Confocals

   There are two Leica SP5 confocals, one in room 42 and the other in room 53.

   One has a resonant scanner for fast imaging and two hybdrid detectors, which are the more sensitive detectors. Both systems are also equipped with standard photo-multiplier tubes (PMT's) detectors. The other difference is one has four laser lines (405, 488, 546 and 647) while the other has five (405, 488, 561, 594 and 647). Otherwise, they are identical.

 

User instructions

Starting up

1.  Switch on the fluorescence lamp.

2.

Switch on all three press switches on the panel on your right (see pic).  In this order:

A - PC/Microscope

B - Scanner

C - Laser power

D - Turn key to "On"

SP5_start_buttons.jpg

3.  Start LAS Software

> (in the resonant confocal) Select whether the system should be operated with the resonant scanner.

Note: the resonant scanner is mostly for live-cell imaging or applications that require fast scanning. For fixed cells and tissues it will result in lower signal and poorer images.

> When prompted, say "no" and do not initialize the stage.

LAS_start.jpg

4.  Laser configuraton

After the hardware and software have started, go to the "Configuraton" tab then click "Laser" and activate the laser lines you will be using depending on the fluorophores you have in your sample.

405 - DAPI, Hoechst

488 - GFP, alexa488

546 - alexa546

561 - mCherry, alexa561

594 - tdTomato

647 - alexa647, Cy5

5. Imaging

beampathsettings.jpg

6. Intensity adjustments

1. Select the laser lines for excitation

> Activate the laser lines according to your fluorophores.
> Start with a laser intensity of around 15 % for mean signal intensities.

 

2. Activate the detectors: PMTs (Photomultiplier Tubes) or HyDs (GaAsP Hybrid Detectors).
Note: HyDs are very sensitive and show low dark noise. Because they are easily oversaturated first set the laser intensity with a conventional PMT and then reduce it to around 50% before you switch to a HyD. Choose the Standard mode.

 

3. Visualize the reference emission curves of your fluorophores in order to get an idea where to detect your signals.
> Check for overlapping spectra of different fluorophores.

4. Set the detection bandwidth.
> Make sure that the edges of the detection window are at least 5-10 nm apart from the excitation lines to prevent imaging the reflection of the laser.
> Open the detection spectrum as much as possible, the larger it is the more signal you will have and the better your image is going to be. Stop at the increase of emission of your next fluorophore.

5. Choose a pseudocolor for each channel.


6. You can save and reload your settings in the Load / Save panel.

The intensity is controlled by:

* the laser intensity

* the gain and offset of the detector

  1. To adjust intensity, set laser parameters (number 4 above) and then click LIVE

  2. Increase the gain until you see an image.

  3. Adjust the focal plane with the z-knob on the control panel until you see the brightest plane or you are happy with the z-plane of your tissue.

  4. Adjust gain:

    1. To adjust the gain in the image choose the LUT "glow-over-under" option

    2. In this display mode the saturated pixels (grey level 256 for 8 bit images) are represented in blue and the black signals (grey level 0) are shown in green.

    3. While scanning adjust the offset and gain so that there are a few green pixel in the background and some blue speckles in the object. The aim is to have the whole range of grey levels in
      the image.

    4. The gain of the PMT is normally adjusted to a value between 700 – 800 V and the one of the HyD to a value between 10 and 100%. For best image quality with the HyD the gain should be set to 10 % with a bit depth of 12 bit (Configuration > Setttings).

  5. Adjust the final intensity in the image by balancing the gain level and the laser
    intensity. 

  6. Note: In order to reduce photobleaching and phototoxicity work with the lowest laser intensity possible but note that the enhancement of gain will increase the noise.

LUT.jpg

7.  Scan parameters

Settings.jpg

8.  Sequential scanning

1. Click on the “seq button” in the Acquisition Mode window. The Sequential Scan control panel appears now at the bottom of the left panel.
2. Configure the sequence:
Define first channel as scan 1. Add (+) scan 2 and define the settings for this second channel and so one for further channels.


