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:
- 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
- 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.
- 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.
- 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.
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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.
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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)
Position Mag NA Immersion Working 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 Name Excitation (nm Mirror (nm) Emission (nm) Fluorophore Dapi_WF 340 - 380 400LP 435 - 485 Dapi
Hoescht
Alexa405
CFPGFP_WF 465 - 495 505LP 515 - 555 FITC
Alexa488
GFPTxRd_WF 528 - 553 565LP 590 - 650 Texas Red
Alexa568
mCherryQuad 425 - 477
503 - 542
571 - 628
661 - 728multiple Turret 2 Cy3/Cy5 572 - 621
660LPCy3
Alexa568
Cy5
Alexa647FITC/TRITC 470 - 490
540 - 565500 - 540
575 - 660503 - 530
580 - 620FITC
Alexa488
TRITC
Alexa568FITC 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.