Manual DELTAVISION deconvolution microscope

Manual DELTAVISION deconvolution microscope
Manual DELTAVISION deconvolution microscope
Switching on the system
1) For live cell imaging experiments, warm up the microscope system a few hours before
acquiring images. This can be done overnight. Switch on the temperature control unit on
the left side of the microscope, and close the cabinet around the microscope. Wait with
the CO2 supply until the cells are placed and you are ready to start the experiment.
But before, placing the cells in the microscope, complete the start up of the system first
and create your experimental set up (‘Creating an image file’).
2) Check if the microscope stage is empty of holders, and that the objectives are in the
lowest position.
3) Start by switching on the following components
a) Powerware 9125
Press the right button for a few seconds until the continuous beeping.
b) Instrument Control Unit
Behind the lid, press the right button.
c) Workstation Unit
Behind the lid, press the right button.
d) Workstation
Press the orange switch, which will turn on.
4) Windows will start (note: no mouse will be detected), and automatically IC50-DV control
window will open.
5) Press F1 and Y.
Note: during the system initialization, errors may occur. Exit the program and open the
DV instrument controller using the arrow keys on the keyboard.
Note: When the program freezes, switch off the following: switch 3d, and the middle
button of 3a. Rerun from point 3 onward.
6) After the system initialization by the DV instrument controller (XY stage coordinates, z
coordinates, filter wheel … starting normal operation), switch on the mercury lamp. The
Burner On lamp should light up.
7) Toggle to Linux using the select button on the D-link box.
8) Login: root
Password: Fogdevreten
9) Start the program SoftWoRX.
10) Position the objective into place manually. Place the object glass holder (or the live cell
imaging holder) on the stage, and tighten the screws. Mount the specimen in the holder.
Note: the 40 and 60x are oil objectives and require oil, which you can find at the
microscope. For live cell imaging, place the plexi-glass cover over the cell chamber and
position the chamber on the stage. Turn on the CO2 supply.
Acquiring an image:
1) Open the different windows required for image acquisition by selecting Acquire
(Resolve3D) in the file menu.
2) In order to look at your samples with the eyepieces, rotate the Beam Selector on the right
side of the eyepieces to the eye symbol:
3) Select the eyepiece filter by rotating the eyepiece filter wheel below the oculars. As you
rotate the filter wheel, on the monitor the filter name next to EP (eyepiece) changes and
the EM and EX (emission and excitation) filters change automatically to match.
Available filters on the microscope:
DAPI
(DAPI , Hoechst)
FITC
(FITC, Alexa488, GFP, CY2)
RD-TR-PE (TRITC, Alexa543, RFP, CY3)
YFP
CFP
4) Open the shutter of the mercury lamp by pressing the EX SHUTTER button in the lower
left corner of the key pad.
5) Rotate the filter wheel to look at different fluorophores. Note: the shutter will
automatically close when you turn the filter wheel, so press the EX SHUTTER again for
excitation light.
6) When your sample is focused and centered through the oculars, close the shutter (press
EX SHUTTER) and rotate the Beam Selector from the eye symbol to the SP position.
This directs the light path to the camera.
7) In the Resolve3D window:
i)
ii)
iii)
Select the appropiate filters for excitation and emission
ND = 100% (you can change this so more light is blocked rather than adjusting
the exposure times which could result in faster acquisition). Use higher ND
(lower %) if your image is very bright, or the camera saturated with lowest
exposure.
Exposure. To start with, enter 0.1000 sec.
iv)
v)
Lens. Under Info, the appropriate lens oil number can be determined (correlated
with temperature and mounting medium).
Bin:1x1
8) Press ‘Acquire’ in Resolve3D to view the cell in the window. Use the XY position
buttons in the middle of the Resolve3D window to center the object of interest. Press
‘Acquire’ to get an image of the cell in the new position. Similar procedure for finding
the best Z-position.
XY:
Z:
9) Adjust the exposure time if needed. You can also find the appropriate exposure time by
clicking the Find button (‘4095’ is saturation of the camera. Try to get as close to the
max, but remain below this value).
10) The settings for this fluorophore are set. Switch to another excitation/emission filters in
the Resolve3D window to optimize the exposure for other fluorophores.
Creating an image file:
1) First define the folder where your images will be saved: click on Settings in Resolve3D
window:
The images will be saved in a folder on the common directory /data1.
2) Open the Design/Run experiment window by clicking Experiment in Resolve3D window.
Here you can enter the file name of the image. Before clicking start scan, the acquisition
protocol needs to be designed.
3) Click the design window…several tabs are available:
- Sectioning:
For acquiring images along the z-axis, you need to define the upper and lower limit
for the image first. This can be done in the Resolve3D window.
First go to the lowest position in the z-direction using the arrow keys. Press the lower
limit button (right). Then go to the upper limit and press the left button.
Now that the range is defined, you can get the sample thickness by clicking on the
corresponding button in the Sectioning setup. Define the number of slices or the
optical section spacing.
-
Channels: Different channels can be selected with the different excitation and
emission filters, exposure time, ND filter. The software uses the settings that were
optimized in the image setup (see above Acquire an image, point 8/9).
-
Time-lapse: The number of time points can be entered and the total time. The timelapse will automatically be calculated. Alternatively, enter the time lapse and the total
time, and the number of time points is automatically calculated.
-
Point visiting: With the point visiting function, you can acquire images on different
XYZ coordinates sequentially. There are two ways to mark points within the sample:
using the keypad or via the Resolve3D window.
With the eyepieces you can find another object of interest. Press the point mark
button on the lower right of the keypad to store the coordinates. Or press in the
software:
The marked points can be seen by opening the Point List:
In the point visiting setup, enter the marked point numbers. For example: 1,2,5, 7-10
4) When the acquisition protocol is designed, go back to the Run Experiment window.
There will be a few windows popping up; press ok for those windows. Start the scan.
Data transfer:
The image files will be saved in a folder on the acquisition computer as described above
Creating an Image File 1). This folder is usually located in /data1. This data can be
analyzed/transferred to data carriers in the analysis computer, next to the deltavision
microscope system.
1) In linux, click StartHere, and select ‘home’. Find the folder /data1 in the folder tree. If
needed use the ‘up’ symbol. Double click on the /data1 and select your own folder.
Keep this window open.
2) Click StartHere again, and according to the same procedure, open the /deltavision2
folder. This is the shared folder for the 2 computers.
3) Drag and drop your folder in /deltavision2 folder.
4) Start the analysis computer (same login/password), click home and open
/deltavision1, the shared folder again. Open your own folder.
5) When image files are double clicked, the Resolve3D software is opened. Here several
image visualization tools can be applied. In addition, the images can be exported
(save as) as TIFF files and/or movies.
6) To copy your data to an external hard disk or USB stick, and right-mouse click on
your folder/image files, select ‘copy to’, ‘browse’, ‘storage media’,…..
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