308	1.063	0.989	0.299±0.004
C4Y	2	2.34	0.290	0.316	1.127	0.867	0.299±0.004
C10Y	1	18.82	0.225	0.234	0.821	0.963	0.228±0.003
C10Y	2	3.44	0.234	0.248	0.940	0.936	0.228±0.003
C40Y	1	27.69	0.159	0.160	0.966	1.094	0.158±0.002
C40Y	2	6.58	0.149	0.149	1.012	1.079	0.158±0.002
C80Y	1	10.809	0.126	0.128	1.017	1.144	0.116±0.002
C80Y	2	6.910	0.118	0.125	1.042	1.083	0.116±0.002

Table 2. Statistical E and R_C values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

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Table 2. Statistical E and R_C values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

Plasmid	R_SN	E		R_C
Plasmid	R_SN	mExEm-spFRET	E-FRET	mExEm-spFRET	E-FRET
C4Y	≤3(39)	0.292±0.019	0.313±0.022	1.034±0.071	1.005±0.080
C4Y	>3(22)	0.294±0.020	0.302±0.017	1.012±0.077	1.027±0.077
C10Y	≤3(25)	0.226±0.021	0.224±0.028	1.023±0.037	0.993±0.059
C10Y	>3(19)	0.237±0.015	0.240±0.018	1.025±0.097	1.016±0.096
C40Y	≤3(23)	0.155±0.010	0.163±0.012	1.086±0.039	1.001±0.029
C40Y	>3(28)	0.157±0.011	0.159±0.017	1.085±0.089	0.897±0.126
C80Y	≤3(32)	0.126±0.018	0.135±0.023	0.989±0.045	0.994±0.034
C80Y	>3(35)	0.123±0.016	0.127±0.010	1.046±0.126	1.083±0.151

Table 3. Statistical E and R_C values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10

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Table 3. Statistical E and R_C values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10

Date	E
	mExEm-spFRET								E-FRET
	C4Y		C10Y		C40Y		C80Y		C4Y		C10Y		C40Y		C80Y
December 12		0.29±0.02		0.23±0.01		0.16±0.02		0.12±0.01		0.30±0.03		0.23±0.04		0.16±0.02		0.12±0.02
March 10		0.32±0.02		0.24±0.03		0.15±0.02		0.10±0.06		0.33±0.02		0.24±0.05		0.18±0.02		0.14±0.12
Date	R_C
	mExEm-spFRET								E-FRET
	C4Y		C10Y		C40Y		C80Y		C4Y		C10Y		C40Y		C80Y
December 12		1.02±0.07		1.02±0.07		1.09±0.06		1.02±0.09		1.05±0.05		1.04±0.09		0.99±0.08		1.07±0.14
March 10		0.98±0.08		1.04±0.10		1.08±0.09		1.04±0.16		1.11±0.10		1.13±0.13		1.17±0.12		1.27±0.53

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Ao Yin, Shixian Zhai, Han Sun, Zhi Liu, Zhengfei Zhuang, Tongsheng Chen. Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells[J]. Chinese Journal of Lasers, 2021, 48(21): 2107001

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Paper Information

Category: biomedical photonics and laser medicine

Received: Mar. 18, 2021

Accepted: Apr. 9, 2021

Published Online: Oct. 27, 2021

The Author Email: Zhuang Zhengfei (zhuangzf@scnu.edu.cn), Chen Tongsheng (chentsh@scnu.edu.cn)

DOI:10.3788/CJL202148.2107001

Topics

laser devices and laser physics

Lasers and Laser Optics

Laser physics

laser manufacturing

Instrumentation, Measurement and Metrology

Table 1. E and RC values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

Table 1. E and RC values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

Table 2. Statistical E and RC values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

Table 2. Statistical E and RC values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

Table 3. Statistical E and RC values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10

Table 3. Statistical E and RC values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10

Table 1. E and R_C values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

Table 1. E and R_C values of cells in Fig.5 measured by mExEm-spFRET and E-FRET methods

Table 2. Statistical E and R_C values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

Table 2. Statistical E and R_C values of the cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods

Table 3. Statistical E and R_C values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10

Table 3. Statistical E and R_C values of cells expressing four different FRET plasmid measured by mExEm-spFRET and E-FRET methods on December 12, March 10