Olympus Fluoview FV1000 Manuale d'uso - Pagina 5

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Olympus FV1000 User guide

August 3, 2011

You can optimize the image quality with Hi‐Lo LUT (Look‐Up Table). Click LUT

button to bring the LUT

control window and select Channel and click Hi‐Lo button (Ctrl‐H

shortcut key will do the same without opening LUT window). Now

the image is displayed using this specific look‐up table where red

pixels represent intensity beyond scale (saturated) and blue pixels

represent 0 pixel value. To maximize the signal‐to‐noise ratio of the

image, adjust the acquisition setting (laser power level, HV, Offset)

such that the image shows a few red and blue pixels while you are

scanning.

Often excitation/emission profiles of fluorophores you are using may

be close, so there could be possible bleed‐through of a fluorophore

emission to neighboring channel. To minimize this, the detecting

range of each channel can be adjusted by changing Spectral Setting.

Click on

, it will bring Spectral Setting window. Change the range

of spectrum for each channel (CHS1 and CHS2) by sliding, widening,

or narrowing the tabs

or arrowheads.

Use Area panel

to rotate and zoom the imaging area (click on 0 and 1 buttons to return to the original

viewing area). To rotate the viewing area, click on the red dot and drag clock‐ or counter‐clockwise. With

zoomed view, you can select the scanning area by moving the blue‐lined box around. To zoom a specific region

in, click

icon and draw a box on the live view area. It will zoom in the area specifically.

Once you are satisfied with the setting, stop scanning. Set the scan speed to slow rates (~10‐12.5us/pixel)

(the slower the speed set, the better the signal‐to‐noise ratio, but the more bleaching). Click XY button

to acquire an image. When acquisition is done, a 2D view window will appear.

E. Z‐Series Image Acquisition

Use this mode to obtain optical section through the depth (z

dimension) of your sample that can be used for 3D visualization.

Click

button to scan. Use the arrowheads

buttons

or the fine focus knob on the remote controller to focus into

b



different Z‐axial planes (large arrowhead buttons shift a full step

size and small ones a half step size). When you find upper limit of

your sample, click End Set button. Bring the objective down until

you find lower limit and click Start Set button. Determine the

Step Size and the number of Slices, which correlate with each

other. It is recommended to set the step size similar to the

optical section thickness of the objective you are using, so that

there is no gap between the optical sections upon projection into

3D. The step size can be fixed by checking the box