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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 Cech lab has reserved hours on the microscope: Wednesday and Friday, from 9:00am - 5:00pm.
  2. If by Monday at 4pm the Cech lab hours for the current week are not reserved, they become open for reservation by all.
  3. The maximum duration of a reservation from 9:00am – 5:00pm is 4 hours, except for on Mondays.  If you need more than 4 hours, please start your session after 5:00pm.
  4. The STORM microscope can be reserved for any amount of time starting at 5:00pm each afternoon (Tuesday – Friday) and all day Saturday, Sunday, and Monday.  Please save the Monday reservations for very long experiments if possible.
  5. If you need to reserve multiple sessions more than two weeks in advance, please contact Joe Dragavon (joseph.dragavon@colorado.edu) to discuss this.
  • 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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