Core Biotechnology Services

Nikon microscope 1

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Specifications

Nikon microscope 1 is a TE300 semi-automatic microscope with automated shutters and filter wheels and manual XY control. The imaging part of the system is automated and controlled by Improvision's Openlab software, running on a Mac (OS X). The system is equipped with an Hamamatsu ORCA-R2 digital camera (pixel size 6.45 x6.45 µm) and an X-cite120 fluorescence illumination system. A TransferMan NK manipulator from Eppendorf is mounted on the microscope stage and can be used with the Eppendorf microinjection system. There is an heated insert to keep the dish with cells on temperature. This system can also be used for imaging of slides. Keep in mind that you have to turn your slide upside down, so the coverslip has to be sealed well.

Nikon objectives

There are a list of objectives available for the Nikon microscopes.

See the Nikon's objectives

Instructions

Switching on

  • Remove dust cover.
  • Turn on the computer in windows mode, NOT Mac.
  • Check the correct objective is on the microscope before using it. (REMEMBER – IF THE OBJECTIVE DOESN”T HAVE THE WORD “OIL” WRITTEN ON IT DON”T USE OIL!!!)
  • Write your name etc in the folder.
  • Turn on the mercury bulb.
  • Turn on Orbit box (this controls the filters and shutters).
  • Turn on the camera controller at the socket. Controller goes into standby (orange light). Press the on switch on the controller till the green light comes on.
  • If you want to use the brightfield bulb or the microinjection equipment switch these on.
  • Open Volocity software.
  • Click on 'connect' - No password required.
  • Select your library or open a new library.
  • Click on video preview. At this point you should have the following controls in the right hand pannel on your screen:
    • Most used
    • Hamamatsu C10600-10B (Orca R2)
    • Dummy Hardware
    • Improvision ORBIT 1 Controller
  • Click on the Dummy Hardware control and click on the objective you will be using. Failing this might result in the wrong calibration for you images!
  • Change microscope nobe to position C. Your image should appear on the screen.

Switching off

Check to see if anyone is booked on the Microscope after you and if so check they definitely still want to use it. If they do just clean the objective with LENS tissue and leave everything on.

If they don’t …

  • Shutdown any software which is open
  • Shutdown computer
  • Switch off camera controller
  • Switch off Orbit box
  • Switch off brightfield bulb and microinjection equipment if you have used these
  • Note down the time you finished and the number of lamp hours in the folder
  • Switch off fluorescence bulb
  • Clean the objective with LENS tissue
  • Replace dust cover making sure it is NOT placed over the bulb houses… else it will melt!

Keypad

This controls the excitation filters that are housed at the back of the microscope in a Filter Wheel. It also controls the Brightfield Shutter.

  1. Blank (use to close shutter)
  2. FITC (Chroma S484/15x [477nm - 492nm]) Alexa488, GFP etc.
  3. TRITC (Chroma S560/15x [553nm - 568nm]) dsREd etc.
  4. DAPI (Chroma S395/10x [390nm - 400nm]) Hoechst etc.

S1: Brightfield Shutter

Filter cubes

Filter blocks are housed in the black box under the objective holder on the right hand side of the microscope. The position of each filter block is numbered and there is a slider underneath to move the filter blocks into position.

The following emission filters are available on the system:

  1. Don't use! Old filter stuck in system.
  2. DAPI/Hoechst (DM400/LP420)
  3. FITC/Alexa488/Cy2/GFP (DM505/LP520)
  4. Triple ex/em block; Chroma 61000v2. (Red, Green and Blue)

Bleu = excitation filter
Green = Dichroic mirror
Red = emission filter

Troubleshooting

I can’t see anything down the eyepiece / My sample looks very faint

  • Is the fluorescence bulb switched on and the bulb symbol on the display constant?
  • Is the blue dial on the fluorescence bulb unit in the up most position?
  • Is the manual shutter (silver rod on the bottom left hand side of the microscope towards the back) pushed in?
  • Are the neutral density filters (silver rods with black ends on the bottom left hand side of the microscope towards the back) both pushed in?
  • Is the dial (on the right hand side of the microscope) in position A, so 100% of the light is going to the eyepiece?
  • Are the correct filters selected?

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