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The Reflector should be in the Rear position unless you are using the NDDs for multiphoton imaging.
Also in this set of controls T-PMT, Ratio, Stage, Focus, Incubator.
Acquisition Mode
- Expand Acquisition Mode. In here you can change a variety of parameters but the main ones are the scan settings:
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- In here you can determine which channels are actually scanned by unchecking the tracks and you can define laser powers and detector settings such as detector gain
- For most purposes you will use Integration Mode, which is the normal mode of using a PMT, but photon counting can be used to quantify signals so that each intensity level corresponds to a detected photoelectron. Photon counting mode is only available for ChS and Ch2.
In Channels, set the Gain (Master) to 700 to 800. Leave Digital Offset on 0 and Digital Gain on 1.0.
Set the Pinhole to 1 AU for optimal confocality (the best optical sectioning performance). The optimal pinhole size will be smaller for bluer wavelengths of light and larger for redder wavelengths. The pinhole can only be resized in Frame mode. When using Line or Frame Fast it will be fixed on whatever the value is for the current Track. Smaller pinhole sizes give better optimal sectioning (Z resolution). Larger pinhole sizes let in more light.
- Click the Live button in the top left and adjust the laser power and gain (Master) so the image is not saturated. Live performs a fast preview scan of the currently selected Track at low resolution. If you have multiple tracks you will only see the dyes in the selected track.
Use the Range Indicator
at the bottom of the screen will indicate saturation and black pixels
- Single Channel
tickbox to help you minimise saturated (red) pixels. In the Range Indicator LUT, blue pixels have a value of zero. The Digital Offset can be used to adjust the ADU conversion factor to increase or decrease the number of zero valued pixels.
- If you click Continuous it will scan using the settings in Acquisition Mode, which will probably be slower.
- If you are able to scan multiple channels at once, the Single Channel tickbox allows you to see just a single channel, which is useful when adjsting adjusting the range. Note that the confocal is still scanning all the channels - it just isn't displaying themLive scans at the maximum possible speed. If you click Continuous it will scan using the settings in Acquisition Mode
- Save by clicking the floppy disk icon in Images on Documents (top right). Delete by clicking the X
Z-Stack
To set up a Z stack, tick the Z stack box.
- Z-Stack optimise scetioning sectioning and step. Diagrams illustrate the degree of sampling, which is also shown in percentage form below. This changes when the step size is changed.
In First/Last you can set up the Start and End position for the Z series. In Center, you set a centre and then do a Z series in a range around that.
You can set the Interval or Number of Slices. Optimal will set the interval to the best resolution achieveable with the lens NA and wavelength.
Use Piezo should be left unticked unless the piezo focus deveice is mounted in the microscope stage.
Optimise sectioning and step. X:Y:Z = 1 matches the interval to the XY resolution (isotropic). Optimal sets the optimal pinhole size for each Track separately. Longer wavelengths will have thicker sections. Match Pinhole will resize the pinhole so each channel is the same section thickness. Longer wavlengths will have less light than optimal. Note, in Line mode the pinhole size will stay the same for each channel anyway.
Refractive index Correction compensates for differences in RI between the immersion and mounting medium by making intervals proportionally smaller of larger depending on whether a low RI is imaging into a high RI or a high RI is imaging into low RI, respectively.
Auto Z Correction bumps up laser power or gain as you go through a thick specimen to compensate for loss of signal through scatter and refraction.
The function Match Pinhole sets the pinhole to keep this optimal interval and in addition sets the slice thickness for all detection channels approximately the same. This typically results in slightly thicker slices for channels detecting the longer wavelength range. In case the channels are assigned to different tracks and a Frame wise Multitracking scheme is applied, the pinhole diameter is set for each track such that the values of the resulting optical sections from the different channels are identical and have double the value of the optimal interval.
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