Transcript Slide 1

Fluorescence filter info
Mono/RGB selection lever
14
2
12
13
Fluorescence filter
controls
3
Focus wheel
14
Condensor turret disk
10
Microscope on/off
Microscope/computer
Selection lever
Camera
6
7
11
8
1
9
4
Stage up/down
Mercury bulb
Objective controls
Stage positioner
Focus wheel
Light on/off
5
Using the CCD microscope for Histochemistry
You should not use this microscope unless you have completed training with Rachel
Gregory or Liz Brooks.
1.
Start up procedure
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Switch on the computer. Fill in the log book (‘no bulb’)
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Turn on the microscope itself via the green switch on the
right-hand side.
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Turn on the camera situated on top of the microscope, you
should be able to see the function lights turn on if you hold your palm above it.
It is essential that the camera be the last thing to be turned on. Power
fluctuations caused by the other equipment may damage the camera!
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Below the camera there is a black circular turret
with ‘RGB’ and ‘Mono’ written on the left-hand
side. Ensure the silver lever underneath is
turned to ‘RGB’.
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Also, ensure the microscope/computer selection lever is
fully inserted.
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You are now ready to start the software. On the desktop you
should see an icon for a shortcut to Volocity, double click on it.
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Fill in username and password.
If you do not have your own username and password you should not be
using the equipment on your own-contact Rachel Gregory or Liz Brooks to
book a session. (if you have forgotten your password we can create you a new
one-no expense!)
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Create a new library and label it accordingly for your samples, save this in your
user folder.
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In the toolbar of Volocity go to ‘window’ and scroll down to select ‘show video
preview’.
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In the control panel that appears on the right-hand side,
ensure ‘colour’ is selected from the drop down
menu.
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The microscope is now set for your imaging.
2. Microscope Controls
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Firstly you will need to select an objective to use. The CCD microscope
has 6 different objectives;
The x5 x10 and x20 are low power objectives and are therefore good for
imaging areas of cells. These objectives do not require oil. The x10 uses
Ph1 and the x20 uses Ph2.
The x40 and x100 are high power objectives and are therefore useful for
imaging single cells or structures within cells. These objectives do require
oil. Only the oil provided (518 F) should be used. Both these
objectives require Ph3.
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Place your slide onto the stage. It should slot between the metal clips,
don’t push it under the objective just yet!
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First ensure there is adequate clearance between the slide and the
objective.
If there is not, move the stage down using the focus wheel. If the stage moves
quickly when turning the focus wheels, you will need to change the setting. .
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The focus wheel has two settings; coarse and fine. We recommend you
always use the fine setting. To switch between coarse and fine, there is
a button on the left-hand side of the microscope, behind the focus wheel
labelled coarse/fine. Depress it to switch between the two settings.
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Using the fine setting will make it easier for you to focus on your sample
and will also help prevent any damage occurring to the microscope and/or your
slide.
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If you are using a non-oil objective, position your sample on the slide
underneath the objective.
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If you are using an oil objective, put a drop of oil on your slide.
You don’t need much!
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It’s now best if you bring the stage down to it’s lowest position.
To do this press the ‘down’ button located towards the front of the
microscope base on the right-hand side.
The stage will automatically
move down.
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Now position your slide so it is directly underneath the objective. Bring the
stage up again by pressing the ‘up’ button.
The stage will automatically move up to it’s original position.
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Positioning your slide under the objective this way allows a clean
connection between the objective and the oil, and reduces the occurrence
of air bubbles in the oil.
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The light adjustments are located on the
right-hand side of the microscope. You
can turn the light on and off by
pressing this button
The light intensity is controlled by the black dial next to the main on/off
switch.
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For histochemisty images it is recommended you use the light at it’s highest
intensity. Press the 3200k button to set the light at the correct intensity for
imaging.
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If the image down the eyepieces has a strange colour to it, the filter wheel may
not have been replaced to it’s proper position.
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Change the filter wheel using the directional buttons located under the focus
wheel on the left-hand side of the microscope until ‘blank’ is displayed on the
black disk underneath the eyepieces.
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If you decide you want to change the objective you’re using, first bring the stage
down to it’s lowered position.
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Move round to the objective you now wish to use before bringing the stage back
up.
IMPORTANT NOTE WHEN CHANGING OBJECTIVES!
It is imperative that no oil touches a non-oil lens. If you are using oil and want to
change the objective to a non-oil objective, make sure you clean the oil off of
your slide first.
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Also check which Ph setting the objective you are using requires (it will be
written on the objective itself. This can affect the quality of the images you are
capturing.
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You must also remember to clean the oil off of the oil-lens using the lens tissue
provided.
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When moving onto a new slide, bring the stage down to its lowered position
before changing the slides. This will prevent any damage being caused to the
objectives.
3. Viewing your sample on the computer
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When you have identified an area you wish to image, pull the silver lever located
on the right-hand side of the microscope out one stop. This will carry the image
from the microscope to the computer.
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The computer imaging controls are found on the right-hand side of the viewing
window.
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There are 4 channels set up for you to use;
FITC/GFP
Rhodamine/RFP
DAPI/CFP
white light/histology
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Select the channel 4.
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Select ‘auto-exposure (AE)’, the computer will calculate the exposure time for
your image. You can alter this by moving the sliding bar or changing the numbers
in the panel.
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Once you have the settings right, right-click on the channel button and select
‘overwrite’. This will save your exposure settings for that channel.
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Next you need to adjust the white balance.
Ensure the ROI tool is selected and use
it to mark an area of your image which should
be pure white (i.e. either lacking in sample or a
piece of white paper can be used if preferred).
Then go to ‘video’ and select ‘auto white balance’ . It will take a short time for
this to be calculated, be patient.
• You can adjust the colour balance manually by using the individual colour slider
bars.
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To capture a snapshot: Just click on the camera button.
• The image on the screen will be automatically saved to your library, you can
alter its name here by double clicking on it.
• If you are taking multiple images of the same sample it is possible to name the
images before you have taken them.
• Double click on the acquisition set up icon
• The acquisition set up window will open. At the very top of this window, fill in the
‘title’ box with your sample identity and click ‘OK’.
Brilliant Image
• Every image taken will now be labelled with the wording in the title window with
numbered increments (1, 2, 3 etc).
•When you wish to return to viewing down the eye pieces, simply push the silver
lever on the right-hand side of the microscope back in.
5. Shutdown procedure
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When you have finished with the microscope you must:
• If you have been using oil, clean oil off of the lens you’ve been using with the
lens tissue provided.
• Move the objective turret round until the 5x objective is in place.
• Ensure the stage is left in the ‘up’ position
• Fully insert the computer/microscope lever.
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Unless you have been asked to leave the microscope on, or the next user is
waiting you must proceed with the rest of the shut down procedure.
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Firstly, it is essential the camera is turned off before any other equipment.
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Turn off the microscope and computer monitor.
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Replace dustcover over microscope.
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Please leave the computer on to allow remote access to Volocity elsewhere in
the building.
Don’t forget to return the room key to the log book on the 4th floor!