User manual | Quick user guide to BD Pathway 855

BD Pathway 855
Quick user guide to the Automatic
fluorescent microscope/HighThroughput Screener (HTS)
Lasse Evensen (March 2012)
Starting up the instrument and
imaging software
Insert the desired objective
Turn on the main switch
Turn on the computer located in the cabinet
Wait for the green ”Ready” lamp on the front of the microscope to
light up (if it doesn’t, re-start the microscope). The ”Ready” lamp assures that all movable parts are in the
correct starting position
When the “Ready” lamp is lit, turn on the mercury lamp(s) (Lamp A
is for GFP, Hoechst and PI. Lamp B is for Tritc, Alexa 647 and Alexa
546)
Type in the password to access the computer
Open Attovision software and choose your username (no password
required)
Never turn the lamps off and directly on again! If the lamps are
turned off you must wait 30 minutes before turning them on again
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Introduction to the
software
BD Pathway 855 BioImager
To perform image acquisition:
Some general info……
The macro is the process that ”tells” the Pathway what to
do
It consists of four main steps: (just as manual microscopy)
Positioning of the objective (on the pathway the plate is stationary and the objective move around underneath)
Finding the focal plane
Chose of filters
Image acquisition
When performing multicolor fluorescence imaging, filter
combinations must be defined and will be demonstrated
later (Slide 15)
If the microscope does not perform as desired there is
most likely something wrong with the macro!
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First, define which objective you
inserted!
1. Click on ”Setup” on the
main menu at the top
2. Click on ”Geometry”
3. From the ”Description”
drop-down menu in the
“Geometry Setup”
window, set the correct
objective
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The Attovision software:
Three important buttons
These three buttons
controls (from the left):
1.
Navigation of the
objective, which wells
are going to be imaged
2.
Dye Setup: Filter control
(chose your filter,
exposure time and gain)
3.
Macro setup: The
window where you
program the microscope
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The Plate Map Setup Window
Controls what plate type you have (96- or 384-well plate) and which wells you want
to image. In manual mode it controls which well you want to navigate the objective
to.
In the navigate mode (red circle), if you click on a
well the objective will locate to that well. For
example, click on C5 and the objective will locate
under C5. On your screen down to the left, the
software will inform you about the location of the
objective
In the select mode (red circle), if you click on a
well it will appear blue with a plus sign in the
middle. That means that you later want to include
that well in the automatic imaging. You can select
single wells or the whole plate
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The Dye Setup Window (signal tab)
Chose dye from the
drop-down menu
Be sure you are on the
signal tab
Adjust exposure time
(seconds) by typing in a
number or click on the arrows.
The same for Gain, choose a
number between 0 and 255.
Autoexposure (AE) sets the
exposure time automatically
Click on the
”Capture”
button to see
signals from
the camera
Dynamic range let you control the distribution of pixel
intensity. Extremes are 0 as Min and 4095 as Max
(widest distribution). Min:200 and Max:2000 means that
pixel intensity is distributed in a smaller range giving you
a more intense image. Auto Range sets the dynamic
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range automatically
The Macro Setup Window
From the drop-down menu,
find your macro
The steps in the macro
performed in each well
selected for imaging
(XY Offset: Positioning of the object, Laser
Autofocus: Finding the focus plane,
Fitc+Hoechst; the selected dye (filter)
combination
Available actions:
Click on each icons to get a
list of available focusing
methods, filter combinations,
objective positions etc. The
list will appear in the window
below. As an example,
available filter combinations
are shown
Montage capture
control (stiching of
tiles to image a
larger viewfield): If
montage capture is
desired it is chosen
here
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Building a macro
(How to instruct the machine
to behave as you want)
Step by step protocol
Program the microscope:
Macro setup – Object position
Open the macro setup window
Click on the XYZ-position icon (red
circle)
Click on XY-offset and click on the
”Insert” button (dotted blue arrow)
Double click on the XY-offset command
line (red arrow) and the ”xyz-position
setup”window opens. Here you can
choose the coordinates in the well the
objective shall travel to during imaging
If zero is typed in for x- and y-axis the
objective will travel to the middle of the
well
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Program the microscope:
Macro setup - Autofocus
Click on the Autofocus action
button (red circle)
Click on Autofocus Coarse – Bin
8 and click insert (blue dotted
line)
Use the arrow buttons on the top
left (red arrow) to set the order of
the actions. XY-offset should be
first followed by Autofocus
Double click on the Autofocus
Coarse – Bin 8 command line
and the “Autofocus Setup”
command window will appear
(Next slide)
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Program the microscope:
Macro setup - Autofocus
Check the box called ”Use dye settings” and choose which
filter you want to use to find the focal plane.
Hint: If cells are stained with Hoechst this is a nice dye to find
the focus
The cartoon explains how the autofocus work:
In the “dye setup” control window (slide 8) click the capture button
and use the focusing wheel to find the focus. If cells are stained
with Hoechst use this filter
During autofocus the objective starts from the z-posistion you left it
in. Based on the number you type in in the box ”Maximum distance
from starting position” (D) the objective travels up the indicated
distance D. In this example it travels 80 µM upwards.
In the box ”Number of intervals on each side” (n) you choose how
many times an image shall be acquired during distance D. In this
example n=8 meaning that 8 images are acquired as the objective
travels distance D.
The operation is repeated below the starting position. The objective
travels 80 µM below the starting position and acquires 8 images.
In total, after autofocus is performed 16 images are acquired and
the computer software uses an algorithm to choose from these
images and picks the one where the cells are in focus.
The chosen image sets the focal plane for acquisition of images in
that well
When adjusting autofocus in order to bring the cells in focus nicely:
Adjust n and D and click the “test”-button until the machine is able
to bring the cells into focus automatically
Test the focus-settings for the wells furthest away from each other.
