Home | CIML - How to use the SP5 confocal microscope · 2018. 3. 10. · 2016 / 02 Mailfert...

2016 / 02 Mailfert Sébastien – Imaging Immunity photonic microscopy facility – Centre d’Immunologie de Marseille-Luminy 1

How to use the SP5 confocal microscope

Mailfert Sébastien ([email protected])

Tel : 9126

Imaging Immunity (ImagImm) photonic microscopy facility

Centre d’Immunologie de Marseille-Luminy

mailto:[email protected]


Table of contents

I. Introduction .......................................................................................................................................... 4

A. Booking ............................................................................................................................................. 4

B. Location ............................................................................................................................................. 5

C. General view ..................................................................................................................................... 5

II. Startup procedure ................................................................................................................................. 6

A. General (general switch box, see page 5) ......................................................................................... 6

B. Fluorescence lamp (see page 5) ........................................................................................................ 6

III. Sample positioning ................................................................................................................................ 8

IV. Simultaneous acquisition of 3 channels ................................................................................................ 9

A. Introduction to acquisition ............................................................................................................... 9

a. Software window .......................................................................................................................... 9

B. QUIDELINES to follow ..................................................................................................................... 10

a. Excitation parameters ................................................................................................................. 10

b. Detection parameters ................................................................................................................. 10

c. Image parameters ....................................................................................................................... 10

C. First channel: DAPI .......................................................................................................................... 11




D. Second channel: AF488 ................................................................................................................... 16




E. Third channel: AF568 ...................................................................................................................... 19

F. Simultaneous three channels imaging ............................................................................................ 20

V. Sequential acquisition of 3 channels .................................................................................................. 21

A. Check the possible problems: switch the different lasers OFF ....................................................... 21

B. Sequential acquisition ..................................................................................................................... 23

VI. Z scan .................................................................................................................................................. 25

VII. Go further ........................................................................................................................................... 26

A. Zoom factor ..................................................................................................................................... 26


B. Pinhole effect .................................................................................................................................. 27

C. White Light Laser: interest??? ........................................................................................................ 28

D. Transmission image (pseudo brightfield)........................................................................................ 29

VIII. Saving files and apply previous settings ............................................................................................. 31

................................................................................................................................................................ 31

IX. Switch OFF procedure ......................................................................................................................... 32


You should cite the microscopy facility and FBI (France Bio Imaging). Please write this sentence: We acknowledge financial support from n° ANR-10-INBS-04-01 France Bio Imaging in your publications containing data obtained with our microscopes

I. Introduction A. Booking

Go to the intranet website http:\\Aton and select “Scientific Platforms” -> “Imaging Immunity” -> “Microscopy booking”

On the left part, select « Microscopy booking »

Sign in with your personal user and password and select Confocal Barry on the listed items

Figure 1 Intranet website

Figure 2 Booking website

http://aton/


B. Location The microscope is located on flour R-1, room 112

C. General view

1. Laser 2. Confocal head 3. Microscope 4. Joystick 5. Fluorescence lamp 6. Control panel 7. General switch box

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2 3

4

5 6

7

Figure 3 Microscope location

Figure 4 General view


II. Startup procedure

A. General (general switch box, see page 5)

• Switch the three green switches ON (PC + microscope, Scanner power, Laser power)

• Turn the key to the ON position

B. Fluorescence lamp (see page 5)

• Switch the fluorescence lamp ON • Choose the lamp intensity with the

rotating button: middle position is normally enough. If your signal is too weak, increase the lamp intensity but photobleaching can occur more rapidly

• NEVER press the small red button, this is to reset the lamp hours

C. Software (on the computer screen) • Double-click on the "PICSL" shortcut • Log to your session in order to be able to

save your data on your restricted shared area on PICSL

• Double-click on the "LAS AF" shortcut (Leica software)


• Click on “OK” (don’t touch the other buttons)

• Click on “Yes” (it will initialize the XY stage)

1. Select the “Configuration” tab 2. Click on “Laser” 3. Activate the checkboxes

- The 405 diode for dyes excitated @ 405nm - the WLL (White Light Laser) for dyes excited

between 470 and 670nm)

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2

1


III. Sample positioning Here we will follow these steps:

• Choose the objective • Position your sample in XYZ in brightfield (white) or widefield (fluorescence)

• Select the “Acquire” tab • The objective turret is not movable directly from the

microscope. You have to choose the objective from the software as indicated

• Be sure it’s an immersion objective before putting a SMALL drop of oil

• If it’s a dry objective, clean your sample

• To look at your sample in brightfield (white lamp), use

the “Tl/IL” button (switches between widefield to brightfield) (bottom left of the microscope)

• To tune the intensity, use the vertical “INT” buttons (bottom left of the microscope)

• Ensure the “STOP” lever is down (top right of the microscope)

• To look at your sample in widefield (fluorescence lamp), select one fluorescence cube (bottom front of the microscope). From left to right: GFP -> Cy3 -> None -> None -> DAPI -> GFP/Cy3

• Press the shutter “button” (first button on the top of the panel)

• You can see the objective selected (and other parameters) on the display panel (bottom front of the microscope, not tactile!)

