Switching On:

1. Turn on the “LASER IN USE” switch on the wall.

2. Follow the order of the labelled switches (check that 0, the compressor is on) from 1 to 3, number 4 is the mini PC, 5 is the Zeiss PC. The Argon ion laser (switches 6 and 7) is needed for laser lines 458 nm, 488 nm and 514 nm. Do not switch it on if you do not intend to use any of those lines. The epifluorescence lamp 8 is only for observing fluorescence by the eyepiece. Do not turn it on if you do not intend to use it.

   

3. Log in with your PPMS account on the mini PC and log in as Zeiss User on the Zeiss PC. Start ZEN Black Edition software.

Notes on the epifluorescence lamp:
Do not switch off the lamp if it has been on for less than 30 min. Do not switch on the lamp if it has been off for less than 15 min. Switch off the lamp if you do not need it for a long time (e.g. acquiring a long time-lapse). The lamp is only for locating the sample using the eyepiece.

Notes on the Argon ion laser:
The laser needs a few minutes to warm up and stabilize (indicating by green LED at the control panel with laser on/idle switch 6). If you interrupt your experiment for a break or there is a gap of less than 1 hour between your and the next user’s time slots, switch the laser to idle (6), do not leave it on or turn it off completely.

Switching Off:

1. Make sure you have saved your data and turn off ZEN software.

2. Move the objective to the escape position and remove your sample.

3. If you have used an immersion objective, wipe it clean. Use the lens cleaning tissue. For cleaning oil immersion, moisten the tissue at the solvent dispenser. Always wipe the objective only once, in one direction. If this is not sufficient, repeat with a new piece of tissue. Never reuse the tissue.

4. Use the mini PC to transfer your data to OMERO or shared network drive. Shut down the computers. Switch off the mini PC first!

5. Turn off the numbered switches in the reverse order, starting from 8 or 7. Important: After turning off the Argon ion laser key 6, wait for a few minutes until the laser fan has stopped (audible) before proceeding to the switches 3 – 1.

6. Cover the microscope with the dust cover.

7. Turn off the “LASER IN USE” switch on the wall.

8. Make sure you leave the microscope room clean. Spray with 70% ethanol and wipe any surfaces that could have been in contact with biological material. Do not leave any samples or any other belongings behind.

Finding sample using the eyepiece:

Locate tab in ZEN software is used to set up observation by the eyepiece. To observe the sample in transmitted light, select the appropriate observation mode (1). The transmitted lamp will turn on (2) and the shutter in front of the lamp will open; you can control its power in the software or using the knob at the base of the microscope body. The objective can be selected in the software (3) or using the touchscreen panel. See an important note on objective selection.

Notes on objectives selection:

Do not use the 100x objective for confocal imaging. Use the 63x objective instead - it has the advantage of larger available filed of view without compromising the resolution (both have the same NA). The "magnification" in confocal scanning is determined by the scanned area rather than the objective. The overlap of 405 nm laser and the other lasers is optimised for the 63x objective and optimal performance cannot be guaranteed for the 100x objective.

 

For observation of fluorescence, select the appropriate fluorescence channel (4) according to the fluorescent labelling of your sample; this will determine the filter cube that will be inserted into the path (5). The fluorescence lamp power can be controlled in the software (6), provided it has been switched on.

If using an immersion objective, apply a drop of immersion on the objective. Place your sample on the stage and focus it using the focusing knobs (course and fine) on the side of the touchscreen panel or the microscope body. Find the location of interest by moving the stage. The button on the top of the joystick lever switches between fast and slow stage movement.

Confocal scanning - setting up channels:

To set up confocal scanning, we will move to Acquisition tab.

We can load an existing experiment setting using the Experiment Manager (E1) drop-down menu or reuse the settings with which a saved image has been acquired by pressing the Reuse button in the Dimensions tab under the open image. In case no suitable settings from the past are available, we can configure the settings from scratch as described here. You can also watch a ZEISS video tutorial on setting up light path (external link).

1. Setting up laser excitation

We can switch on the lasers we want to use in the Laser panel (E4). The 405 nm diode laser because of its fast response is switched on automatically only when needed (during image acquisition only). The Argon laser is switched on manually as a part of the switching-on procedure. The software receives no feedback whether the laser is on or off, thus it will give no error message if you select a line of the Argon laser in your channel settings without having turned the laser on. However no excitation light would reach the sample.

The desired laser line(s) need to be selected also in the Channels panel (C1, 561 nm is selected in the example). The slider below the check boxes allows setting the laser power (in % of the full power); refer to the table of the actual laser powers at sample plane.

