Leica SP5 confocal User manual

1

Title: Leica SP5 Confocal User Manual

Date of first issue: 23/10/2015

Date of review:

Version: Admin

For assistance or to report an issue

Office: CG07 or 05

Email: Igmm-[email protected]

Website: www.igmm-imaging.com

Download a PDF copy of manual:

\\igmm-smbhost\microscope-users\microscope user manuals\Leica

SP5

Facility Usage Policy

1. You must have the relevant Risk Assessment/COSHH form for the

work you are undertaking before using imaging facility resources

2. Users must be trained before using facility equipment

3. Please leave the microscope clean and tidy for the next user

4. Please report any issue, even if it seems minor, to facility staff

5. Any clinical waste must be placed in the orange bins provided

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* See the labelled image below for location of each switch

1. Mercury-Metal Halide Fluorescence Lamp

2. Green confocal control switches found on the right side of the desk (from left to right)

-PC/Microscope, Scanner Power and Laser Power

Turn the Laser Emission key to the ON position

3. The PC will boot automatically, login using your windows user account. If you have

been trained on the system a user account will have been created for you.

4. Launch LAS AF using the Desktop icon

5. The software initialises devices during start up, when messages appear click OK to

proceed.

6. Select the Configuration tab and laser. Tick the boxes of the lasers you want to turn

on. Move the Argon slide bar to 50% output

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1. Check the booking system to see if anyone is booked on the system within the

next hour. If not shutdown.

2. Select Configuration>Laser and drop the Argon slider back to standby

3. Close the LAS-AF program and shut down the PC

4. Turn the Laser Emission key to the horizontal position

5. DO NOT SWITCH OFF THE LASER POWER SWITCH UNTIL

THE ARGON FAN HAS STOPPED. Continue to next step while

waiting.

6. Switch off Scanner Power

7. Switch off PC/Microscope (Ensure PC is off before doing this)

8. Switch off the Mercury-Metal Halide Fluorescence Lamp via the power switch

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1. Ensure the correct stage insert is mounted (see facility staff if

unsure)

2. Remember to invert your slide so that the cover glass is the closest

surface to the lens

3. Select the objective lens either using the two buttons behind the right focus

wheel or in the software within the Acquire tab then select the Objective text to

see a drop down list of lenses

4. Use the joystick to move the stage if necessary. Change the XY speed

of the stage by pressing the black buttons on the left side of the joystick

base. The black buttons on the right change the speed in Z.

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1. Choose a fluorescence filter cube via the buttons on the front of the microscope.

Push the shutter button to toggle open/close shutter.

I3 = FITC

N2.1 = TxRd

A = DAPI

2. Once the sample is in focus you can record this Z

position by holding the two outer Set Z buttons on the

right side of the microscope. This sets this Z position to

X

Y

Z

Fluorescence

shutter

Filters

Focus speed

(course/fine)

Observation

mode BF/FL

BF intensity

(Controller on

LHS of microscope

Z position

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zero. Pressing the lower Z set button drops the lens to its lowest position. This

is useful when you want to change to a different cover glass on the same slide if

you’re using an oil immersion lens. Dropping the lens is always good practice

when changing slides too.

3. To view brightfield select an empty filter cube position using the buttons on the

front of the microscope.

4. Switch on the transmitted light using the TL/IL button on the left side of the

microscope. Adjust the intensity up/down with the INT buttons.

5. At this point you can either change to a higher magnification lens if required or

begin the scanning process

6. Lenses 60x and above always require a drop of oil between the lens and the

coverglass. 40x lenses can be either a dry or oil type. The software menu used

to select the lenses tells you if the lens requires oil.

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1. Select Configuration tab>Laser

2. Tick the boxes next to the lasers you want to use

3. If using the Argon laser (458/488/514 lines) set the percentage output to

50% in the text box next to the power slide bar then press enter

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1. Select Configuration tab>Settings

2. From the Image Resolution drop down list select 12bit

3. The detectors have a 12bit analogue to digital converter so if you select

12bit image resolution this ensures the full dynamic range of the

detector can be utilised

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1. Under the Beam Path Settings heading select the drop down list

and scroll down to the heading “User Settings”, here you will find

the personal beam path settings you have already configured.

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1. To see a continuously scanned live image of the sample press the Live button at

the bottom of the screen. You can change any of the capture parameters with or

without Live mode enabled.

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1. Activate the laser lines required by moving the vertical slide bars to >0%, the

laser line will be added to the spectral diagram

2. Activate as many detectors as required using the tick box below each detector

3. Select the emission spectrum label for each channel, choose the

fluorophore/protein you’re using if possible. This will add the emission spectrum

of the dye to the diagram.

4. By moving the spectral sliders you can adjust the detectors detection bandwidth.

Click and drag a slider to move it. Hover the cursor over the edge of a slider to

allow the width to be adjusted. Try to match the width of the slider to the

emission spectrum of the dye. Avoid overlapping the spectral sliders with the

excitation laser lines (5nm min separation).

