Apotome - Robarts Research Institute

TRAINING MANUAL
Zeiss Apotome
Wide Field Microscope
July 2014
TABLE OF CONTENTS
A. Start-Up Procedure ...................................................................................... 1
B. Image Acquisition ..... ................................................................................... 4
Single fluorophore image acquisition........................................................................
Multidimensional Acquisition ...................................................................................
Saving your Image......................................................................................................
ROI .............................................................................................................................
Z-stack........................................................................................................................
4
6
8
8
8
C. Transmitted Light ........................................................................................ 9
D. Display Image Properties ........................................................................... 10
E. Exporting your Image................................................................................... 10
F. Apotome ........................................................................................................14
Principles.................................................................................................................... 11
Acquiring an Image ................................................................................................... 12
Configurations ........................................................................................................... 14
G. Shut Down Procedure ...................................................................................15
A. Start-Up Procedure
1. Turn on the mercury burner (#1)
2. Turn the stage box on (MCU) - back right (#2)
3
3. If you are using the Apotome function, turn the Apotome
bon on (#3). If not, go to the next step.
1
4. Turn the microscope on (at the right side of the microscope)
2
5. Now turn the computer on and log in
6. Start the AxioVision Software by double clicking on the
icon in the desktop
Main screen:
tool bar
work area
Image Window
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition
This system has the capability to acquire multichannel, Z-stack and time-lapse images. There are
pre-set channels that you can modify it.
1- After turning all the components of the microscope on, select the objective that you want to use
Here are the specifications of the objectives in this system:
Objective
NA
Immersion
Working
Distance
Coverglass
thickness
10X Plan Neofluar
0.3
Dry
5.2mm
0.17mm
20X Plan Neofluar
0.5
Dry
2.0mm
0.17mm
40X LD Plan Neofluar
0.6
Dry
2.9mm
0 -1.5mm
63X Apochromat
1.4
Oil
0.19mm
0.17mm
2- Add the appropriate immersion (see table above) and load your sample with the coverslip facing
down
3- At the toll bar, select the appropriate filter cube according to your fluorophore (green, red or
DAPI)
4- Select EYEPIECE to visualize your image in the microscope
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition Continued...
If you want to change the filter cube to visualize a different dye, you can do it at the main
tool bar in the AxioVision software (see step #3 in previous page) or by pressing the reflector
button in the microscope (last 2 buttons on the right back side of the microscope)
4- After focusing your sample, close the fluorescence shutter to protect your sample from
bleaching. You can do this by pressing the FL button (first button on the right side of the
microscope) or in the software by clicking on the SHUTTER icon (toolbar)
5- To switch to the camera, click on CAMERA in the software (toolbar)
6- Click on LIVE in the main tool bar to visualize the image at the computer screen
To Adjust the brightness:
If you want to adjust the exposure time
(brightness) for your dye, select CAMERAS AxioCamHR in the work area window.
You can click on MEASURE to let the software find
the best exposure time for your image or change it
yourself by using the Exposure bars.
Measure is always too dark or too bright for your
purposes you can adjust the level e.g. set to 90%
for a darker image (underexposed) or 110% to
overexpose using the lower slider control. If you
have Auto Snap box checked the exposure will be
adjusted automatically before each single snap.
Checking the Auto Live box enables continuous
exposure time adjustment in the live image.
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition Continued...
To Adjust the number of pixels:
If you want to change the number of pixels of your image, go to
CAMERAS - AXIOCAMHR in the work area, select the FRAME tab and
choose the number of pixels that suit your experiments. We usually use
1388X1040 pixels as default.
Click on LAST to go back to the same number of pixels of your
previous image.
For single fluorophore images:
If you only have one dye in your sample, at this point all you have to do is
click on SNAP at the main tool bar. The image will be storage and you
must save it before closing the software.
For samples with multiple labels, use the Multidimensional acquisition
function.
Multidimensional Acquisition
The multidimensional acquisition was designed to
acquire multichannel, Z-stack and time lapse
images.
1-In the work area window, select
MULTIDIMENSIONAL ACQUISITION
2- In the Experiment tab, select the type of
image you want to acquire (Z-stack, time lapse...).
