The FV10-ASW 4.2 confocal image acquisition software is very modular and the main panels are:
1. Acquisition Setting: allows you to set up the parameters for the confocal image acquisition
2. Image Acquisition Control: here you can set up the lightpath for your experiment and start an image preview or the image acquisition
3. Live View: shows as live preview of your sample
4. Data Manager: displays properties of selected image files
5. Explorer: here you can access saved files and load acquisition parameters of previous experiments
First you need to set up the light path including lasers and detectors using the Dye List menu. This can be accessed by clicking the on the Dye List icon located on the right hand side of the Image Acquisition Control panel.
In the Dye List menu (shown below) drag the dyes you would like to use into the Selected Dyes box or double-click to select them. If you want to remove any dyes you can drag them out of the list individually or click clear all to remove all dyes and start over. Click Apply and the lightpath for the selected dye combination will be configured automatically (includes activation of lasers and selection of detectors and filters)
Please bear in mind that you cannot select more than three dyes at a time and that certain dye combinations are not available such as green, red and far red. If you would still like to image this dye combination this can be achieved by using the Virtual Channels tool. How to set this up is described in the section USING VIRTUAL CHANNELS TO ACQUIRE MULTICHANNEL CONFOCAL IMAGES.
If you would like to check that the light path is configured correctly or want to manually adjust it you can do so by clicking the Light Path & Dyes icon.
When you have finished with the lightpath configuration the dyes you have selected will be shown in the Image Acquisition Control window and the lasers you are using for fluorescence excitation will be ticked in the Acquisition Setting menu. Click the XY Repeat button to open a Live View window to preview your specimen. Clicking the Focus x2 or Focus x4 button also initiates a Live View, but these high-speed scans will have a lower resolution. You can use them to focus easily on your specimen and adjust laser power, gain etc.
The Live View window below shows a saturated fluorescent root section. You will need to adjust the acquisition parameters until there is no more saturation in your images or if your sample is very dim so that you detect sufficient fluorescence signal.
Click the LUT (Look Up Table) button in the Live View window to open the LUT panel. Select the channel for which you would like to change the LUT, i.e. CH1, then select a LUT on the right. To check for saturation click Hi-Lo. Saturated areas in the image will be shown in red and non-saturated areas in white (see step 9). The image Offset (minimal signal intensity detected by the sensor) can be adjusted by changing the Offset value in the Image Acquisition Control window. Adjust the value until no or only few blue pixels are displayed.
There are several ways to adjust the intensity of your images. Panel 1 below shows a saturated fluorescence image of a root section at the top and an adjusted image at the bottom. You can adjust the laser power (2), gain or detector voltage HV (3, lower it if saturation, increase if signal too low) for each channel (dye). Be careful not to increase the laser power too much, as this may lead to photobleaching or photodamage of your sample. Make sure to do these adjustments for all channels that you would like to image. Then change the LUT back to the colour you would like to view your channel in.
To stop the Live View click the RepeatStop button in the Image Acquisition Control window.
Before starting the image there are a few more parameters you can adjust in the Acquisition Setting panel:
1. In Mode you can pick between the unidirectional (Oneway) or bidirectional (Roundtrip) scan mode. Roundtrip on this microscope tends to display unwanted phase shifts in the images. It is recommended to use the Oneway rather than the Roundtrip scan mode on this machine. If you select one of the ROIs to the right of the scan mode you can acquire images with special formats such as Lines, Points or Rectangles. The pixel dwell time can be adjusted using the slider and dragging it to the desired scan speed. Decreasing the pixel dwell time allows faster scanning at the expense of signal to noise ratio.
2. In the Size section the frame format (number of pixels that will be imaged) can be changed by adjusting the slider position.
3. In the Area section the field that is being imaged can be changed or rotated around. You can also zoom using the slider on the right to look at a smaller field of view.
Some other image acquisition parameters can be adjusted in the Image Acquisition Control window:
1. The SU slider can be used to change the confocal pinhole size. Usually it is set to Auto, so it will be the optimal size to reject out-of-focus light without losing too much signal.
2. Below the channels you will find a Kalman filter checkbox. Selecting this will allow you to turn on Line or Frame averaging to improve the signal to noise ratio in the images.
3. To the right of the Kalman filtering you can turn on Hard Disk Recording (equivalent to Auto Save in other softwares) by pressing the button and selecting a folder to save the data in.
4. Check the Sequential checkbox to make sure that the different colour channels in your images are acquired sequentially rather than simultaneously. Be aware that when sequential is selected only the currently selected channel will be refreshed during the Live View.
5. On the bottom left of the window you can follow the progress of your experiments.
When all imaging parameters are set up press the XY-Acquisition button to start the image acquisition.
When the image acquisition has finished a new window with the recorded image file will open. To save the image highlight it, go to file Save as…, enter a filename and click save. Files are best saved as .oif or .oib, which are Olympus’ own file formats containing all acquisition metadata. You should save all data on the PC’s D-drive when acquiring and later copy them to a safe location such as your project folder on the UCL Active Directory. Instructions how to connect to your project folder can be found here.
A Z-stack acquisition can be configured in the Acquisition Settings panel under Microscope. Focus on the plane where you would like to start the Z-stack and press Set under Start, then focus on the last plane of the stack and press Set under End. Set a Step size in μm by typing in a value or press Op. for the microscope to set the optimal step size for the objective lens currently being used. If the Z-stack menu is hidden just double-click on Microscope to display it. Before pressing the XY button make sure that Depth is selected. The Z on the XY-button turns black and if you press XY-Start a Z-stack will be acquired.
When the Z-stack acquisition has finished press Series Done to open a window displaying the Z-stack and save it or press Append Next to acquire another Z-stack and append it to the previous one.
To acquire a simple time series select Time under the XY-Start button (the t on this button turns black) to activate it. You need to do this before setting up the actual acquisition interval as the software does reset it to 0 when the Time button is pressed.
At the bottom of the Acqusition Setting panel under Time Scan enter the Interval time in the 00 (h):00 (min):00 (s).0 (ms). In the Num (number) enter how often you would like this interval to be repeated.
Make sure that the interval is not shorter than the time it actually takes to acquire the image. Click on the Time Info button (see picture above) and you will get an estimate of how long a single scan will take.
If you are planning to acquire images over a long time period, e.g. overnight we recommend to turn the hard disk recording on (bottom right in Image Acquisition Control window).
Press the XY-Start button to start acquiring a time-series. When the acquisition of the time series has finished press the Series Done button to obtain the final data set in a new window and save it or press Append Next to acquire another time series and append it to the previous one.
It is also possible to acquire data sets containing a combination of a Z- and temporal information. For this select Depth and Time beneath the XY-Start button.
For more advanced experiments such as multichannel acquisitions you can have a look at the sections USING VIRTUAL CHANNELS TO ACQUIRE MULTICHANNEL CONFOCAL IMAGES or MULTI-AREA TIME-LAPSE IMAGING.

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