Cancer Institute Microscopy Core Facility
Ocular Viewer
Main Stage
Mercury Lamp
(Reflected light)
Control Box
Microscope Power
Supply Box
Microscope
control
touch-screen
Viewing
Slide
1st Focusing
Wheel
ZEISS AXIO IMAGER A1 AND M1
A USERS GUIDE
This is intended to provide a basic coverage of use of the Zeiss Axio Imager M1 for multichannelled fluorescent acquisition. For more complex usages please contact
Fig.1 – Front View of Microscope
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Axiocam
CCD image
capturer
Viewing
Slider
Objectives
Main
Stage
XY
adjust
RL Natural
Density filter
wheels
RL aperture
control
Reflected light
(RL) centering
bulb
Slide
Holder
RL Glare
block
Transmitted
Light (TL)
adjustment
wheel
Ocular
Viewer
height adjust
1) SWITCHING ON
CHECK MICROSCOPE LOG BOOK OR WITH PREVIOUS USER PRIOR TO SWITCHING ON. 30 MINUTES MUST
HAVE PASSED BETWEEN TURNING OFF REFLECTED LIGHT LAMP AND TURNING BACK ON IN ORDER FOR
MERCURY BULB TO COOL.
a) Switch on mercury lamp control and Microscope power boxes located
proximally to main microscope stage. Turn on Microscope switch (located
behind 1st focusing wheel)
b) Turn on computer and Activate AxioVision Program
Fig 2 – Side View of Microscope
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2nd Focusing
Wheel
TL aperature
controls
Stage Returning
Button
Stage Dropping
Button
Fig 3 – Side View of Lower Microscope
2) LOCATE FOCAL PLANE OF SAMPLE
a) Using the microscope touch-screen open TL (Transmitted light) filter
b) Reduce light level using TL adjustment wheel
c) Place sample slide in slide holder.
d) Select magnification
if using x40 or x60 addition of droplet of oil is necessary. This is
achieved by pressing stage dropping button and adding a tear sized
drop on top of slide (so cover slips are necessary) and returning
stage by pressing stage returning button. Make sure at this point to
press ‘set working position’ on the touch screen control.
e) Using Ocular Viewer (viewing slide must be fully inserted) focus the sample
using Focusing Wheel. (For finer focus use focussing 1st focusing wheel)
f) Switch TL filter to closed on touch-screen controls
3) ACQUISITION OF IMAGES
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It is possible to choose various methods of acquisition of an image and most
actions capable from the software are possible. This will show you how to collect a
Z-Stack. If you only desire a single multichannel image you can leave out to portions
about Z-stack. Remember to not select a Z-stack (section f) )
Fig 4 – Axiovision Tool Bar
a) Select Workarea tool bar if workarea is not showing – Fig 4
Fig 5 – Work area
b) Select Objective of choice (ensuring
oil is used as previously described
above in 2c) or leave as presently
chosen - Fig 5
c) Select ‘Multidimensional Acquisition’
sub tab (as shown also in Fig 6)
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Fig 6 – Multidimensional Acquisition Experiment Tab
d) Select Experiment tab and click on
ReUse button – Fig 6
e) Select a .zvx file normally found in
directory;
‘my documents/Carl Zeiss/data/experiment’
These files are labelled with the
combination of channels that will be
examined by microscope if chosen.
For example in the Experiment box
shown in Fig 6 the name shown is
‘dapi-gfp-rhod-phase*’ – This file will
make the microscope acquire images
through all 4 channels
It is also possible to reselect settings
from a saved image to replicate the
settings from that. This is achieved in
the ‘ReUse’ drop down menu and
choosing to load from image.
f) If you desire to create a Z-Stack click
the Z-Stack button in the ‘Options’
box
g) Select the C tab – Fig 6
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Condenser
aperture
slider
Condenser
filter wheel
Fig 7 – C tab of Multidimensional Acquisition
h) Ensure the correct condenser filter is
selected for application – Fig 8
H – Brightfield
2 – Phase contrast in x20 objective
3 – Phase Contrast in x40 and x60
Objectives
I,II and III are for DIC applications which are
not currently installed, treat as brightfield
1 is intended for phase contrast using an x10
objective which is not currently installed
i) It is now possible to select the optimal
settings for each channel by clicking
on each channel separately and
selecting measure in the exposure
box – This will automatically select an
optimal exposure time, if you desire a
known exposure time to generate
comparable images select the desired
exposure time. The Exposure
Measurement window will open.