5. Select the Channel colour box for each detector if you’d like to channel the

image colour (Lookup table)

6. Press a detectors PMT text to access that detectors Gain and Offset values

7. Use the Save button to store the adjusted beam path setting. This new setting

will appear below the User Settings heading at the bottom of the drop down list.

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1. The process of setting up the beam path setting above describes the setup for

simultaneous scanning. This means that all active laser lines and detectors

scan and acquire simultaneously. This has the advantage of speed but the

disadvantage is that fluorescence cross-talk (signal from one fluorophore

recorded in more than one detector) is more likely.

2. In Sequential scanning mode only one detector/laser is active at any one time,

making cross-talk much less likely but at the expense of speed.

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1. From the left hand margin of the screen under Acquisition

Mode select the Seq button to open the sequential mode

window

2. Select the + button which will add a scan. Each scan

corresponds to a single laser/detector combination.

Therefore if you are imaging in 2 channels then you will need

to add 2 sequential scans.

3. For a GFP-RFP setup for example you would

select the newly added Scan 1 button then

configure the beam path setup so that only the

488nm laser and the detector assigned to GFP

are active. Use the vertical slide bars to turn all

other laser lines off. Uncheck the other detectors

to inactivate them.

4. Now select the Scan 2 button and repeat the

procedure but this time enable the laser line and

detector for RFP and switch off the first detector

and 488 laser by setting to 0% power.

5. Now when you scan the lasers/detectors are

sequentially turned on and off rapidly to avoid cross talk.

6. Note that there are 3 sequential scan options:

Between lines- (Recommended) The laser currently scanning switches on a line

by line basis. I.e. one line of the GFP channel is imaged, then one line of the

RFP channel etc.

Between frames-An entire image of one channel is created first, then the laser

switches and acquires an image of the second channel.

Between stacks-If acquiring a Z stack, then the stack of Scan 1 (GFP) is

acquired first then the stack of Scan 2 is acquired (RFP).

7. Press the Save button in the sequential scan window and save to your folder on

the D: Disk. Include which wavelengths the sequential scan settings relate too,

to make it easy to select another time.

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Acquisition Settings

1. Select the XY window in the left hand margin

2. Format changes the size of the image in pixels. For

better xy sampling choose 1024x1024. For speed

choose 512x512.

3. Speed determines how long the laser spot dwells over

any particular part of the sample. The higher the

frequency the lower the dwell time and thus the lower

the exposure. Optimal values can range between 400-

100Hz.

4. Tick the Pinhole box which will expand the Pinhole window. Press the 1 Airy

button. This selects the best compromise between light throughout, resolution

and noise. It is also responsible for determining the optical section thickness

with higher values increasing the thickness of the optical section.

5. Use the Zoom Factor slider to increase the image magnification. This can

increase resolution because the pixel size decreases (microns per pixel).

6. Line/Frame Averaging can be used to reduce the image noise level by

scanning each channel a number of times and averaging the resulting signal.

Only use if you have a noisy image. Image capture time is increased as is the

likelihood of phoobleaching.

7. Rotation allows the whole scanned field of view to be rotated, if you need to

rotate your image while scanning.

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1. Start with a format of 512x512 and a speed of 400Hz. Set the laser power

between 5-10% for a each channel. Do not apply zoom or averaging at this

stage.

2. Press the Live button hopefully you will see an image of each channel displayed

on the right hand monitor. Each channel has its own window by default and you

can toggle them off/on using the panel on the far right of the image monitor. The

lowest button pictured here toggles the merged view on/off.

3. Check the detector gain settings for each channel by clicking on the PMT text at

the top of each detector window. Gain is used to amplify the signal thus the

image brightness will increase.

4. However, high gain levels will increase the noise level in

the images therefore achieving optimum exposure is

always a compromise between laser power, gain and scan

speed.

11

5. The Offset is also found in the PMT settings menu and controls the image black

level. As an example setting an offset of zero means that pixel intensity values

between 1-4095 have a grey scale value and will be visible in the image. If you

set the offset to -10% this means that any pixel with an intensity lower than 409.5

(4095 x 0.10) will be set to black (zero). You can use this to reduce background

fluorescence but be aware that you can cancel out specific signal if not set

correctly.

6. You can determine if the signal fills the dynamic range of the detectors by using

the saturation indicator which is found in the toolbar to the left of the images.

Press the button once and the indicator will be activated. Now any pixels which

have an intensity of 4095 (max) will be coloured blue.

7. The aim is to set each channel image so that the pixel intensities are just under

saturation level (4095). It’s not a concern if there are a few blue pixels. Press

the saturation indicator button again twice to see the channel images in their

original colours.

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1. If you have followed the sections above you just need to

decide if the image is to be captured simultaneously or

sequentially.

2. Press the Capture Image button at the bottom right of the left monitor and a

single scan of each channel will be acquired.

