Quantitative Analysis of Membrane Ordering of Living Industrial Model Microorganisms

doi:10.13523/j.cb.2008140

China Biotechnology

2021, Vol. 41

Issue (1): 20-29 DOI: 10.13523/j.cb.2008140

Quantitative Analysis of Membrane Ordering of Living Industrial Model Microorganisms

LV Xue-qin,JIN Ke,LIU Jia-heng,CUI Shi-xiu,LI Jiang-hua,DU Guo-cheng,LIU Long(

)

Science Center for Future Foods, Jiangnan University, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Wuxi 214122, China

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Abstract

As a phase sensitive fluorescent probe, Di-4-ANEPPDHQ can specifically label the ordered phase and disordered phase of membrane. Therefore, in theory, the probe can be used to quantitatively image the order of cell membrane. By combining Di-4-ANEPPDHQ and laser scanning confocal microscopy, the ordered phase and disordered phase live-cell imaging of a variety of representative industrial model microorganisms were carried out. Combined with the statistical comparison of polarity normalization values, the quantitative analysis of the cell membrane ordering of the above industrial model microorganisms is finally realized. The above study provides an intuitive and rapid detection method of living cells for cell membrane engineering.

Key words： Industrial model microorganisms Di-4-ANEPPDHQ Membrane ordering Live-cell imaging

Received: 27 August 2020 Published: 09 February 2021

ZTFLH:

Q819

Corresponding Authors: Long LIU E-mail: liulong@jiangnan.edu.cn

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	LV Xue-qin
	JIN Ke
	LIU Jia-heng
	CUI Shi-xiu
	LI Jiang-hua
	DU Guo-cheng
	LIU Long

Cite this article:

LV Xue-qin, JIN Ke, LIU Jia-heng, CUI Shi-xiu, LI Jiang-hua, DU Guo-cheng, LIU Long. Quantitative Analysis of Membrane Ordering of Living Industrial Model Microorganisms. China Biotechnology, 2021, 41(1): 20-29.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2008140 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I1/20

Fig.1 The structural formula of di-4-ANEPPDHQ

Fig.2 Emission spectrum analysis of Di-4-ANEPPDHQ in Saccharomyces cerevisiae (a) Raw images taken in the lambda-mode using confocal laser scanning microscope, Bar=5μm (b) ROIs selection: 3~6 cells were randomly selected, and 8 regions with the size of 12×12 pixels were selected in each cell (c) Original intensities were calculated from eight ROIs (ROI1-4 selected from plasma membrane, ROI5-8 selected from intracellular vesicular organelles) and built into profiles (d) Mean intensities of ROIs were calculated and built into profiles

Fig.3 Analysis of membrane order degree of Saccharomyces cerevisiae (a) Images of di-4-ANEPPDHQ-labelled S. cerevisiae cells. The images from the green channel (500~580nm) and red channel (620~750nm) are on the left. The merged image and Hueesaturationebrightness (HSB) image are on the right. Bar=5μm (b) The enlargement of the white box labeled cells in (a) (c-f) The distribution of GP values of different membrane structures. More than 3 000 pixels were counted for each group

Fig.4 Analysis of dyeing effect of Di-4-ANEPPDHQ in Escherichia coli (a) Images of di-4-ANEPPDHQ-labelled S. cerevisiae cells (top) and E. coli cells stained for 5 min (middle) and 20min (bottom). The bars were 5μm, 2μm and 2μm, respectively (b) The average fluorescence intensity of di-4-ANEPPDHQ in different cells (in each group, 25~30 cells were randomly selected for detection)

Fig.5 Emission spectra of di-4-ANEPPDHQ and membrane ordering in Bacillus subtilis (a) Raw images taken in the lambda-mode using confocal laser scanning microscope (b) Emission spectra of di-4-ANEPPDHQ in B. subtilis (c) Images of di-4-ANEPPDHQ-labelled B. subtilis (top) and S. cerevisiae cells (bottom). The images from the green channel (500~580 nm) and red channel (620~750 nm) are on the left. The merged images and the HSB images are on the right. Bar = 2 μm (d,e) The distribution of GP values of different cells. More than 3 000 pixels were counted for each group

Fig.6 Analysis and comparison of di-4-ANEPPDHQ emission spectra of Bacillus megaterium and Corynebacterium glutamicum (a,b) Raw confocal images of B. megaterium (a) and C. glutamicum (b) taken in the lambda-mode (c,d) Based on the image stack taken in the lambda-mode, fluorescence intensity was measured to draw the emission profiles (9 cells were randomly selected from each group) (b) Original intensities (c) Normalised intensities

Fig.7 Analysis and comparison of membrane order degree among three gram-positive bacteria (a) Confocal Images of di-4-ANEPPDHQ-labelled B. subtilis (top), B. megaterium (middle) and C. glutamicum (bottom), Bar=2μm (b) The GP values of different cells, more than 3 000 pixels were counted for each group (^** P<0.01)

Fig.8 MK-7 titer in BS-168 and BS-MK7 strains (n=3 times; ^** P<0.01)

Fig.9 Analysis of membrane ordering degree of different Bacillus subtilis (a) Confocal images of di-4-ANEPPDHQ-labelled BS168 (top), mBS168 (middle) and BS-MK7 (bottom), Bar=2μm (b) The distribution of GP values of different cells, more than 3 000 pixels were counted for each group (c) Histogram of GP peak values in different cells (^** P<0.01)


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