Skip to content. | Skip to navigation

Personal tools

Navigation

You are here: Home / Imaging / Microscopes / Nikon A1R Laser Scanning Confocal and TIRF

Nikon A1R Laser Scanning Confocal and TIRF

The Nikon A1R Laser Scanning Confocal and TIRF is a powerful and greatly used microscope. It is capable of imaging up to 4 fluorophores simultaneously. While ideally used for fixed samples, live-cell imaging is possible on the if the environmental chamber is used. Combined with advanced 3D reconstruction software (Imaris), the Nikon A1R allows for the high-resolution multidimensional visualization of almost any sample.

The Nikon A1R Laser Scanning Confocal user manual can be found here.

The objective-based Total Internal Reflectance Fluorescence (TIRF) attachment can be used in widefield mode.

Rules of Use:

  1. The maximum duration of a reservation from 9:00am – 5:00pm is 3 hours. If you need more than 3 hours, like for an overnight experiment, please start your session after 4:00pm.
  2. If you run an overnight experiment, you are not allowed to reserve a session during peak hours (9:00am - 5:00pm) the following day.  Please end your overnight session by 9:00am.
  3. If you would like to do an experiment that lasts for more than 17 hours, you are allowed to start this on Friday afternoon and continue until Monday morning at 9:00am.
  4. The microscope can be reserved for any amount of time starting at 4:00pm each afternoon and all day Saturday and Sunday.
  5. University holidays and cancellations (i.e. snow days) will be treated like a Saturday or a Sunday.
  • Lasers: 405, 457, 476, 488, 514, 561, 638
  • Objectives:
    PositionMagNAImmersionWorking Distance (µm)Characteristics
    1 10x 0.5 Air 4000 Plan Apo I
    2 20x 0.75 Air 1000 Plan Apo I
    3 40x 0.75 Air
    4 40x 1.3 Oil 240 Plan Fluor DIC H N2
    5 100x 1.45 Oil 130 Plan Apo I
    6 100x TIRF 1.45 Oil 130 Plan Fluor DIC H N2
  • Light Source for widefield: Sola Light Engine
  • Filter Cubes (for widefield and TIRF):
    NameExcitation (nmMirror (nm)Emission (nm)Fluorophore
    405 340 - 380 400LP 435 - 485 Dapi
    Hoescht
    Alexa405
    CFP
    488 465 - 495 505LP 515 - 555 FITC
    Alexa488
    GFP
    561 532 - 587 595LP 608 - 683 Texas Red
    Alexa568
    mCherry
    Quad 425 - 477
    503 - 542
    571 - 628
    661 - 728
    multiple
  • Default Pixel size for Confocal:
    ObjectiveNumber of Steps
    Mag NA 64 128 256 512 1024 2048 4096
    10 0.5 19.89 9.94 4.97 2.49 1.24 0.62 0.31
    20 0.75 9.94 4.97 2.49 1.24 0.62 0.31 0.16
    40 0.75 4.97 2.49 1.24 0.62 0.31 0.16 0.08
    40 1.3 4.97 2.49 1.24 0.62 0.31 0.16 0.08
    100 1.45 1.99 0.99 0.50 0.25 0.12 0.06 0.03
    100 1.45 1.99 0.99 0.50 0.25 0.12 0.06 0.03 
  • Nyquist step size for Confocal (for a good explination of Nyquist sampling, click here):
    ObjectiveLaser
    Mag NA 405 457 476 488 514 561 638
    10 0.5 0.26 0.29 0.30 0.31 0.33 0.35 0.40
    20 0.75 0.16 0.18 0.18 0.19 0.20 0.21 0.24
    40 0.75 0.16 0.18 0.18 0.19 0.20 0.21 0.24
    40 1.3 0.09 0.10 0.11 0.11 0.11 0.12 0.14
    100 1.45 0.09 0.10 0.10 0.10 0.11 0.12 0.13
    100 1.45 0.09 0.10 0.10 0.10 0.11 0.12 0.13
  • Pixel Dwell Times for common Scan Sizes on the Confocal:
    Scan SizeDwell Time (us)
    64 14.1 33.9 80.2
    128 7.8 18.2 45.6 99.4
    256 4.1 9.5 23.8 52.8 110
    512 2.2 4.8 12.1 27.2 57.2 87.8 117
    1024 1.1 2.4 6.2 13.8 21.6 28.8
    2048 1.2 3.2 5.1 6.9
    4096 1 1.5
  • Frames per second for common Scan Sizes on the Confocal:
    Scan SizeFrames per Second
    64 8 4.2 1.8
    128 3.9 2.1 0.971 0.475
    256 1.9 1.0 0.499 0.244 0.122
    512 0.953 0.506 0.247 0.125 0.061 0.040 0.031
    1024 0.481 0.255 0.125 0.062 0.041 0.031
    2048 0.128 0.061 0.041 0.031
    4096 0.043 0.032
  • Camera (For widefield and TIRF): Andor Ixon 3 DU897E - C50
  • Key Specs:
    Active Pixels 512 x 512
    Pixel Size 16 x 16 µm
    Image Area pixel well depth 180,000 e-
    Maximum Readout Rate 10MHz
    Maximum Frame Rate 35 - 7,980 fps
    Read Noise < 1 e- with EM gain
    Maximum Cooling -100 ˚C
  • Pixel Size on Image: (Click here for a nice overview)
    Objective
    Lens Magnification EMCCD Pixel Size 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
  • 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.
This is Pacific Theme