2. Setting up dichroic mirrors

Next we select the dichroic mirrors to suit the lasers in the Imaging Setup panel (I1). There are two laser ports, each using a different dichroic. One, labelled Invisible Light, is used only by the 405 nm laser. The other port, Visible Light, by the remaining lasers. Select a suitable dichroic from the drop-down menu. There are multi-band dichroics that work several laser lines (e.g. the one in the example for 488 nm, 561 nm and 633 nm), together with the 405 nm laser coming by the Invisible Light port, we can have up to 4 laser lines reaching the sample simultaneously.

Not all combinations of laser lines have a dichroic mirror available. There is for example no dichroic for 514 nm and 633 nm simulatenously. Those lasers cannot be sent to the sample simultaneously, can be used only sequentially using the feature of Tracks.

Important: If you are not using one of the ports (e.g. not using 405 nm laser), select the option "Plate" for that port. A glass plate of the same thickness as the mirrors is inserted to the optical path to maintain the beam path identical as when a dichroic mirror is there. Selecting option "None" would change the path of the beam as it would not undergo the shift caused by refraction on air/glass interfaces.

3. Setting up detection channels

Then we set up the detectors for each channel in the Imaging Setup panel in Channel mode (I0). There are two types of photomultiplier tubes - standard multi-alkali PMTs (Ch1 and Ch2) and more sensitive GaAsP PMTs (ChS). While Ch1 and Ch2 are each a single PMT, ChS (S stands for "spectral") is an array of 32 detectors covering a fixed spectral range from 410 nm to 695 nm (8.9 nm band/detector in array). The array of 32 detectors can be divided into several non-overlapping detection channels. New channels are added by pressing the "+" sign (I2). 4 channels are shown in the example. Spectra of fluorophores from the database in ZEN (I3) can be displayed and used as a guideline for selecting the spectral ranges of individual detection channels. Lookup tables (colours) can be defined for each channel (I4).

ChS has the advantage of higher sensitivity, making it the recommended detector choice. The standard PMTs Ch1 and Ch2 have the possibility to detect beyond the spectral range accessible by ChS on both the short wavelength side (not very useful when the shortest excitation laser is 405 nm) and long wavelength side (up to 740 nm for NIR emitting dyes).

A transmitted light PMT can be also selected (I5), which detects the laser light that has passed through the sample. It can be then used to create a brightfield or DIC (depending on the setting of the condenser) image of the sample. This image comes at no additional cost in terms of acquisition time or exposure of the sample to light and provides information complementary to the fluorescence channels. For optimal transmitted light images quality, the condenser needs to be aligned for Koehler illumination.

Setting up DIC:

Use the buttons on the right-hand side of the condenser (labelled as "Rev") to switch between modes of transmitted light illumination. The current state of the condenser can be seen at the status bar of the touchscreen panel (blue box in the picture), brightfield (BF) in the example. There are multiple DIC prisms in the condenser turret (DIC I - III), select the appropriate one according to the objective as indicated in the objective description (yellow box in the status bar of the touchscreen panel, DIC II for the objective in the example).   Check that there is a DIC prism slotted under the objective and make sure the analyzer in a rotating holder (labelled as P in the picture below) is swung to the optical path above the objective. The DIC image can be optimized by rotating the analyzer and shifting the DIC prism under the objective by turning the small knob.

Checking the "Reflection" checkbox (I6) removes laser blocking filters from the detection path to allow detecting reflected laser light rather than fluorescence. The detection channels need to be set to contain the laser line wavelength.

The detector gain is set in the Channels panel (C2). Gains in the range 500 - 800 V are typically needed for fluorescence, lower values (100 - 300 V usually) are sufficient for transmitted light. Digital gain is just a multiplicative factor and as such does not affect the image quality (signal-to-noise ratio) unlike the real gain. Setting to values higher than 1 only increases the risk of exceeding the dynamic range (bit depth) of the digital image. The elements of the spectral detector ChS do not have independent gain, thus the same value of gain will apply to all channels using the detector array.

4. Setting up pinhole size

Pinhole size is set in Channels panel (C3). The usual setting is 1 AU (smaller pinhole size results in a significant loss of signal, while lager sizes compromise optical sectioning. Note that Airy units are relative to emission wavelength, thus the actual pinhole size depends on detection channels setting. The approximate section thickness is shown below the slider.

5. Setting up scanning

We move to Acquisition Mode panel now. We need to define the scanning speed (A1), the size of the scanned area (Zoom) (A2) and sampling of the image (Frame Size - into how many pixels will the scanned image be divided) (A3).