You don’t need to select anything if all you are
doing is acquiring a multichannel image
3-Click on the C tab to open the available
channels
By default, the software has 5 different channels
already ready to use. If you don’t need some of
them, just right click on the channel. This
command will inactivate the channel selected.
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition Continued...
Multidimensional Acquisition continued...
Channel
Filter Set
Excitation
Emission
Beam
Spliter
Designed for
Blue
488001-0000000
BP 365/12
LP 397
FT 395
DAPI, Hoechst, Alexa Fluor
405 and 350
FITC
488010-0000000
BP 450-490
BP 515-565 FT 510
GFP, FITC, Alexa-Fluor 488
Rhodamin
(Cy3)
488020-0000000
BP 546/12
BP 575-640 FT 560
Rhodamin, Cy3, Alexa
Fluor546, 555, 568
Far-Red
(Cy5)
488050-0000000
BP 640/30
BP 690/50
Cy5, Alexa Fluor 633, 647
Transmitted
light
424931-0000000
-
-
FT 660
-
Bright field image
The first thing you should do it to adjust the exposure time for each individual channel
before acquire the final image. To do this, select the channel you want to adjust, for example
DAPI, click on MEASURE. The camera will find the optimum exposure level for your sample. If
you are happy with the result, click on OK. If not, change the exposure time manually.
Do the same procedure for all the
channels you have and then press
START to acquire a multi channel
image.
AxioVision will display the overlay
image with all the channels. If you
want to visualize only some of
theses channels, on the bottom left
on the image window, click on the
channel(s) that you want to
inactivate. If you want to change
the color of the channel, right click
on it and choose a different color.
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition Continued...
To save an image
To save this image, go to FILE-SAVE AS,
choose the format of the image (zvi, tif,
jpeg or bmp) and save it. You can then
restart the whole process to acquire new
images.
To find out all the information about your
image, click on INFOVIEW in the bottom
left tab of the image window.
To capture an ROI
If you want to capture a region of interest (ROI) rather than the full
frame, click on LIVE and then REFRESH OVERVIEW in the frame tab.
A small preview of your image will show up in the frame tab window. Use
this image as your reference and change the size of the selected area
(green window) to crop your image. To go back to the regular full size
image, click on FULL.
To capture an Z-stack
To acquire a Z-stack image, go to MULTIDIMENSIONAL
ACQUISITION and select Z-stack under the experiment tab.
Select the appropriate filter cube for the dye you want to use as a
reference.
Under the Z-tack tab, select start/stop as MODE.
Now, press the ... button on the right side of the STOP button. A
window displaying your live image will be open. Change the focus all
the way down (counter-clock wise) and click on OK. This command
will set the bottom of your image.
Now click on the ... button on the right side of the START
button. Change the focus all the way up (clock wise) and click on
OK. This command will set the top of your image.
Robarts Research Institute
Apotome Training Manual
July 2014
B. Image Acquisition Continued...
To capture an Z-stack continued...
How far up and down you should go is a matter of your judgment. Try to avoid the planes with high
background (for example the plane closer to the coverslip).
After selecting the bottom and top of your sample click on OPTIMAL DISTANCE to set the
optimal interval between the stacks according to the optics you are using. If you prefer you can
enter this number manually. Click START to acquire a Z-stack image.
Tip: If you are combining multichannel and Z-Stack acquisition,
AxioVision will normally acquire all the z images for channel 1 then all the
z images for channel 2 and so on. This gives the fastest acquisition but
may suffer from slight z registration problems. However if you check All
Channels per Slice AxioVision will acquire all channels for the first z
position and then move on to the next z position and so on. This will give
exact z registration but usually increases acquisition time.
C. Transmitted light
If you want to take pictures of only bright field images, select TRANSMITTED at the main tool
bar and click live.
Adjust the exposure time or click on measure in the camera menu (workarea - left side). If after
clicking measure you think your image is not brigh or dark enough, you can change the percentage
(AxioCAmHR: Exposure) to a higher or lower number, for example 120% instead 100%. Make sure
to also select the Auto-snap option. When you are ready to acquire the final image (SNAP), the
software will adjust the exposure time to the percentage you have selected.
