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- The easiest way to create suitable laser and detector settings in the FV10-ASW software is by using the Dye List. 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)
Note You cannot select more than three dyes at a time using the Dye List and certain dye combinations are not possible because the dichroic mirror combinations do not allow them. If you would like to image such a forbidden 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.
- The combination of detectors activated in the Image Acquisition Control window (CHS1, CHS2, CH3, etc.) depends on the dyes you chose. The lasers you need for fluorescence excitation will be automatically ticked in the Acquisition Setting menu. Click the XY Repeat button to open a Live View window to preview your specimen. You can stop the scan by clicking RepeatStop. Clicking the Focus x2 or Focus x4 button will also initiate a live scan. These are high-speed scans that have a lower resolution but you can use them to focus easily on your specimen and adjust laser power, gain etc.
- The Live View below shows a green and red fluorescenct section of plant root. The signal in both channels is saturated, meaning that the light is too bright and the detector is measuring maximum signal in large parts of the image. It would be impossible to quantify fluorescence within the saturated regions because the intensity value is off the scale. In such a case it is necessary to adjust the imaging settings to reduce the intensity to a measurable level.
- Before adjusting the settings it can be useful to change the image colour look-up table (LUT) to Hi-Lo. This is a greyscale LUT with the brightest and dimmest pixel values (usually 255 and 0) coloured red and blue, respectively. 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 Hi-Lo on the right. There is a choice of other LUTs if you want to change the appearance of your image later on.
- Increasing the laser power (2 below) will increase the fluorescence emitted by your specimen but will also increase the bleach rate and phototoxicity. Also, the intensity will no longer increase if all the fluorophores in your specimen are already in the excited state. The detector voltage HV (3 below) increases or decreases the brightness by changing the amplification of the signal. This won't result in bleaching but it does amplify the photon noise to the same degree as the signal and also adds some multiplicative noise. Gain is a 'digital gain' that simply applies a multiplication factor to the digital value of all the pixels. Offset changes the black level of the image. Increasing the offset will make the background darker by setting more pixel values at the lower end of the scale to zero. Make these adjustments for all channels that you would like to image, then change the LUT to whatever colour you want.
In the Acquisition Setting panel you can change the Mode from Oneway (unidirectional) to Roundtrip (bidirectional) scanning. In most cases it is better to use Oneway rather than Roundtrip scan mode on this machine. Roundtrip mode can be used for high speed imaging if some adjustments are made to align the two scan directions. You can acquire images with special formats such as Lines, Points or Rectangles by selecting one of the ROI buttons to the right of Mode. In practice, most users will not want to change this. The pixel dwell time can be adjusted by dragging the slider between <<Fast and Slow>> and setting it to the desired scan speed (e.g. 2 µs/pixel). Decreasing the pixel dwell time allows faster scanning but with a lower signal to noise ratio.
Note The pixel dwell times can be set to different values for 2x, 4x and XY Repeat, so watch out for that. You can adjust the dwell time so it is the same for all these conditions. The dwell time for XY Repeat will be the one used for any subsequent image acquisitions.
In the Size section the frame format (number of pixels that will be imaged) can be changed by adjusting the slider position. 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.
In the Image Acquisition Control window 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. It can be opened wider to increase signal at the expense of axial resolution. The Kalman checkbox in Filter Mode will turn on Line or Frame averaging to improve the signal to noise ratio in images. To the right of Filter Mode 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. This should be used for any acquisition where the data generated is likely to exceed the maximum capacity allowed by the acquisition software (e.g. overnight time series or large tiled 3D images). You will have to define a file format (see below)
Acquiring and saving images
- When all imaging parameters are set up press the XY 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
Acquiring and saving images
- When all imaging parameters are set up press the XY 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.
To the right of Filter Mode 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. This should be used for any acquisition where the data generated is likely to exceed the maximum capacity allowed by the acquisition software (e.g. overnight time series or large tiled 3D images). You will have to define a file format in which to save the data.
Info OIB is the newer of the Olympus image file formatformats. In contrast to OIF, where image data is stored as many separate TIFF files in which images are stored as a folder of individual TIFFs with an OIF database index file as an index, the OIB file stores all data in one file. Some offline image processing software might not be compatible with OIB. Volocity, for example, will only read OIF filesimages and metadata in a single file. If you intend to use Volocity software for 3D reconstruction, deconvolution or measurement then you will need to use OIF because Volocity cannot read the OIB format.
Simultaneous and sequential acquisition
Check the Sequential checkbox to make sure that the different colour channels in your images are acquired sequentially rather than simultaneously. Sequential imaging is slower than simultaneous but reduces crosstalk when using fluorophores with overlapping excitation or emission spectra.
You can define which channels are scanned simultaneously and which are scanned sequentially by dragging them into different Groups. In the example below Alexa Fluor 488 and Alexa Fluor 568 are in different groups and will be scanned sequentially. If I was also imaging Alexa Fluor 647 I could have that in a third group or I could shorten the scan time by dragging it into Group 1 to be scanned simultaneously with Alexa Fluor 488. It is possible to scan a Live View in sequential mode but when Frame sequential is selected only the currently selected channel will be refreshed.
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