Wellcome Trust Centre for Human Genetics
Molecular Cytogenetics and Microscopy Core
The Nikon TE2000 Inverted Microscope
Fluorescence image acquisition using
NIS-Elements [basic research] and the
B&W Hamamatsu Orca C4742-95 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 :
-
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, typically 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 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.
-
The filter wheel located underneath the objective wheel should be on the empty
position indicated by the double arrow symbol (↔).
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-
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
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illumination . Ask core staff for help with this if necessary.
Please take extra care to avoid sharp impacts when switching between
objectives. You should drop the height of the objective and then refocus.
-
Have the condenser dial 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 objective place slides cover-slip down]. Focus
with the coarse focusing wheel and revolve to objective 20x or 40x if required.
-
Adjust the 20x, 40x or 60x objective correction collar for the optimal resolution .
-
A 60x oil objective and a 1x air objective are also available if required [see
Appendix One]. These must be fitted by the Core staff. If you need to use either
objective, please ask a member of core staff to set up the microscope correctly for
you.
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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 two white arrow markers on
the condenser to show the approximate height position for Koehler illumination.
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With the 20x to 60x 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 collar settings are given
in the ‘objectives’ info sheet and on-line].
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The 60x oil objective with DIC [Differential Interference Contrast]
Microscopy:
- Ask a member of core staff to attach the 60x oil objective and set up the
transmitted light DIC optics [if required].
- Have the condenser dial set to DIC H.
- Slide in the polariser P filter [located above the condenser turret] to the left.
- Push in the black T-A analyser slider [located under the objectives & filter wheel,
to the left of the microscope base].
-
Place microscope slide cover-slip side down. Move a low power objective (4x or
10x) into the light path. When you have found an area of interest carefully revolve
to the 60x oil objective.
-
Place a drop of oil on the objective. Make sure you use the correct oil. Only
Cargille oil should be used with this objective.
-
For correct DIC you should have set the condenser height correctly for Koehler
illumination. You then rotate the analyser and slide in/out the DIC prism [using
the mini-slider under the 60x objective] to give the best contrast enhanced image.
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Epi-Fluorescence Microscopy [with the Hamamatsu Camera ]
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-
You can use IP Labs software with the B&W Hamamatsu Orca camera as well for
fluorescence imaging. IP Labs already has a large user base in the Centre and you
may find it an easier program to use. However NIS Elements can also capture
fluorescence images using the sensitive [and low noise] B&W Hamamatsu Orca
camera, and recently there have been many useful updates to NISElements.
-
Use this guide if you intend to use NIS Elements for fluorescence imaging. There
are separate in-house guides for fluorescence imaging using IP labs software with
the Hamamatsu camera and for full colour imaging using NIS-Elements [Basic
research] with the Nikon DS-5M colour camera.
You can use the Nikon Colour camera for fluorescence image capture, but the image quality
may be very poor if the fluorescence signal is weak. Only the Hamamatsu B&W camera can
capture fluorescence images with low noise (no speckles) and it’s the recommended choice this
– fluorescence ‘colour’ is applied by look-up tables [LUTs].
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The mercury fluorescence arc lamp
-
Note the last switch-off time in the notebook beside the microscope. The arc lamp
must be left off for at least 30 minutes to cool down before it is switched on
again.
-
Switch on the illuminated power switch on the front panel of the mercury lamp
power supply box (on the bench on the right side of the microscope) and make
sure the "power" monitor light (green) is on. Always switch the mercury lamp
on and off with the PC, LCD display, microscope and camera switched OFF.
-
Press the ignition button for 2-3 seconds to light the lamp. Hold it down until the
orange "lamp ready" light stays on. Leave the lamp to warm up for few minutes.
Leave the mercury lamp on for at least 30 minutes before switching it off.
-
Unless you also capturing phase contrast or transmission images as well, the
height and settings of the condenser above the stage are irrelevant for epifluorescence imaging.
Fluorsescence Filter Sets
Optical filters are required in the microscope to ensure appropriate excitation of the
fluorophore. These dichroic filter sets are moved into the fluorescent light path by rotating
the fluorescence filter wheel, which is located underneath the objective wheel [nosepiece],
on the right side of the microscope base.