Robarts Research Institute
Apotome Training Manual
July 2014
D. Image Display Properties
You may want to adjust the contrast and brightness
of the displayed image. For that, click on the Image
Properties function and select DISPLAY tab.
You use this window to modify the way in which your
image is displayed. The histogram shows the
number of pixels at each intensity level for each
color (RGB). Contrast, brightness and gamma can be
controlled via the sliders or by dragging the curve
on the histogram.
Click on Linear for the default display curve.
Try selecting Min/Max to stretch the contrast
to the histogram maximum and minimum.
Tip: Best Fit is similar to Min/Max but it will
stretch the contrast a little further since it
ignores the extreme tails of the histogram. This
is set at 5% by default but can be changed.
Image Properties
E. Exporting your images
If you just want to export a single image then you can simply use Save As in the File menu and
select the file format. However if you want to export multidimensional images it becomes more
complicated as each image in the ZVI file must be exported as a separate image. In this case you
need to use the function described below.
First ensure that the image to be exported is the active document (i.e. click on the bar at the top)
then use Export in the File menu. Refer to the online help for a detailed description of this
function, we will just cover the main points.
Insert a Base name and a Save in location – all images contained within the ZVI will be exported to
a file with a name starting with the base name. If you click Create project folder a folder will be
created with the Base name which will contain all exported images.
Mode allows you to export some of the image e.g. a specified channel, time, z position or all images.
Generate merged images will combine multichannel images as overlaid color images.
File Type - select the output file format.
Convert to 8 bit (only seen when saving as a *.tif) will convert a higher bit image down to an 8 bit
TIFF.
Gray scale will convert a color image to a black and white image.
Apply display mappings will retain the current display property settings
Robarts Research Institute
Apotome Training Manual
July 2014
F. Apotome
Principles
In thick specimens, the signal from the focal
plane is compromised by the out of focus light
coming from planes above and bellows this
plane. The result is a blurred, low contrast
image. In this condition, is not possible to
render a three-dimensional projection of the
specimen.
Some techniques were developed to remove the
out of focus light, either optically (Confocal) or
mathematically (Apotome).
In the Apotome, a grid is inserted in the illumination pathway of a conventional fluorescence
microscope. A plane-parallel glass plate is placed in front of the grid and it tilts back and forth
projecting the grid in the image in 3 different positions. For each depth, 3 raw images with the
projected grid superimposed in 3 different positions are taken. The software uses the projected
images of the grid to calculate and removes the out of focus light. The principle is, if the grid is
visible that information is in focus. If not, that signal corresponds to out of focus light and it is
removed from the final image. The software combines the 3 images into 1 for each stack.
GRID
LIGHT
SOURCE
Image 1
sample
plane-parallel
plate
GRID
LIGHT
SOURCE
Image 2
sample
plane-parallel
plate
GRID
Image 3
LIGHT
SOURCE
sample
Robarts Research Institute
Apotome Training Manual
July 2014
plane-parallel
plate
F. Apotome
Acquiring an image using the ApoTome slider
As seen in the principles of the structure illumination method, the idea of removing the out of
focus light of wield field images is specially helpful for Z-stack images. Therefore, the first step is
setting the configurations for your Z-stack (bottom and top, interval and how many channels).
You can do this part without inserting the Apotome Slider in but in order to calculate the best
exposure time, you MUST insert the slider. Since the software removes the out of focus light of
the processed image, this image will look much dimmer that the non-processed one (regular wide
field). You need to compensate for that by measuring the best exposure time with the grid in.
1- Gently insert the apotome slider (move all the way in). You will hear a beep
2- Open the Apotome window (main tool bar)
if you click on MEASURE, the software will calculate the
best exposure time for your sample
if you click on Refresh Overview, your image will be displayed
in this window. You can then crop it.
select GRID VISIBLE
select OPTICAL SECTIONING
VH = for inverted and high magnification objectives
VL = for inverted and low magnification objectives
Our Apotome is calibrated for the 63X objective. Therefore,
you should always select the VH grid option. If you wish to
use another objective, the grid needs to be changed and a
new calibration needs to be done.
