Centre Optical Instrumentation Laboratory

Deltavision Elite switch on and set up procedure

Switch On Procedure

1. Turn on hardware using the button on the lower left of the electronics box (labelled A), then turn on the Windows XP computer using the silver button on top left of the electronics box (labelled B).

DV On Buttons

2. Log in to the linux computer.

3. Open SoftWoRx

DV Start Icon

4. Check that the scope objective has been lowered out of the way of the stage. This is important as its possible for the stage to hit the objective which will potentially damage both.

5. Select File then Aquire/Resolve 3D.

File Icon

6. Double check that no objectives are obstructing the stage movement and then initialize the scope.

 

Basic Imaging Set Up

Settings A. Set the Excitation filter. Note the filter choices available are dependent on which Set you selected selected within the settings tab. The DV software will remember all of your settings based on the Excitation filter
B. Emission Filter selection
C. Polychroic (See the list below for which one to use
D. This is the % excitation light reaching the sample from the lightsource. This can be reduced if the camera is oversaturating and Exposure time is already at the lowest permissable value (0.035sec)
E. This sets the illumination source, usually EX for fluorescence imaging or TRANS for brightfield
F. Exposure time in seconds, this can't be set lower than 0.035
G. Objective in use, make sure you have the correct one selected as this has a significant effect on any deconvolution or measurements that are made. If its wrong its a lot of work to go back through the images to correct the log files.
H. This sets camera Bin. For normal use 1x1 is OK, if speed is required this can be changed to 2x2 or even 3x3 but this significantly reduces image resolution as pixels are summed together. Its also a very good way of increasing image intensity as pixels are summed but it will be at the expense of resolution.
Lower Panel A. Click Icon to take image
B. Autofocus button, this will very effectively focus an image but it gives a very high light dose so isn't recommended for live cells or easily bleached fluorophores.
C. Icon sets the bottom limit of a Z series
D. Icon sets the top limit of a Z series
E. Icon used to centre a particular image feature, eg. Click icon then click a cell that you would like in the centre of the image. If the correct mag and Aux mag are being used the cell should immediately move to the centre of the field.
F. Icon selects the points list used for point visiting
G. Used to set the zoom level of the slide map. Every time an image is taken a mini thumbnail is taken and added to the black area. This iccon allows the thumbnail to be zoomed in or out to give an overview of the slide.
H. One of the actual thumbnails
I. Dragging and dropping the horizontal bar moves the Z focus position, this is usually used in conjunction with items C and D
J. When taking images for deconvolution its very important to take notice of this histogram as it shows the spread of grey values within your image. The greater the spread of grey values (in simple terms the amount of light gathered) the more accurate the deconvolution algoritims, also, any intensity measurements are much easier the greater the spread. As a general rule you need a difference of at least 1000 between the Min and Max values.

 

Step by Step Image collection for Dual colour, Z, time and multipoint

  1. Set First colour channel eg DAPI excitation and emission, set exposure time and click camera to acquire image.
  2. Check histogram to make sure suffcient grey values have been collected, adjust exposure time and %T accordingly. Make sure that the correct objective is selected and that Aux Mag is set correctly.
  3. Set Second colour channel in the same way as the first.
  4. Adjust image so that feature of interest is centred using centring tool
  5. Set Z stack by dragging and dropping Z bar until the top of the specimen is reached then click set top icon , then drag the bar in the other direction to set the botttom using set bottom icon . Its important to set the focus to the middle of your specimen to get the full Z stack, this is done using the set middle icon .
  6. Set the point from the point list, this can be opened by clicking the point list icon
  7. The point list dialogue looks as follows every time a new point is added its position in x,y,z is recorded. NOTE once a point has been added you cannot use the focus on the microscope body, this is not motorised and therefore not linked to the software. If you touch this focus all Z points will be rendered useless. To focus whilst adding points use the arrow icons this works down the eyepieces or when using the camera and it maintains the Z position information. Points can be visited, deleted and replaced as required.
  8. You now the basic information required to set up an imaging experiment so click Experiment
  9. Click Sectioning Tab it should be ticked by default , make sure that the focus point is set to middle of sample. Click on Get thickness and it uses the top and bottom of sample that you marked previously to set the Z range. Choose the optical section spacing required and the software will automatically calculate the number of sections.
  10. Click the Channels Tab and put a tick in the box next to as many channels as you set up previously, remember the channel settings are linked to the Excitation filter so all you need to do is select the Excitation filter and the rest of the settings are automatically filled in for you. Check that the ccorrect Polychroic is being used for your filter set.
  11. Click the Timelapse Tab and put a tick in the Time-Lapse box to activate it. The Time-lapse boxes are to set the gap between images and Total Time sets the length of the timelapse experiment. Be careful not to set a timelapse interval shorter than the time it takes to take all of your image poins or Z stack. If the timelapse is likely to be a long experiment its worth using Ultimate focus to prevent focus drift. Set it up for 0.1um movement threshold and a maximum of 6 iterations.
  12. Click Points Tab and put a tick in the Visit Point List to activate points. Add a comma seperated list of the points you would like to visit in the dialogue box. Make sure that Ultimate Focus option is ticked and properly set up.
  13. To start the experiment click the Play icon

NOTE The only essential parts are the Sectioning and Channels tabs the others are optional and don't need to be set if your experiment doesn't require it.