The following filter sets are available:





DAPI
Hoechst
FITC
GFP, Alexa-488
TRITC Rhodamine , Alexa-546
CY5
CY5, Alexa-633
↔
Transmitted light only
- Blue emission colour [violet excitation]
- Green emission colour [blue excitation]
- Red emission colour [green excitation]
- Far Red emission colour [red excitation]
- Fluorescence off [Phase Contrast]
For “transmitted light” the filter wheel is normally set to the empty position indicated by
the double arrow symbol (↔). Note: the excitation light colour is seen at the specimen
and the emission light colour is seen down the microscope via the eye-pieces , although
CY5 emission can only seen by the microscope’s Hamamatsu fluorescence camera.
-
During fluorescence microscopy, turn off the bright-field halogen light by using
either the switch on the left side of the microscope main body, or by rotating the
transmitted light intensity knob anti-clockwise to zero.
-
Rotate the fluorescence filter wheel to select the desired excitation filter set
[DAPI, FITC, TRITC or CY5].
-
There is a fluorescence light shutter underneath the filter wheel (at the right hand
side) that needs to be opened. Set to the O [open] position for the fluorescence
light to pass to the objectives. In the C position the shutter is closed. The shutter
should be kept closed [C] during transmitted light microscopy.
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-
2)
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Adjust the fluorescence light brightness by inserting the ND filters (ND4 and
ND8) into the optical path. These are located near the rear UV lamp housing.
TO START THE HAMMATSU CAMERA AND CAPTURE IMAGES:
-
Switch on the Hamamatsu camera by pressing the switch on the large camera
power supply box next the 24” computer monitor. Turn on the computer (PC on
the floor under the bench) and its 24” LCD monitor (button top right edge).
-
If you are a “Well” user log on to the system and network with your usual
username and password. Alternatively, log on directly to the PC as “User” with the
password “cyto02” or as ‘generic’.
-
Double-click on the NIS Elements [Basic Research]
-
Scroll down the pop-up camera driver window and choose the “Hamamatsu’ for
fluorescence B&W image capture. You can also select the ‘Nikon DS-U1’ for
capturing colour images, or ‘none’ just to view saved images. Click OK [any
camera selected must be switched on].
-
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 Port 5 for the Hamamatsu camera [Port 5 is
camera only, with no light to the microscope eye-pieces].
-
To view the sample via the microscope eye-pieces again, return the Port to 1.
-
You should see the Orca-285 Settings control window (camera controls differ
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depending on the camera selected ). If that window isn’t there, right click over the
non-image area and select ‘Acquisition Controls, Orca-285 Settings
[Ctrl_Alt_C] to activate it. Or you can also use View, Acquisition Controls,
Orca-285 Settings within the main menu.
-
From the main menu, click the Live (+)
button, or select Acquire, Live Fast
or Live Quality to open up a live image window on the screen. The “live image”
window appears. This live video window is intended to allow you to set the photo
exposure time and put the image in focus while viewing it on the computer
monitor.
-
You can modify the live image by altering the Orca-285 Settings options (image
acquisition parameters), e.g. hardware gain, exposure, command and advanced
setup.
icon on the desktop:
Note, if you use the Nikon DS-U1 colour camera with NIS Elements, you will get a very
different set of options under Camera Settings. See the specific Nikon colour camera
in-house Core guide for more details.
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-
Select the correct nosepiece objective
via NISElements
software using the Manual Microscope Pad [cntrl-alt-M]. Set the microscope
zoom to 1.0x or 1.5x using the
tab in the top menu. This will calibrate the
image in m/pixels allowing a scale bar to be added - using the Show Scale
button on the left of the live image window.
-
The Phase, DAPI GFP, YFP, TRITC and CY5 buttons
below the main menu bar can select and add the correct colour overlay [LUT] to
the B&W image, e.g. green for GFP and grey for Phase.
In addition the LUT colour can be switched to grey or back to colour via the LUTs
panel tab [Cntrl-Alt-L].
-
The bright grey colour scheme of the NISElements software can be changed to a
dark graphite colour if wished, using the sun/moon tab
at the top right of
the screen.
Exposure settings
-
Adjust the camera Manual exposure time as required via the drop-down. The time
is listed in milliseconds to seconds. A slower exposure time will brighten the
image. You can set the exposure using the Auto Exposure button.
-
With Manual exposure, set the camera hardware Gain to 1.0.
-
Clicking the “Pixel Saturation Indication (Cntrl-Shift-S)” button
will enable
you to see pixels that are saturated [over-exposed & overlaid bright red]. Click the
View, Visualization Controls, LUTs to bring up the LUTs panel.