If focus is found in these two wells it is most likely that it will be
found for all the wells in between.
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Program the microscope:
Macro setup – Choice of filters
Click on the dye setup button
(red circle)
Click on FITC+Hoechst and
click insert (to make new filter
combinations see next slide)
Use the arrows on the top left
to put the imaging action after
autofocus
Make sure that it is indicated
that you want to acquire ”1
datapoints” (red arrow)
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How to set up new filter combinations
(Probe Cycle)
Click on the symbol in the red circle
In the Probe Cycle Setup window: Give
the filter cobination a name
Delete any filters that were listed from
before (blue circle)
From the drop-down menu in ”Probe
Cycle Setup” window, find the filters you
want to include in the probe cycle.
Click ”Insert” button
Arrange the order of the filters by using
the green arrows on the left in the ”Probe
Cycle Setup” window. Hint: If you want to
use Hoechst as filter to find focus,
Hoechst should be the first filter in the
probe cycle
By clicking the ”Macro Setup” button
(slide 6) the ”Macro Setup” window will
appear. The new filter combination is now
available for use in your macro.
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The Macro
The macro is now complete. The
position of the objective is programmed,
the autofocus step brings the cells into
focus and since cells express GFP and
are stained with Hoechst the filter
combination FITC+Hoechst is chosen
The macro is only generated once for
each imaging task. Once you have
made a macro you use it over and over
again. You only need to check the
autofocus everytime you use the imager.
The macro is a program instructing the
microscope of what to do in each well
you want to image
What you have not told the microscope
yet is which wells that are going to be
imaged
See next slide for instructions of how to
import well locations into your macro
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How to import well locations into your macro
Open the ”multi-well plate map setup
window” (red circle)
To choose all the wells on a plate: In the
select mode click and drag the mouse
pointer along the plate while you holde down
the left button on the mouse.
To choose single wells: Click on the wells
you want to include in your macro
From the drop-down menu at the bottom of
the window find the name of your macro and
click ”create compound macro”
The ”Select Macro” window will appear and a
new macro with the extension “96-well” will
be generated. This is how you prevent to
overwrite the “mother macro”. You can
always go back to the “mother macro” and
import well locations, hence, you do not need
to build a new macro every time you are
going to do imaging
Click “OK” in the “Select Macro” window and
your well location will be imported into the
macro
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Steps in the macro
The objective will now travel to well B4
It will move within B4 to the coordinates
you typed in.
It will use the autofocus settings and find
the focal plane of the cells
Finally, it will acquire images using the
FITC and Hoechst filters
Then it will move to well B9 and repeat
the process there
It will repeat the process for all the wells
you have programmed the microscope to
image
In this example, separate images for FITC
and Hoechst will be saved and they can
later be quantified separately
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How to start imaging
Click on the ”Play” button (red circle)
The ”Run Macro” window will appear. Give your experiment a name
(Data type). The data will be saved in a folder with the same name
as you type in the ”Data type” box.
If not already done, choose which objective you are using
Click ”Play” symbol in the ”Run Macro” window
Imaging will start after about 30 seconds
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Export of images
How to obtain pseudocolours
and merge different channels
to one image
Open the experiment containing
the images of interest
1.
2.
3.
4.
5.
Click on the open folder icon and
locate your experiment
In the experiment folder open a
random well folder
In the random well folder choose
*.adf as filetype (see upper image)
Open the channels you want to
merge. In this example the channels
are GFP515LP and Hoechst (lower
image). Choose ”GFP515LP.adf” and
click open
To open more than one channel
repeat step 3 and 4. In this example,
to open Hoechst images in addition
to the GFP515 images choose
”Hoechst.adf” and click open
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After you opened the channels you want to merge this screen will appear.
The images are thumbnails and gives you an overview of the plate
imaged. You can get the overview for the different channels by clicking on
the tabs containing the name of the dyes (see red circle)
Next, to open all the
images acquired for one
single well do the
following:
Hold down shift+ctrl and
click on the well you want
to export images from.
For example: hold down
shift+ctrl and click on well
E5. In this example two
images are acquired in
this well and they will
appear on the screen
(see next slide).
22
How to merge two channels into
one image
As indicated with the red
circle, click on the ”Image”
tab at the main menu and
choose ”Merge”. The
window beneath will
appear:
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Merging two channels
Check the boxes for the colours you want
on your image (pseudocolors). In this
example the cells are stained with Hoechst
and express GFP. Therefore, check the
boxes for ”Blue” and ”Green”
Choose which image that are going to the
two channels. In this example GFP515LP is
put in the green channel and Hoechst is put
in the blue channel.
Intensity of the image can be adjusted by
chosing either subtract, add, divide etc in
the operation box and type in a number in
the value box. Click on apply to check your
settings and click OK when you are
satisfied with the settings.
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When you click ”Apply” the lower screen will appear like
Panel 1 below. If you want to clean up the image (in
this example there is to high intensity in the blue
channel), do as showed in the ”Image Merge” box to
the right. The result is shown below in Panel 2.
Panel 1
Panel 2
Remember, the high-throughput imager is not meant for acquisition of pretty pictures but rather imaging of a high number of cell cultures in a short
amount of time. Most importantly, the images must be of high enough quality to be quantified. Therefore, export only images representing the different
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conditions and not all the images on the plate since the image export operation would be far too time consuming.
Quick check list for
experienced users
When you have learned the
software and built a macro
imaging is easy
Check list
Is the correct objective inserted?
Is the correct objective selected in the software?
Adjust exposure time for the filters you want to use
Find your macro and check that your focusing method
are able to find the focal plane in the wells located the
furthest away from each other
Choose which wells that you want to image
Import the selected wells into your macro
Give your experiment a name and push ”Play”
That's it!
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