Nikon TE2000 Instructions Manual

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Wellcome Trust Centre for Human Genetics

Molecular Cytogenetics and Microscopy Core

Using the Nikon TE2000 Inverted Microscope

Colour image acquisition using NIS-Elements

[Basic Research] software and the Nikon
Digital Sight DS-5M Colour Camera

INSTRUCTIONS

PLEASE HANDLE CAREFULLY!

Handle the microscope gently, taking care to avoid sharp impacts.

1) TO SWITCH-ON THE MICROSCOPE AND LOOK AT YOUR SAMPLE:

Bright-field Microscopy Imaging [with the Colour Camera]:

- Turn on the transmitted light by turning on the switch on the Nikon power

supply box on the bench to the left side of the microscope.

- Press “lamp on/off” button at the base of the microscope on the left.

- Adjust the light intensity by rotating the control dial situated on the left side of

the microscope, below the on/off switch.

- For bright-field [non-fluorescence] transmission imaging: Check that the “D”

(Diffuser) and “HA” (Heat Absorber) filters at the top left of the microscope are
pushed in. Select the optional neutral density ND filters as necessary to dim the
light intensity [top right]: ND16 and ND2 – push both in for maximum light
attenuation, normally the best setting.

- The “eyepiece turret” dial should be set to O (open), when set to C it is closed.

- The "port/optical path" dial on the right side of the base should be set to port 1.

All the light now goes to the microscope eyepieces and none to the cameras.

- The "reticule in/out" lever at front of the base to the right should be turned

clockwise and set to a 2 o’clock position. This lever is under the colour camera.

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- The fluorescence filter wheel located underneath the objective wheel should be on

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the empty position indicated by the double arrow symbol (↔).

- Place the culture vessel or glass slide on the stage.

- Move a low power objective (e.g. 4x or 10x) into the light path.

- Focus on the specimen. Adjust the condenser to the correct height for Koehler

illumination1. Ask core staff for help with this if necessary.

- Have the phase ring set to A for stained cells/tissue, or to Ph1 for phase contrast

when using the 4x, 10x and 20x objectives or to Ph2 for phase when using the 40x
and 60x objectives.

- If microscope slides are to be viewed, they can be placed with the cover-slip side

either up or down [with the 60x objectives place slides cover-slip down].

Using the 20x, 40x and 60x objectives

- Focus using a low power 4x or 10x objective and the coarse focusing wheel. Then

revolve to the 20x, 40x or 60x objective as required.

- Please take extra care to avoid sharp impacts when switching

between objectives.

- Adjust the 20x, 40x or 60x objective correction collar for the optimal resolution2.

- A 60x oil objective and a 1x air objective are also available if required. These

must be fitted by the Core staff. If you need to use either of these objectives,
please ask a member of core staff to set up the microscope correctly for you

With multi-well plates Koehler illumination may not be possible as the condenser might have to

be moved higher to prevent it hitting the top of the plate. There are twin white markers on the
condenser to show the approximate position for Koehler illumination.

With the 20x, 40x and 60x LD air objectives the correction collar should be set to the thickness

of the support material: e.g. approx. 2mm for slides (cover-slip up) or 0.17mm for the thickness
of a 0.17mm cover-slip (cover-slip facing down). The correction collars compensate for
differences in vessel base or slide/cover-slip thickness. The objective collars can be adjusted by
rotating them, and used in conjunction with the normal focus, an optimal image can be
obtained. Please ask Core staff for help with adjusting correction collars. Details of the
correction collars are given on the nearby shelf and on-line.

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2) TO START THE NIKON COLOUR CAMERA AND CAPTURE IMAGES:

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- Switch on the colour camera power supply by pressing the button on the box

labelled Nikon Sight DS-U1. The Nikon DS-5M colour camera is on the front
port of the Nikon microscope.

- Turn on the HP computer (PC on the floor under the microscope workbench).

- If you are a “Well” user log on to the system and network with your usual

username and password. Alternatively, log on to the PC as “User” using “cyto02”
as password or log in as ‘generic’.

- Double-click on the NIS Elements [Basic Research] icon on the desktop:

- Scroll down the pop-up camera driver window and choose the ‘Nikon DS-U1’ for

capturing colour images. You can also select the “Hamamatsu ORCA’ for
fluorescence B&W image capture3, or ‘none’ just to view saved images. Click OK

[any camera selected must be switched on].

- When using the Nikon colour camera, Nikon recommend setting the halogen lamp

brightness to ‘12V100W [camera]’ using the lamp control dial4.

- Once you have found an image down the microscope that you wish to take a

picture of, the port/optical path dial (on the right side of the base of the
microscope) needs to be moved to “4” for the colour camera [Port 4 is split
50:50 with the eyepieces]. To view the sample via the microscope eye-pieces only,
return the port to “1”.

- You should see a Camera DS-5M Settings control window (camera controls

differ depending on the camera selected). If that window isn’t there, right click
over the non-image area and select ‘Acquisition Controls, DS-5M Settings
[Ctrl_Alt_C] to activate it. Or you can also use View, Aquisition Controls, DS-

5M Settings on the main menu.

- From the main menu, click Live (+) , or select Acquire, Live Fast to open

up a live image window on the screen. The “Live - Fast” dialog box appears. This
Live Fast window is intended to allow you to set the photo exposure time and put
the image in focus while viewing it on the computer monitor5.

Some WTCHG users use IPLabs software with the B&W Hamamatsu Orca for fluorescent

specimens. There is a separate in-house core guide for using this camera with IPLabs software.

This sets the lamp to a ‘bright’ setting. Too low a halogen bulb voltage and the camera image

will appear brown (more red and not enough blue light). For quantitative [comparative] colour
imaging ensure that the halogen lamp brightness is always set to the same level. If the lamp is
too bright push in the [top right] ND16 and ND2 filters to reduce its intensity – this won’t affect
the lamps spectral emission [light colour].

‘Live Fast’ can be set to 640x480 pixels or 1280x960 pixels under the resolution tab in DS-5M

settings ‘Fast focus. Although video refresh rates are higher at 640x480, the greater resolution

of 1280x960 is more useful for focussing and is often the preferred choice in bright-field.