-
Use a consistent manual exposure time if you want to compare samples (or the
software might automatically brighten/darken inherently dark/light images). Also
ensure other settings (e.g. Gain, Binning) are consistent [Orca-285 Settings]. Make
a note of your settings – the camera settings will be saved with the image if you
save in formats such as tiff or nd2, but not with jpg.
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Capturing a bright-field image
-
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View a live image using the Live (+) button
below the top menu bar. Capture
the image using the Capture button
(cntrl+ or -).
Avoid increasing the camera gain too high as it will introduce excessive noise. With brightfield images you can increase the halogen lamp brightness instead. Higher camera Gain can
help with dim fluorescence samples by reducing camera exposure time (faster video refresh).
You can also ‘average frames’ to reduce noise using Acquire, Average.
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-
On capture the “Freeze” button
below the main menu will freeze the video
preview without closing the window. Clicking the Live(+)
button again will
return the window to a live image for focussing and exposure adjustment.
-
To capture a transmitted light [bright-field] picture, click on the camera icon
[Capture
(Ctrl+)] in the Live window. The ‘Phase’ button
should be
selected.
-
This will acquire a high resolution image at the Orca-285 Settings, Format for
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Capture resolution e.g. 1344x1025 pixels [no binning] or 672x512 pixels with
2x2 binning. The live image is may be set to a lower resolution, i.e. 672x512
pixels [2x2 binning] to make the live image more responsive during focussing
[Format for Live under Orca-285 Settings].
-
Capturing an image freezes the ‘Live-Fast’ window, so click the video camera
Live(+) button
to restore the live video image.
-
If both the Nikon colour and Hamamatsu B&W cameras are switched on, you can
swap between them via the main menu Acquire, Select Driver.
TO CAPTURE AND SAVE B&W FLUORESCENCE CAMERA IMAGES:
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-
Ensure the [phase contrast] bright-field halogen lamp is switched off.
-
Right side, under the microscope objectives: Move to the correct fluorescence
filter set: DAPI (uV - Blue), GFP (Green), TRITC (Red) or Cy5 (Far Red). Move
the mercury lamp shutter to O [Open]. Focus on the specimen [Port set to “1”].
-
Switch to the Hamamatsu Orca B&W camera at Port “5”, which sends all the
light to the camera.
-
Click the Live
button (+), or select Acquire, Live Fast to open up a live
image window on the screen. The “Live - Fast” dialog box appears. Set the photo
exposure time [Orca-285 Settings] and put the image in focus while viewing it on
the computer monitor.
-
You can use Acquire, Capture Multichannel Image, Multichannel setup to set
the software for the sequence of fluorescence images your wish to acquire, e.g.
tick DAPI [blue], FITC [green], YFP [yellow], TRITC [red] or CY5 [purple] as
required – you only need to do this once.
The Hamamatsu camera captures 12-bit B&W (4,096 grey levels) images by default, with a
maximum resolution of 1344 x 1025 pixels [with binning set to 1x1, i.e. no binning]. This
can be reduced to 672 x 512 pixels with binning set to 2x2 – where every four pixels of the
CCD are combined, doubling camera sensitivity and halving resolution. Camera binning is
adjusted in the Orca 285 window, under the Format for capture tab. Generally leave binning
to 1x [the camera’s maximum resolution]
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-
Acquire a fluorescence image sequence using the Acquire, Capture
Multichannel Image, Capture Manually menu option. The Live-Fast window
will open. Click the Camera [Capture (cntrl +)]
icon. The software will
acquire a phase and fluorescence image sequence, pausing at each channel
[fluorochrome] to allow you adjust the camera settings [exposure] and focus. Use
the ‘Next multichannel capture button’ to capture the next image. A multichannel
image will be manually captured, e.g. a colour combined Phase, DAPI, FITC, and
TRITC fluorescence image [All].
-
Select the ‘In colour’ button under LUTs can apply the correct fluorescence
colour overlay (e.g. green for GFP) or to view the image in B&W grey.
-
You can alternatively capture a single channel fluorescence B&W image using the
method outlined on the previous page for capturing a bright-field image. Simply
click the Phase, DAPI, GFP, YFP, TRITC or CY5
button below the main menu bar and the live view will be set for that
fluorochrome and add the correct colour overlay [LUTs] to the B&W image, e.g.
green for GFP and grey for Phase. Use the Camera icon
[Capture (Ctrl+)] to
capture an image. Capturing an image freezes the ‘Live-Fast’ window, so click the
video camera Live(+) button
to restore the live video image.