Robarts Research Institute
Apotome Training Manual
July 2014
F. Apotome
Acquiring an image using the ApoTome slider
Make sure that in the tab EXTRAS, the DISPLAY NORMALIZATION option is checked. This
option will rescale how the image is displayed, adjusting the intensity.
X
You are now ready to acquire your image. Press START in the Z-stack tab.
The system will acquire 3 images for each focal plane, changing the position of the grid (3
different positions). These images will be used to calculate the out of focus light and remove it
from the final image.
If you want to continue using the Apotome mode for your next images, just keep the slider in, find
a different cell/area and repeat the process.
If you want to go back to the widefield mode, pull the Apotome slider out (gently) and close the
Apotome window. You will have to set new exposure time for your channels.
Regarding the Apotome Filter and Averaging, if your
final processed image shows the grids, you can apply a
weak filter and noise reduction of minimum 2 to
improve it.
WIDE FIELD X APOTOME
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Apotome Training Manual
July 2014
F. Apotome
Configurations
How the configuration affects your final image:
Acquisition Mode
These options influence the way the image acquisition is done.
Ø No Processing: In this mode, only one image is acquired, with the grid in its current position. No
processing takes place. This function is particularly important if, with automated multichannel
image acquisition, you want to acquire an additional image in transmitted light. In this case you need
to deactivate the acquisition of three raw images with subsequent processing, as fringe projection
does not take place in transmitted light.
Ø Optical Sectioning: In this mode three images are acquired, combined online into an optical
section, and displayed.
Ø Conventional Fluorescence: In this mode three raw images are acquired and combined into an
image resembling the widefield situation. This mode allows comparisons between widefield image
and optical section.
Ø Raw Data Mode: In this mode all raw images (i.e. the individual images with the imprinted grid
positions) are kept after acquisition. The advantage of this mode is that it allows you to switch
between the algorithms (Optical Sectioning or Conventional Fluorescence) after image acquisition.
Switching takes place in the Properties window. In addition, the different filter operations or
image- vs. display normalization (usually gives result images with better dynamic range) or even
further improve resolution by deconvolution can be done after acquisition.
Filter
Depending on the sample, bleaching rates of the fluorescent dye, exposure time, vibrations in the
setup etc., residual stripes may be visible in the resulting image. These lines can be removed using
the filter. The options Off, Low, Middle and Strong are available.
Any repetitive lines or stripes appear as points along a vertical line in Fourier space. The filter can
remove a defined group of these points out of the Fourier spectrum.
· Off: No filter operation
· Weak: Suppression of the 3rd and 6th order of the fundamental grid frequency
· Medium: Suppression of the fundamental, the 3rd and 6th order of the fundamental grid
frequency
· Strong: Suppression of the fundamental, 2nd, 3rd and 6th order of the fundamental grid
frequency
Start with the setting Low. If residual lines can still be identified in the image, increase the
setting step by step.
Robarts Research Institute
Apotome Training Manual
July 2014
F. Apotome
Configurations continued...
Averaging (Noise Reduction)
This function can be used to achieve noise suppression through the sequential acquisition of several
images and the subsequent averaging of the gray values. It is possible to acquire and average
between two and five images. This ApoTome 2 function not only acquires and averages a number of
images, but also displaces the position of the grid lines in each case. This means that the function
also enables you to minimize residual lines in the resulting image when working with samples that
bleach out easily.
G. Shut Down Procedure
1- Save all your images
2- Before shutting everything down, check the online calendar to see if there is anybody coming
after you. If the next user is coming within 1 hour after you, close the software, log off and leave
the other components on. If the next user is coming with more than 1 hour after you, proceed with
the shut down steps listed bellow.
2- Close the software and shut computer down
3- Remove your sample and clean the objective
4-Turn the microscope off
5- Turn the Apotome box off (if used)
6- Turn the Fluorecence lamp off
7- Turn the stage box off (in the back)
8- Cover the microscope
9- Sign the user sheet
Robarts Research Institute
Apotome Training Manual
July 2014