3. Select the mode of scanning:
Between lines: Only the settings of the laser lines are changed. The detection ranges must not overlap and are not adjusted between lines. Fastest method, lowest movement-artifacts.
> Between frames: Maximal cross-talk prevention, complete changing of settings between channels, movement artifacts possible. Mandatory if the detection windows have to be moved between the sequences or if a detector has to be used multiple times.
> Between stacks: Speeds up the acquisition of z-stacks but might lead to movement artifacts and z- aberrations between channels.
4. The sequential settings can be saved and reloaded.

Sequential.jpg
Clipboard.jpg

9.  Acquiring a Z-stack

Open the z-stack window . The cube represents the specimen. The yellow plane marks the current z-position. This position is specified in the field below the cube (z-position μm).
 

1.Set the z-stack range:
Select the bottom first then the top of the sample by moving the z-position knob of the control panel and define the limits by clicking into the black Begin and End arrow (will turn red when fixed).

 

2.Set the z-step size:
By default the software calculates a z-step size and slice number. However, often such a high z-imaging frequency is not necessary. In this case define the interval of a z-stack manually (0.5 or 1μm recommended).

 

Note: Do not forget to set back the number of steps to 1 if you don’t want to record
stacks anymore.

Z-stack.jpg

1. Save your images on Brutus or on Scratch
2. Turn off the lasers in the software
3. Quit software
4. Clean the objective if you used immersion media: Moisten a soft tissue with ethanol or isopropanol
for cleaning oil, with distilled water for glycerol or residues of embedding media. Clean first the metal body and then sweep gently over the lens with a new tissue moistend with the cleaning solution.
5. Check in the online Resource EMBL Calender (Bookings in the page head)
if somebody uses the microscope after you (in the next 2 hours).

If yes
> Log off and leave the microscope and fluorescence lamp on.

 

If no
> Turn off the laser key D on the panel.
> Shut down the computer
> Switch off the fluorescence lamp
> Switch off the buttons A (PC/Mic) and B (Scanner) on the panel. Wait for 5 min unless you switch off the laser button C.

 

Note: It is mandatory to leave the microscope and room in a clean state.Especially make sure that all immersion oil is wiped off from the microscope and the surrounding. Thank you !
 

SP5_start_buttons.jpg

Shutting down

Most commonly used:

- xyz to image a volume

- Format either 512x512 (to search sample around) or 1024x1024 (for image acquisiton)

- Speed: 400 Hz for search field of view, 100-200 Hz for image acquisition

- Line and frame average and accumulation, use to reduce noise (it will increase acquistion time and possibly bleaching)

- Zoom, use this with care. Smallest pixel size is set by the NA of your objective. E.g. At 60x (NA 1.3) smallest pixels size should be about 100 nm. Pixel smaller than the optimal will result in oversampling, increased bleaching,increased noise and no extra spatial information. Pixel larger than the optimal will result in lower spatial resolution than given by the optics.

- Do not change: Pinhole (set to 1 AU)

 

Frequently Encountered problems

How should I correctly set the spectral detectors?

     The bar under the spectrum should be set as to cover as much as possible of the emission spectrum of the dye (or fluorescent protein) you are using. Ideally you would like to detect all of it. In practice there are two main considerations:

a. Do not let laser light into your detector.

b. Open the bar as much as possible and only limit this by the emission of your next fluorophore. This means that if you are using DAPI and 488, you can open the collection for the 488 until the end of the tail of the spectrum. If you have say 4 colors (405-DAPI. 488, 543 or 561 and 640) then you will not collect all but limit your detector until the next emission (see fig).

How much laser power should I use?

  • Are you imaging fixed cells or tissues labeled with organic (alexa, atto, etc) dyes? Then, don't worry too much and put laser (say 50% or even more). Roughly speaking, the amount of laser you set will be proportional to the amount of signal you will obtain. This is, the final quality of your image will depend on the amount of signal collected, which will be higher the more laser you put. As your sample is fixed and your goal is to get the prettiest picture possible you could even destroy all the fluorescence of your sample while you image it, as you would have gotten all the possible signal out. The only concern is if you have to image large Z-stacks, then be a bit conservative (say 30-40%).

  • Are you imaging live cells and you need to make a time-lapse movie? Then yes, you do need to care for the laser power (~10%) as your sample has to provide signal for all that time.

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