-
The colour look-up-tables [LUTs] are applied to a 12-bit 4,096 grey levels B&W
image. If you save it as a Tiff file the image will still be 12-bit and appear black in
Photoshop – saving a jpg will convert the image to 8-bit [256 grey levels] and
overcome the problem. Alternatively you can use freebie NISViewer, ImageJ or
MetaMorph to view to the 12-bit B&W image. You can convert the image from a
12-bit B&W to an 8-bit B&W or RGB colour image within NISElements using the
‘Image, Convert’ menu options (e.g. Change bit depth, Convert to RGB). Ask if
you need assistance.
-
You can adjust the image contrast by grabbing the grey level histogram sliders in
the LUTs panel
. This can remove background fluorescence in the dark
regions of the image by pushing dark grey pixels to black. To make the contrast
adjustment permanent click on the LUTs ‘Modify Image Through LUTs’
button. Load the LUTs panel using Cntrl-Alt-L.
TO SAVE THE FLUORESCENCE CAMERA IMAGES:
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-
Go to File, Save As. Save in either TIFF [uncompressed for archiving, editing,
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image analysis] or jpg [compressed for email, PowerPoint] . If you save in
JPEG2000 or ND2 probably only NIS Elements can read the format.
-
Save images in your own folder in “User Images” on the E:\ [User Data] drive.
Images are 12-bit black and white (4,096 grey levels) for this Hamamatsu camera. You can
use ImageJ: Image, Type, to convert these 12-bit images to 8-bit [256 grey levels] B&W, if
you want to view them correctly in PhotoShop or in PowerPoint. JPEG2000 is a new [more
efficient compression] licensed jpg format that may not be read by some programs - Photohop
CS4 requires a free plug-in.
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TO FINISH (…and go home):
-
Close NIS Elements. Switch off the camera power supplies. Press the halogen
transmission lamp on/off button at the base of the microscope on the left and turn
off the switch on the lamp power box.
-
Switch off the computer before you switch off the mercury lamp. Leave the 24”
LCD monitor in standby.
-
Before turning off the mercury lamp allow the fluorescence mercury bulb to run
for a minimum of 30 minutes and check with the on-line booking that nobody
else will be using the microscope in the next few hours.
-
Switch off the power switch on the mercury lamp power box and note your name,
the time off, date and indicated hours, in the notebook provided.
-
If you used the 60x oil objective please remember to wipe off the oil and inform a
member of core staff when you have finished so that the objective can be removed.
The 60x microscope oil objective should only be cleaned with the lens tissues
provided. The standard Nikon air objectives don’t need cleaning after use.
___________________________________________________________
If you have any microscope/imaging problems or queries, just ask a member of the
Microscopy Core staff for help. There is additional help and advice on our Core webpages: http://www.well.ox.ac.uk/microscopy.
©WTCHG “Fluorescence image acquisition using NIS-Elements [basic research] and the B&W
Hamamatsu Orca C4742-95 camera”.
This version updated: November 2012.
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TROUBLESHOOTING
If you get imaging problems, check the following:
Is there a shadow in the image with transmitted light illumination?
If you are using bright-field microscopy the condenser must be adjusted
correctly to Kohler illumination [otherwise you might see the dark shadow of
the phase rings in the centre of the image and get poor image quality]. Ask a
core member how to set up Kohler illumination on the microscope.
Alternatively an incorrectly set filter slider or other component may be
obscuring the light path.
Is the mercury bulb or transmission bulb turned on?
Check the orange and green lights on the power boxes and the microscope
white button/brightness control. Check light can be seen at the specimen.
Can you see a coloured light passing through the bright field [phasecontrast] sample?
Move the filter wheel to the bright field double arrow (↔) position.
Is the fluorescence filter block in place? Are you using the correct filter
block – do you need it ?
Rotate the filter wheel and see if any light of the right colour appears.
Have you checked the dial below the eyepiece?
It should be set to O.
Is the objective correctly positioned?
The objective wheel should gently click into position.
Is the port/optical path in the correct position?
Port 1 is for observation down the microscope. Port 5 is for the Hamamatsu
B&W camera only. Port 4 is split 50/50 between the microscope and the
Nikon colour camera.
Is the transmission light too faint or too bright?
Remove/Add the neutral density filters. Ensure the top ‘p↔’ polariser and
the black T-A polariser slider [left side, under the objectives] aren’t pushed
into the light path unless you need them.
FOR ANY QUERIES OR DOUBTS, JUST ASK
A MEMBER OF THE CORE STAFF
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