Zoom (A2) is what defines the scanned area as well as the effective magnification of the confocal image. The smallest zoom factor is smaller than 1 (the largest possible scanned area; consider zoom factor 1 as the smallest recommended zoom (largest recommended area) in terms of maximising image uniformity and minimising aberrations. The actual size of the scanned area (Image Size) in μm is displayed together with the pixel size (the scan size divided by the number of pixels).

If you want to zoom to a particular feature which is not necessarily in the centre of the field of view, you can use the Crop button in the Dimensions tab under the image, which will allow you to define a custom scanned area.

The pixel size thus depends on the Zoom as well as on the Frame Size (A3). When too large (too few pixels), they limit the resolution of the image (pixelated image). On the other hand too high number of pixels (too small pixels) will either increase the acquisition time or reduce the pixel dwell time (compromising signal-to-noise ratio). The optimal (Nyquist) sampling is such that the pixel is slightly smaller than half of the theoretical optical resolution limit (diffraction limited). You can set it automatically by pressing "Optimal". Do not forget to optimise the Frame Size every time you change Zoom.

Pixel Dwell time and Scan Time (A1) are determined by scanning speed and Frame Size. Slower scanning increases Pixel Dwell and therefore the amount of signal collected in each pixel. For live samples is may be advisable to avoid very slow scanning (prolonged continuous exposure of the same area in the sample to laser light), but rather to increase the effective time per pixel by repeatedly scanning each line or the whole frame (A4).

Other setting (A5) available in this panel include the bit depth of the image and direction of scanning (mono- or bi-directional). Scan Time can be reduced twice by using bi-directional scanning (collecting data when the beam moves from left to right as well as from right to left). Scan artefacts (odd and even lines are shifted with respect to each other) may appear in his case. They can be  corrected by adjusting CorrX and CorrY; this is an topic recommended only for adventurous users.

6. Scanning an image

We are now set to scan a multi-channel image by pressing either Live, Continuous or Snap (E2). The difference is that Live, being intended for a fast preview (e.g. during focusing or stage movement) uses high scanning speed, 512 x 512 pixels frame and no line or frame averaging, regardless of what you set in Acquisition Mode panel. Continuous, on the other hand, continuously scans using the full settings, while Snap scans a single frame (using the full settings).

The image is displayed in the main window. The display of the image can be controlled in the panels below it and along its left-hand side. The left-hand side panel allows toggling between overlay of channels (2D) and split view. Dimensions tab allow selecting which channels are displayed (the magenta channel is off in the example), Graphics panel allows adding annotations to the image (e.g. a scalebar). The image histogram for all channels is also shown.

 

In case the histogram shows that the signal is low (all pixel values are close to zero, only a small fraction of the dynamic range is utilised) consider some of the following approaches to increase signal:

Notes on improving signal:

Be cautious when increasing laser power, especially when working with live samples. Fluorescence intensity saturates, meaning that when excitation intensity exceeds a certain threshold, there will be no further increase in fluorescence intensity  with increasing excitation intensity. In some cases, fluorophore bleaching and/or phototoxicity may become and issue even below the saturation threshold. Avoid using excessive detector gain. At very high gain levels, noise will be over-amplified and signal-to-noise ratio (SNR) compromised. The weaker the signal, the higher the gain that gives the best SNR, but in general gain over 800 V is not likely improve the image quality. Line or frame averaging is in general a good strategy to improve signal, unless imaging speed is a concern.

Confocal scanning - setting up tracks:

So far we have set up all channels to be acquired simultaneously. This is not always the optimal approach. For example DAPI spectrum as shown in the example of Imaging Setup panel overlaps strongly with the spectrum of EGFP and to some extend even with DsRed spectrum. Thus if all laser lines are on at the same time, the EGFP channel will receive a large contribution from DAPI, making it impossible to separate those two fluorophores. In such cases sequential excitation can help. DAPI is excited only when the signal in the DAPI channel is collected and not when EGFP is excited and the signal in EGFP channel collected.

This can be implemented using a feature called Tracks (T1). New Tracks can be added by the "+" button. 4 Tracks are shown in the example. Track 1 is active, thus the settings we currently see and can edit apply to Track 1. Different laser lines and detection channels can be on in each Track. In this case DAPI channel is on in Track 1 and that means that most likely only 405 nm laser will be on.

Tracks can be switched every line, every frame or every stack (in case Z-stacks are acquired) (T2). Switching every line is the fastest and the images in individual Tracks are acquired nearly simultaneously. If there is any movement in the sample, the resulting shifts between channels would be minimal. On the other hand because of the requirement to switch the Tracks very fast, no mechanical movements of components are possible when switching. That limits the differences in settings between individual Tracks when switching every line. Permissible are for example switching on and off laser lines and switching on and off detection channels; prohibited are for example switching dichroics (here the multi-band dichroics come handy) or changing the detection ranges of  individual channels. Being the fastest, switching every line is recommended unless not possible because e.g. each Track requires a different dichroic. When Z-stacks are acquired, switching tracks every stack is generally faster than switching every frame.

