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)
  

  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 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.