Before you start

Heating and CO2 for the incubator

The CO2 Module S and TempModule S are normally switched off at the back. If temperature and/or CO2 control is required, these must be switched on.

Coherent Vision II multiphoton laser

If you need the Coherent Chameleon Vision II Ti Sapphire laser for multiphoton imaging or ablation, you must turn the key on the Coherent power supply from Standby to On and wait for the power to ramp up until the Status says OK (takes a minute or two). You can continue with the start-up of the rest of the system while the laser ramps up.

Main switch laser safety key

The key on the side of the keypad must always be left in the horizontal position (see image below), even when the system is powered down. However, occasionally I find that it has been turned to the vertical position, which means it is switched off. Make sure it is in the horizontal position, otherwise the lasers and anything else connected to the "Components" switch will not come on.

If you get an error message saying "Unexpected error during hardware control task. An attempt to move the filter-set "ShutterArgon" to position 1 failed", it is because the key is in the wrong position.

If this happens, you should quit Zen, turn on the key and then open Zen again.

Switching On

Turn on the "Main switch" on the keypad.
Turn on "Systems/PC" on the keypad.
Wait for computer to boot up and enter the Windows account credentials if prompted.
Turn on the "Components" switch on the keypad.
Double-click the Zen Black icon on the desktop.
Click the "Start System" button.
If CO2 Module S and TempModule S are off, you will see messages warning you about this in the Error Log in the bottom left corner. This is normal and can be ignored.
Click the" Acquisition" tab and expand the "Laser" menu. Turn on the lasers you need. (Some of the lasers might show a red background initially, which means it is not ready to be used. This will go away after a couple of minutes when the laser is warmed up).

Locating your Specimen

Click the "Locate" tab and expand the "Microscope Control" drop-down menu.
Select the objective lens you'd like to use - either via the menu in locate, or via the touchscreen controller next to the screen.
Select a suitable fluorescence filter. Only the first three should be used for fluorescence; the rest are special positions for the confocal and multiphoton, but you can use BS-MP positions 4 and 5 for transmitted light.
Put your specimen on the microscope and open either the transmitted light shutter or the fluorescence shutter depending on which mode you want to use to find your sample.
Turn on the lamp by clicking its icon and pressing the On button.

Open the fluorescence shutter to let light through.

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

Imaging 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.

Click the + button and pick dyes from the list, and Smart Setup will arrange the light path to optimally capture those dyes with three possible options:
- Fastest (simultaneous acquisition). Fast but with a risk of crosstalk.
- 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. This reduces crosstalk and is faster than Best Signal.

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. Switching between the tracks takes time because physical 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, 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 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.
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 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 adjusting the range. Note that the confocal is still scanning all the channels - it just isn't displaying them.
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 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.