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TIRF Imaging of Changes in Membrane Morphology and Molecular Dynamics

Total Internal Reflection Fluorescence (TIRF) Imaging of Changes in Membrane Morphology and Molecular Dynamics under the Cell Membrane with Olympus’ Z-drift Compensation System

Introduction

One important issue in current cell biology research is to understand the mechanism of physiological phenomena associated with the intercellular communication between adjacent cells. A promising step toward this goal is live cell microscopy that enables researchers to monitor changes in cell membrane morphology and the dynamics of localized molecules at the intercellular adhesion site. Figure 1 illustrates how high-precision TIRF imaging is enabling new types of advanced cellular research. The images, captured using an Olympus motorized inverted microscope IX series, show changes in the membrane morphology and molecular dynamics under the cell membrane. The Olympus Z-drift compensator maintained a sharp focus on the cells over a long period of time enabling these images to be captured in such high quality. This process demonstrates the importance of TIRF and the Olympus Z-drift compensator to advanced live cell imaging.

Figure 1. Time-lapse images of a Cos-1 cell co-expressing GFP-17 and Lifeact-mCherry.

Examination of whether the recruitment of FBP17 to the plasma membrane is dependent on transient reduction of membrane tension caused by myosin based contraction force. FBP17 acutely disappeared from the cell edge after treatment with the myosin inhibitor blebbistatin (175 sec). This ef fect can be rescued by subsequent reduction of membrane tension induced by hypertonic buffer (260 sec), indicating that the FBP17 senses the membrane tension to assemble at the plasma membrane.

Time-lapse movie of a Cos-1 cell co-expressing GFP-17 and Lifeact-mCherry.

Imaging System;
Microscope: Research Inverted Microscope IX81
Objective: PlanApo 100XOTIRFM(100X, N.A.1.45)
CCD camera: Cascade II cooled CCD camera (Photometrics)
Z-drift Compensation System: IX-ZDC

Image data courtesy of;
Kazuya Tsujita, Ph.D., Toshiki Itoh, Ph.D.
Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University

Reference;
Nat Cell Biol. 2015 Jun;17(6):749-58. doi: 10.1038/ncb3162.
J Cell Sci. 2013 May 15;126(Pt 10):2267-78. doi: 10.1242/jcs.12251

Polarisation of FBP17 is induced by PM tension increase.
COS-1cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy upon hypotonic buffer. The movie was taken at 1 frame per 5 seconds and played at 15 fps.

Polarisation of FBP17 is disrupted by PM tension decrease.
COS-1 cell co-expressing GFP-FBP17and Lifeact-mCherry was observed by time-lapse microscopy upon addition of hypertonic buffer. The movie was taken at 1 frame per 5 seconds and played at 15 fps.

Polarisation of FBP17 is induced by PtdIns(4,5)P2 liberation.
COS-1 cell co-expressing GFP-FBP17, CFP-FKBP-PLC δ1 PH domain, and mRFP-FRB-MoA was observed by time-lapse microscopy upon addition of rapamycin. The movie was taken at 1 frame per 5 seconds and played at 15 fps.

Polarisation of FBP17 is disrupted by PtdIns(4,5)P2 depletion.
COS-1 cell co-expressing GFP-FBP17, CFP-PM-anchored FRB domain, and mRFP-FKBP-5-phosphatase domain was observed by time-lapse microscopy upon addition of rapamycin. The movie was taken at 1 frame per 5 seconds and played at 15 fps.

Dynamics of FBP17 at the leading edge. COS-1 cell co-expressing GFP-FBP17and Lifeact-mCherry was observed by time-lapse microscopy. The movie was taken at 1 frame per 5 seconds and played at 15 fps.

Acute disruption of FBP17 polarity by N-WASP inhibition. COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy upon addition of wiskostatin. The movie was taken at 1 frame per 10 seconds and played at 15 fps.

Acute disruption of FBP17 polarity by Arp2/3 complex inhibition.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy upon addition of CK-666. The movie was taken at 1 frame per 10 seconds and played at 15 fps.

Conclusion

Olympus’ live cell imaging solutions and Z-drift compensator facilitate long-term imaging studies of cellular processes. The Z-drift compensator utilizes low phototoxicity infrared (IR) light to detect the correct focus position, to make automatic focal adjustments, and to maintain precise focusing over time by avoiding focus drift due to factors such as temperature changes. The type of experiment described above cannot be accomplished using conventional microscopy because the images captured over time would be out of focus because of focus drift. The Z-drift compensator enables images to be captured without loss of focus. This facilitates chronological, high-precision tracking of dynamic changes of FBP17 and the Lifeact actin marker under the cell membrane.

Produtos usados nesta aplicação

Sistema de microscópio para reflexão interna total (TIRF)

IXplore IX83 TIRF

Para experimentos de dinâmica de membrana, detecção de molécula única e colocalização, o sistema IXplore TIRF possibilita a formação de imagem TIRF (fluorescência de reflexão interna total) multicolorida simultânea e sensível de até quatro cores. O sistema cellTIRF da Olympus fornece um controle de ângulo do laser individual motorizado e estável, proporcionando uma penetração de onda evanescente igual para imagens de baixo ruído e alto contraste. Nossas objetivas TIRF contam com uma alta relação sinal-ruído, alta AN e colares de correção para ajustar a espessura e a temperatura das lamínulas.

Colocalização exata de até quatro marcadores graças ao controle de profundidade de penetração individual
Aproveite ao máximo a objetiva TIRF da Olympus com a maior abertura numérica (AN) do mundo, de 1,7*
Configuração intuitiva de experimentos complexos com o Gerenciador de experimento gráfico (GEM), cellFRAP e U-RTCE

* Em 25 de julho de 2017. De acordo com a pesquisa da Olympus.

Saiba mais

Objetivas de alta resolução para superresolução/TIRF

APON-TIRF/UAPON-TIRF/UPLAPO-HR

Oferecendo os nossos maiores valores de abertura numérica, essas objetivas apocromáticas são otimizadas para TIRF de alto contraste e formação de imagem de super-resolução. Obtenha um nivelamento amplo com a alta AN das objetivas UPLAPO-HR, possibilitando uma formação de imagem de super-resolução em tempo real de células vivas e micro-organelas.

Alta AN para criar um campo de onda evanescente para imagens TIRF de alto contraste ou super-resolução
A série HR são as primeiras objetivas apocromáticas planas do mundo com AN de 1,5 que alcançam um amplo nivelamento

_{* Em novembro de 2018. De acordo com a pesquisa da Olympus.}

Saiba mais