Adjust exposure here or click ok
Figure 8 – Condenser Controls.
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If you do not desire to collect a Z-stack ignore sections j) to o)
j) Open the Z tab – Fig 9
Fig 9 – Multidimensional Acquisition Z tab
k) if you desire a collection of images
through the Z axis click on Z button at
top of tab – as shown fig 10. Select
Start/Stop mode as shown.
l) Using ocular viewer focus (slide
viewing slider in) to the top of your
sample and click on start button – the
computer now marks where imaging
will begin
m) Focus to beneath sample and click on
stop
n) Select a slice distance so that z slices
are at desired distance from each
other.
o) Return Viewing slider to the original
position (pulled out)
p) Click on Start – located at bottom of
screen near left side.
q) Once the series of images (or image)
is collected it is prudent to save
immediately. Saving as a .zvi file is
recommended as ALL data about how
it was collected by the microscope is
saved. These files are larger but you
can return to the file and process to a
much greater proportion to other file
formats. It is possible once you have
finished creating desired images to
export as a smaller file type of choice.
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a)
b)
c)
4) IMAGE PROCESSING AND ANNOTATION
VIEWING YOUR IMAGE OR Z-STACK
You should now have a Z-Stack of you sample in every channel desired - Fig 10. In this
format the data is still raw and is best saved to be processed separately afterwards.
Fig 10 – Z-stack Window
a) You can turn each channel collected on and off using the channel buttons.
b) Scroll through the images top to bottom using the Z scroll bar or use the play button
to animate the scrolling.
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c) Choose a view with each channel tiled separately using gallery view.
DECONVOLUTION OF Z-STACK
Fig 11 – Deconvolution Workarea
Axiovision software comes with a
deconvolution image processing option.
The function of deconvolution is to ‘clean up’
images to remove positional and chromatic
aberrations. There are a choice of 3
algorithms which each function in their own
way. Simply click on the image you want to
process and then click on start when
deconvolution is selected. This process can
take a long time as it is very processing
dependant. More slices will take a lot longer
to deconvolve, as will more channels.
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ORTHOVIEW
Fig 12 – Orthoview Workarea
The Orthoview option will take a Z-Stack and
average and overlay images so that all the
information from the Z-Stack can be viewed
at once in a 2D image.
a) select image to process
b) select initial and final frame of view
desired and enter into parameters
section of workarea
(StartPos and EndPos – Fig 12)
c) Select dimension of view
(default of ZY)
d) Click Start to create new image.
Fig 13 – Example of Orthoview projection from Z-Stack shown above
If you compare this image to Fig 10 you can
see that more background green tone is
present. This image was orthoview-
processed but not deconvolved. The
combination of both can produce much
clearer images.
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ANNOTATIONS
Axiovision can annotate images for publication. Things like Text, Arrows, Scale bars
and geometric shapes can be added to your choice of colour, size and style. These can be
chosen from the annotations tab in the tool bar – Fig 4
Fig 14 – Scaling Tab
a) When adding a scale bar the scale of
the pictures is not automatically
chosen to be the right magnification.
When you choose to annotate with a
scale bar a scaling tab needs to be
opened. This allows you to set the
scale of the image
b) Select scale and click apply to image
or both activate and apply to image
for multiple images of same
magnification.
c) Draw scale bar on image as desired
Fig 15 – Example of Annotated Image
Size of scale bars can be chosen by clicking
On end of bar and dragging it to a larger
size
Other annotations such a text or geometric
Shapes do not require setting to use.
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Fig 16 – Annotation / Image Properties Window
Figure 16 shows the properties window that can be accessed for any image by right clicking
on the image and selecting properties. It is in this window that the Attributes tab can be
selected where properties of annotations can be changed. This is particularly useful when
annotation colour is not contrasting enough to background.
In the Display tab image properties such as brightness, contrast and gamma can be altered.
These are useful when trying to bring out morphological data in an image.
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