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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.

Produits utilisés pour cette application

Système de microscope pour imagerie TIRF

IXplore IX83 TIRF

Pour les expériences de dynamique membranaire, de détection monomoléculaire et de colocalisation, le système TIRF IXplore permet de réaliser de l’imagerie TIRF (fluorescence par réflexion totale interne) multicolore simultanée d’une grande sensibilité avec jusqu’à quatre couleurs. Le système cellTIRF d’Olympus comprend un contrôle motorisé de l’angle de chaque laser d’une grande stabilité, ce qui assure une pénétration égale des ondes évanescentes pour produire des images à contraste élevé et à faible bruit. Nos objectifs TIRF présentent un rapport signal sur bruit élevé et une grande ouverture numérique et sont dotés de bagues de correction pour ajuster leur performance à l’épaisseur de la lamelle couvre-objet et à la température.

Colocalisation exacte d’un maximum de quatre marqueurs grâce au contrôle individuel de la profondeur de pénétration
Profitez de l’objectif TIRF d’Olympus doté de la plus grande ouverture numérique au monde, à 1,7*
Configuration intuitive des expériences complexes avec le gestionnaire d’expériences graphique (GEM), cellFRAP et U-RTCE

* En date du 25 juillet 2017. À la connaissance d’Olympus.

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Objectifs haute résolution pour la super résolution/TIRF

APON-TIRF/UAPON-TIRF/UPLAPO-HR

Dotés des valeurs d’ouverture numérique les plus élevées parmi les objectifs que nous offrons, ces objectifs apochromatiques sont optimisés pour l’imagerie à super-résolution et l’imagerie TIRF à fort contraste. Obtenez une planéité étendue avec la grande ouverture numérique des objectifs UPLAPO-HR, lesquels permettent une imagerie en super-résolution et en temps réel des cellules vivantes et des micro-organites.

Grande ouverture numérique permettant de créer un champ d’ondes évanescentes pour des images à super-résolution ou des images TIRF à fort contraste
Série HR : les premiers objectifs planapochromatiques au monde* possédant une ouverture numérique de 1,5 qui permet l’obtention d’une planéité étendue

_{* En date de novembre 2018. Selon les recherches d’Olympus.}

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