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• When your specimen is in focus, close the shutter and click the "Acquisition" tab.

Imaging Setup

Smart Setup

The easiest way to set up imaging conditions is to use Smart Setup.

Click on the Smart Setup button and add the dyes you want to image.

Choose from Fastest, Best Signal or Smartest proposals. Basically, Fastest will be simultaneous (one Track), Best Signal will capture all channels separately, moving the filters each time to optimise signal (which is slow). Smartest optimises the filter positions so that they don't have to move, but still captures the channels sequentially.

The applicable lasers will automatically turn on and the filters will automatically move into the positions programmed for that dye combination and configuration.

Manual Setup

• Expand Imaging Setup
• Choose LSM and Channel
• (Non Descanned is for multiphoton only)
• (Lambda Mode and Online Fingerprinting are for spectral imaging)

Smart Setup

• Use Smart Setup if you want to easily set up your experimental settings (e.g. filter and detector combinations) depending on your dyes.

• Pick Click the + button and pick dyes from the list, and Smart Setup will arrange the light path to optimally capture those dyes with three combinationspossible options:
  • Fastest (simultaneous acquisition). Fast but with a risk of crosstalk).
  • Best Signal (Sequential acquisition with the spectral windows optimised for each dye in turn to maximise signal, but may be slow)
  • Smartest (Line sequential with the spectral windows balanced to get the best signal with the best speed)

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• You can manually setup or adjust the light paths in imaging Setup

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• • will capture all channels separately moving the filters each time to optimise signal (which is slow).
  • Smartest optimises the filter positions so that they don't have to move, but still captures the channels sequentially. This reduces crosstalk and is faster than Best Signal.

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• In the example image above, Fastest shows all three colours being excited and captured simultaneously in one Track (Track 1). Sequential acquisitions would require more than one Track.
• Best Signal creates three tracks for the three selected dyes, one for each channel so they are all captured separately but switching . Switching between the tracks takes time because the components (e.g. dichroic mirrors) movephysical components must move.
• Smartest creates two tracks, with spectrally separated dyes in the same track as each other. Also, the physical components don't move to save time, which saves time.
• Note that the optimal settings for other dye combinations may look different.
• NB lasers are always set to 2% in Smart Setup - but you can change them afterwards
• Click Live to get a live scan.

If you have multiple tracks then you can define whether you switch them every Line (only suitable when the light path is set up in the Smartest manner), Frame or Frame Fast (only suitable when the light path is set up in the Smartest manner).

You can edit the imaging setup after loading the Smart Setup configuration, or alternatively you can create an imaging setup manually from scratch without using Smart Setup.

Detectors

The microscope has three main detectors (internal aka descanned detectors) suitable for both confocal and multiphoton imaging, and a further four for multiphoton only (non descanned detectors).

Two of the detectors (Ch1 and Ch2) are high sensitivity GaAsP detectors and a third (ChS1) is a Zeiss Quasar detector made up of a 32-channel GaAsP array. This array can be configured as multiple detectors or can be used in the Lambda and Online Fingerprinting modes to generate a spectral image of the sample.

You can use Ch1, Ch2 or up to 32 individual channels from ChS1 (or some combination of these) as detectors.

You can change the broadness of the filter band by dragging it or clicking the down arrow to type in numbers.

You can add new ChS detectors using the + button and delete them using the - button.

Primary and Secondary Dichroic Mirrors

Select suitable primary dichroic mirrors for the visible and invisible lines (405 or multiphoton).

If the Mirror is in position the light will be sent to the Ch1, Ch2 and ChS detectors. If another position is selected then some portion of the light (up to 100%) will be sent to the Airyscan detector. See Airyscan for details.

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:

• • Scan Mode: Whether the scan switches channels in Frame or Line mode (frame by frame or line by line)laser scans 2D frames, lines or spots. Most of the time you will have this set to Frame.
  • Frame Size = Scan Format or the number of spots that the laser scans on the sample (e.g., 512 x 512, 1024 x 1024, etc.).
  • Line Step: Increases scan speed but lowers the resolution in one dimension by skipping lines in the scan.
  • Scan Speed: the line frequency. Expressed in pixel dwell time as opposed to in Hz.
  • Averaging, including number of scans and whether it should be done line by line or frame by frame. A method by which photon noise can be reduced in images.
  • Method: Mean = Average, Sum = Accumulate.
  • Bit Depth: 8 bit is default.
  • Direction: unidirectional or bidirectional (Stick to unidirectional).
  • Scan Area: Zoom and Scan Field Rotation.

Channels

• Expand Channels
  

• 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.
• Range Indicator at the bottom of the screen will indicate saturation and black pixels

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• Single Channel

  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.

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• 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 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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