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1. Captured images will be added to the experiment. You can

access the experiment images by selecting the Experiments

tab in the top left corner of the left monitor.

2. Images are given a default name “Image” followed by an

incremental number unless you uncheck the “Default image

basename” option.

3. You can rename an image by right clicking on it then select rename.

4. Images in the experiment are stored temporarily so you must ensure you save

the experiment to disk at regular intervals and at the end of the session using the

Save All option at the bottom of the experiment window.

5. Images are stored as Leica LIF format by default which can be opened with

FIJI’s Bio-Formats plugin.

12

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1. Adjust scan parameters as for 2D image capture.

2. When optimising the image intensity for a z stack, move

through focus while live scanning to find the focal plane with

the highest signal intensity. Turn on the pixel saturation

indicator to aid in this process.

3. Ensure the Z stack panel is visible. View the live image and

move in one direction through focus using the joystick Z

control until you find the top/bottom of your sample. Click on

the Begin arrow on the diagram to record that position.

Then move to the opposite end of the sample and click the

End arrow. Stop scanning.

4. You can change the number of steps or step size by typing in the relevant boxes.

It is recommended that you use the System Optimised option. This will

calculated the step size and number of steps automatically according to the

Nyquist-Shannon sampling theorem.

5. Click Start button to acquire the Z stack.

6. Z stacks appear in the experiment folder with the name “series” by default.

13

Capturing a Tile Scan Image (Image Stitching)

1. Enable the tile scan option in the Acquisition mode window.

The tile scan window will appear.

2. Here you can see an image of the stages travel range. The cross hairs point to a

small square box which represents your current XY position. Click on the +

magnifying glass to zoom in on the box.

3. Clicking the mouse anywhere within the scaled region will

cause the stage to move to that position, USE WITH

CAUTION. Only click small distances from the current

position (indicated by cross hair).

4. The Scan Field box allows you to define how many images

should be captured in XY. For example type “2x2” for a

square grid of 4 tiles.

5. Click on the rubbish bin button to remove the scan field

setting.

6. You can customise the region to be tile scanned further by

using the Mark Position button. For example to define your

own xy grid, begin live scanning and move to the top left

corner of the region you want to scan by using the xy joystick.

Your current position is updated within the tile scan window.

Click Mark Position, then move to the bottom right corner of

the region you want to scan, click Mark Position again.

7. The software will calculate how many xy tiles are required to scan the area you

have defined. Once finished the tiles will be stitched together.

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1. Enable the Mark and Find option in the Acquisition Mode

window. The mark and find window will appear.

2. Here you can see an image of the stages travel range. The cross hairs point to a

small square box which represents your current XY position. Click on the +

magnifying glass to zoom in on the box.

3. Clicking the mouse anywhere within the scaled region will cause the stage to

move to that position, USE WITH CAUTION. Only click small distances from the

current position (indicated by cross hair).

4. To mark a field of view to be captured click the Mark Position button.

5. Continue to add further positions to be scanned by using the joystick to move

around. The positions are added to a dropdown list, selecting a position from the

list will move the stage to that position.

6. The Show All Positions button will highlight within the stage area the fields of

view you have marked for scanning.

7. You can delete a single position using the Clear Position button or delete all

using the rubbish bin button.

8. If acquiring a Z stack at each position you can either use the same stack

dimensions as defined for one position at all positions or define individual Z

stacks per position. This is set using the Same Stack For All tick box.

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1. Select a beam path setting and configure each wavelength channel as required.

Follow the sections above to add a tile scan, mark and find etc.

2. In the Acquisition Mode window choose the relevant time

experiment from the dropdown list xt, xyt, xyzt etc.

3. This will open the Time window. Define the total

duration of the time lapse and the frequency of

captures in the Interval box.

4. The minimise tick box will set the shortest possible

delay based on your scan speed etc.

5. The Stacks option allows you to choose how many time

points you collect instead of having to input a duration +

interval.

15

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Adding a scale bar to an image

1. On an open image select the Scale icon from the set of tools left of the

displayed image

2. Hold the left mouse button and drag across the image to draw a scale

bar

3. To edit the length or position of the bar select the Cursor icon then click on one

end of the scale bar to adjust its length or in the middle of the scale bar to move

it within the image

4. To delete the scale bar select the bar using the cursor tool then select the Delete

button

5. To save a copy of the image with the scale bar, right click on the

image then select “Snapshot all”

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1. To export an experiment or single image, right click on it in the Experiments view

then select Export>As TIFF.

2. Select the destination folder for the exported images.

3. The Overlay Channels option will save a single file

(RGB colour) that contains all of the wavelength

channels captured. This is not the raw data and

cannot be used for quantitative intensity analysis.

4. If the Overlay Channels option is not ticked then a

Raw data check box appears, select this option. The

greyscale 16bit images will be saved as individual

TIFF files.

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Leica SP5 confocal User manual

Leica SP5 confocal User manual

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