The Tracks (and the Channels associated with them) are shown also in Channels panel (C4). The checkboxes allow you to switch Tracks on and off (e.g. when you want to focus or move the stage you would keep a single Track on to increase speed and/or prevent photobleaching of more sensitive fluorophores).

Z-stacks:

Z-stacks are undoubtedly the most common of the more complex experiment types that can be selected in the upper part of the Acquisition tab (E3). The Z-stack is the defined in the Z-stack panel. You can define the stack by either selecting the first and last slice (Z1) or by selecting the central slice and defining the range. The latter approach is useful when using autofocus. In the first approach, focus to the plane where you want to start the stack (doesn't matter if it's the uppermost or the lowermost slice) and press Set First (Z2). Then focus to the last plane and press Set Last (Z3). To have the optimal spacing of slices based on the sectioning capability (the combination of the objective and pinhole size) press the suggested spacing (Z4). Start acquisition by pressing "Start Experiment". You can also watch a ZEISS video tutorial on setting up Z-stacks (external link).

Once a Z-stack is acquired it can be viewed using orthogonal  projections or 3D rendering (using the menu along the left hand side of the image). It is strongly recommended to save your data before doing a 3D rendering. Videos of rotating 3D object can be exported.

 

Multi-position experiments and time-lapse:

There are two basic types of multi-position acquisitions: Tile Scans (moving the stage is used to capture an image of a continuous area larger than the maximum area accessible to the scanner) and acquisitions at discreet Positions (useful in combination with time-lapse acquisition when multiple location in the sample can be revisited at each time point). Both can be selected in the upper part of the Acquisition tab (E3). They are set analogously to multi-position experiments at widefield microscopes. You can also watch a ZEISS video tutorial on setting up tile acquisition (external link).

Time Series can be also selected in the Acquisition tab (E3). The setting is straightforward be selecting the interval (30 s in the example) and the number of repetitions (100000 in the example).

Z-stacks, multi-position and time-lapse acquisitions can be combined together into larger scale experiments. See below how to use focusing based on reflection from coverglass surface to keep the sample in focus during such multi-dimensional experiment:

Long-term multi-dimensional acquisition with autofocus:
For each position focus to the plane you want to be in the centre of the stack and press "Center" in Z-stack panel. Update the position in Positions panel Press "Find selected position offset " in Positions panel.

The other types of experiments (Bleaching and Regions) are useful in setting up FRAP (fluorescence recovery after photobleaching) experiments to study mobility of molecules. Approach ABIF staff if you are interested in such experiments.

Spectral imaging:

So far we have used only the Channel mode in Imaging Setup (I0). Lambda mode can be used to acquire spectral images leveraging the 32-element array of the spectral detector (ChS). A spectral image with spectral resolution of 8.9 nm (or multiples of it) can be acquired in a single scan. It is possible to acquire spectral images with higher spectral resolution; those however require sequential acquisition, one image per each wavelength.

In Lambda mode with 8.9 nm spectral resolution, each of the 32 detectors forms a separate channel, thus providing up to 32-channel image (for the full spectral range). The scanning settings are analogous to those in Channel mode. The spectral images can be visualized in spectral mode as a false-colour image where the signal from each of the 32 detectors is represented by a different colour. Spectra from a ROI in the image can be plotted in the Unmixing tab. As the name suggest, spectral unmixing can be performed to separate signals from fluorophores with overlapping spectra. Approach ABIF staff if you need assistance with spectral umixing.

Saving images:

Acquired images are listed in the image explorer panel at the right-hand side of ZEN window. For each image the name, size, thumbnail and icons indicating image type are shown. In this example we see a multi-channel (C) Z-stack and a spectral image (λ). The yellow warning sign indicates the image hasn't been  saved yet. Image (selected by clicking on it) can be saved by clicking the saving button (second from left in the bottom row) with the symbol of a floppy disk (an ancient data storage medium still remembered by members of the older generation).

This is not very convenient for saving large numbers of images. If you want to acquire many images, it is useful to set up Auto Save option in a dedicated panel. The images will be then saved automatically to the selected folder and given the selected name ("test" in the example); individual images will be differentiated by numbers appended to the name.

Note that Auto Save applies only to images acquired by pressing "Start Experiment" button, not to images acquired by "Snap". If you want to capture individual images (not stacks or other multi-dimensional experiments), use the following trick to benefit from Auto Save: set a Time Series with a single cycle.

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