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Nikon N-STORM

The Nikon N-STORM has the most powerful lasers and most sensitive cameras within the facility and is ideal for single particle detection using TIRF and STORM, with the ability to image 2 colors simultaneously, The microscope was acquired by Professor Tom Cech through HHMI and is fully enclosed within an environmental chamber allowing longitudinal live cell imaging.

The user manual written by Nikon can be found here.

Be careful when preparing your samples. Most conventional fluorophores are not STORM compatible. Nikon has prepared some protocols (click here) that are a good starting point. However, real success will come from some trial and error.

Rules of Use:

  1. The maximum duration of a reservation from 8:00am to 6:00pm, Tuesday through Friday, is 4 hours. If you need more than 4 hours, please start your session after 4pm
  2. The STORM microscope may be reserved for any amount of time starting at 6:00pm each afternoon (Tuesday - Friday) and all day Saturday, Sunday, and Monday. Please save the Monday reservations for very long experiments if possible.
  3. Please refrain from using the environmental chamber (heat) on Sunday, Monday, and Tuesday, as the residual heat causes stage drift that greatly disrupts super resolution STORM experiments. The environmental chamber can be used at will Wednesday - Saturday.
  4. If you need to reserve multiple sessions more than two weeks in advance, or for any other modifications or exemptions to the reservation guidelines, please contact the facility director to discuss your needs.
    If you require more than 4 hours of microscope access during prime use hours, you must complete the Reservation Limit Exemption Form at least one week in advance. If you do not complete this form, your session may be cancelled.
    Weekend-long sessions are permissible, but you must complete the Entire-Weekend Imaging Request Form at least 2 weeks in advance. If you do not complete this form, your session may be cancelled.
    • Lasers: The Agilent Technologies MLC400B includes the 405, 488, 561, and 647 laser lines.
    • Objectives: (click here for a nice overview of microscope objective specifications)
      PositionMagNAImmersionWorking Distance (µm)Characteristics
      1 20x 0.8 Air 1000 Plan Apo VC DIC N2
      2 Empty
      3 60xA 1.2 Water 310 Plan Apo VC DIC N2
      4 Empty
      5 100x TIRF 1.5 Oil 130 Plan Apo DIC H N2
      6 Empty
    • Light Source for widefield: Excelitas X-Cite 120LED
    • Filter Cubes for widefield, TIRF, and STORM:
      Turret 1
      NameExcitation (nmMirror (nm)Emission (nm)Fluorophore
      Dapi_WF 340 - 380 400LP 435 - 485 Dapi
      Hoescht
      Alexa405
      CFP
      GFP_WF 465 - 495 505LP 515 - 555 FITC
      Alexa488
      GFP
      TxRd_WF 528 - 553 565LP 590 - 650 Texas Red
      Alexa568
      mCherry
      Quad 425 - 477
      503 - 542
      571 - 628
      661 - 728      		 multiple
      Turret 2
      Cy3/Cy5 572 - 621
      660LP      		 Cy3
      Alexa568
      Cy5
      Alexa647
      FITC/TRITC 470 - 490
      540 - 565      		 500 - 540
      575 - 660      		 503 - 530
      580 - 620      		 FITC
      Alexa488
      TRITC
      Alexa568
      FITC 465 - 495 505LP 515 - 555 FITC
      Alexa488
      GFP

    • Cameras: 2x Andor Ixon Ultra DU897U - C50 and Hamamatsu ORCA-Flash4.0 V2 C11440.
    • Andor Ixon Ultra DU897U - C50 EMCCD Key Specs: (click here for the product pdf)
      Active Pixels 512 x 512
      Pixel Size 16 x 16 µm
      Image Area pixel well depth 180,000 e-
      Maximum Readout Rate 17MHz
      Maximum Frame Rate 56 - 11,074 fps
      Read Noise < 1 e- with EM gain
      Maximum Cooling -100 ˚C

     

    • Hamamatsu ORCA-Flash4.0 V2 C11440 sCMOS Key Specs: (click here for the product pdf)
      Active Pixels 2048 x 2048
      Pixel Size 6.5 x 6.5 µm
      Image Area Pixel Well Depth 30,000 e-
      Maximum Readout Rate 1000Hz
      Maximum Frame Rate 30 - 25.655 fps
      Read Noise 1.4 e-
      Maximum Cooling -10 ˚C

    • Pixel Size on Image: (Click here for a nice overview)
      Objective
      Lens Magnification Pixel Size (nm)
      		 10x 20x 60x 100x
      1 16000 x 16000 1600 800 266.67 160
      1.5 16000 x 16000 106.67 533.33 177.78 106.67
      1 6500 x 6500 650 325 108.33 65
      1.5 6500 x 6500 433.33 216.67 72.22 43.33
    • Image pixel size = (Camera pixel size x bin factor)/(objective magnification x lens magnification x C mount magnification).
    • Pay attention to your units! The units in the table are nm.
    • Most often the lens magnification and C mount magnification = 1.

     

     

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