GSD SR microscopy

 

Introduction
 

Visualizing the localization of molecules and cellular structures with high precision is essential to gaining a thorough understanding of cellular processes. Ground state depletion (GSD) super-resolution (SR) microscopy, using methods to identify the localization of single molecules (STORM, PALM, fPALM), offers the maximum possible resolution (x,y ~20 nm) in wide-field 2D microscopy. This imaging system is also equipped with an automated TIRF module and incubator that is suitable for use in living cells or for advanced fluorescence imaging.
Please contact the Core staff for details.

Services include:

  • Access to GSD SR fluorescence microscopy, live imaging microscopy, and total internal reflection fluorescence microscopy (TIRFM)
  • User training for super-resolution image acquisition and analysis.
  • Imaging platform consultation
 
 
 
 
Equipment Manager:Po-Yen Lin     Tel-9526  
 

Items

Equipment

Brand/Model

Recommended Fluorophores

Status

1

Leica SR GSD system

Microscope

Leica DMI60000 B TIRFM

Light sources

Fiber laser: 488 nm, 532 nm, 642nm

Diode Laser: 405nm

Leica EL6000 white light

Objectives

HCX PL AOP 10x/0.40 DRY

HCX PL FLUOTAR L 40x/0.6 DRY

HCX APO 100x/1.47 OIL

Camera

Andor iXon DU-897 EMCCD

  •  AlexaFluor 488, Atto 488, YFP
  •  AlexaFluor 532, Atto 532, Rhodamine 6G 
  •  AlexaFluo 546, Atto 546 
  •  AlexaFluor 647 

Two color GSD imaging

  •  AlexaFluor 532 +  AlexaFluor 647
  •  AlexaFluor 488, or Atto 488, +  AlexaFluor 647

 

OK

NOTE: All photo-switching probes (photo-activated fluorescent proteins, quantum dots, dyes, etc.) can be used in GSDIM imaging.

 

Applications

  • Super-resolution imaging
  • TIRF
  • Live cell imaging

Pricing

  • Days 9 AM - 6 PM          $ 300 / hr
  • Nights and weekends       $ 150 / hr

References:

An important key to obtaining high quality SR images is choosing the right switchable probes. Here we provide references to selected articles that should be useful to prepare users for obtaining their first SR images.

Fluorescent probes selection

  • Fernandez-Suarez, M. and Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells.  Nature Reviews Molecular Cell Biology 9: 929-943(2008).
  • Lippincott-Schwartz, J. and Patterson, G. H. Photoactivatible fluorescent proteins for diffraction-limited and super-resolution imaging.  Trends in Cell Biology 19: 555-565 (2009).
  • Graham T Dempsey,   Joshua C Vaughan, Kok Hao Chen, Mark Bates & Xiaowei Zhuang. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature Methods 8, 1027–1036 (2011)

Sample preparation

  • László Barna, Barna Dudok, Vivien Miczán, András Horváth, Zsófia I László & István Katona "Correlated confocal and super-resolution imaging by VividSTORM"  Nature Protocols 11, 163–183 (2016)
  • Charlotte Kaplan & Helge Ewers “Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast” pp1007 - 1021 doi:10.1038/nprot.2015.060
  • L. Barna, B. Dudok, V. Miczán, A. Horváth, Z.I. László, and I. Katona, "Correlated confocal and super-resolution imaging by VividSTORM", Nat Protoc, vol. 11, pp. 163-183, 2015. http://dx.doi.org/10.1038/nprot.2016.002
  • Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010)
  • David Baddeley, David Crossman, Sabrina Rossberger, Juliette E. Cheyne, Johanna M. Montgomery, Isuru D. Jayasinghe, Christoph Cremer, Mark B. Cannell, Christian Soeller, "4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues" DOI: 10.1371/journal.pone.0020645 (2011)

 

 

 
 
Policies Manager:Po-Yen Lin    Tel-9526  
 
  • The GSD SR microscope may be reserved through the ICOB reservation system. If you are more than 20 minutes late to your reserved time, your time slot may be canceled.
  • Please cancel reservations at least 1 hour in advance of scheduled booking. Otherwise, charges will be applied based on the time reserved.
  • All users must use their own badge to enter the GSD SR microscope room.
  • Users must have received training and certification as independent users before using the instrument. After certification, users can apply to access the GSD SR microscope room (R109).
  • For users without training, core staff will assist you in operating the GSD microscope. The price is $500 / hr.
  • All users must clearly sign the work log with user name, PI name and usage time for billing.
  • All users are obligated to keep the facilities (including all objectives and the environment) clean, especially after using immersed objectives.
  • If users encounter any questions or experience trouble using the instrument, please contact Core staff immediately. Replacing any accessories or changing the settings of the control software are not allowed.
  • No drinking and eating are allowed in the GSD SR microscope room. Experimental consumables and packages of food must be removed from the laboratory and cannot be placed in the trash can.
  • Anyone who violates the above policies will have their right to access the instrument revoked and their PI will be informed.
 
Acknowledgement of the Core Facility

All users of the Imaging Core Facility should acknowledge the facility in publications and presentations that include data generated in the facility. Please also acknowledge the macro editor who writes the customized macro or script. We also appreciate receiving an electronic version of publications that include data gathered in our facility.

Acknowledgment template

We thank the Imaging Core Facility at the Institute of Cellular and Organismic Biology, Academia Sinica, for the technical support in image acquisition and analysis.

 

 

 
Usage protocol Manager:Po-Yen Lin     Tel-9526  
 

Service object
   ICOB members

Apply for access

  1. Contact the Core staff to arrange a training course.
  2. Contact the Core staff to become certified as an independent user.
  3. Fill out the application form. (Download)
  4. Submit a printed and filled out copy to the computer room

How to use

  1. Make a reservation online. The reservation system allows scheduling 13 days in advance. (Link)
  2. Log in online before use.(Link)
  3. Fill out the work log on the desk.
  4. Log out online after use. (Link)

 

 

Contact us  
 

Po-Yen Lin, Ph.D.
Phone: 886-2-27899526
Email: pylin@gate.sinica.edu.tw

 

 

 

 

 

 

 

128 Sec.2, Academia Rd, Nankang, Taipei 115 Taiwan, R.O.C Tel: 02-27899515 Fax: 02-27858059

icob@gate.sinica.edu.tw Copyright © ICOB 2013. All rights reserved.