• To move the objective faster (or slower), press the small button on the bottom right of the microscope

• To move the XY stage, use the joystick (X/Y with the first knobs and Z with the one on the back)

• To move the stage faster, press the “coarse” button and slower with the “fine” buttons at the base of the joystick


Multi-channel acquisition can be either simultaneous (faster) or sequential (slower but with less crosstalk)

IV. Simultaneous acquisition of 3 channels A. Introduction to acquisition

a. Software window

In this document:

- Laser and laser lines refer to Excitation - Channels refer to detection

General

settings

Excitation

Detection Images


B. QUIDELINES to follow

General steps that you need to follow. We will describe them more precisely in the rest of the document.

a. Excitation parameters • Switch the laser ON with its own shutter • Set the wavelength (for the WLL) • Set the laser power

b. Detection parameters • Activate the channel • Select the fluorophore (if it’s listed) • Display the emission (w or w/o the excitation) spectrum • Set the detection range • Set the PhotoMultiplicator Tube detector (PMT) Gain/Offset • Set the false color of the channel

c. Image parameters • Set the pinhole size • Set the directional mode (bi- or mono-) • Set the image format (number of pixels) • Set the scan speed • Set the number of averages • Set the zoom factor

In the next pages, we will begin with one fluorophore (DAPI) and add other channels. You can begin with any kind of fluorophore.


C. First channel: DAPI a. Excitation parameters

• Select the “Acquire” tab • Switch the 405 laser ON with its own shutter • Set the laser power (10 to 20% at the beginning)

b. Detection parameters • Activate the channel (click on the “Active” checkbox) • Click on “None” to open the drop-down window and

select the fluorophore (if it’s listed). If you don’t find your specific fluorophore, select one fluorophore with the same characteristics or select none. This is not required for the acquisition, it’s just to help you to set properly the excitation/detection parameters

• To display the emission (w or w/o the excitation) spectrum: right click on the rainbow image

• Set the detection range: move the slide bar entirely with the mouse or change the lower and upper limit with the mouse to define your own detection range. You can also set precisely the detection range by a double-click on the slide bar

• The detection range has to be shifted to the right of the laser line (i.e. if the laser is @ 405nm, begin to detect the DAPI @ 415nm)

• If the fluorescence is high (you will see later a saturated image), you can reduce the detection range

• If the signal is weak, enlarge this range. • If there is a potential overlap with a second

fluorophore, reduce the detection range


• Set the PMT Gain/Offset: click on the “PMT1” button • Set the Gain to 800 at the beginning • Set the offset to 0 at the beginning

• Set the false color of the channel: click on the color image on the right of the “PMT1” button and choose the rendering color of your final image

• It’s not a real color: you can choose any kind of LUT (Look Up Table)

• Don’t forget that your eyes are not really able to see the contrast between black and blue and prefers cyan/green/red/yellow/white contrasts

c. Image parameters Pinhole

• Click on the arrow on the right part of the Acquisition panel to display the panel entirely

• Select the “pinhole” checkbox

• A small widow appears where you can set the pinhole size

• If you increase this value, the thickness of the plane of interest will increase: more photons are detected (good!) but the resolution decreases (bad)!

• The pinhole size has to be equal to 1 AU (Airy Unit) if you want to obtain the best Z resolution


• The pinhole size is linked to the NA (numerical aperture) of your objective and the wavelength

• You can choose to display the pinhole size in µm or in AU

• Use the “Airy 1” button to set directly the pinhole size to 1

• The “section thickness” of the plane you image is automatically calculated and displayed

Scan direction, zoom factor and scan speed

• Set the directional mode (bi- or mono-) • Bi-directional save half of the acquisition time

because the laser scans your sample like a snake (from left to right then from right to left, etc.)

• Don’t change the correction factor which should be equal to -31.3

• Set the zoom factor: it’s not a numerical zoom, it’s really the laser which scans a smaller region when you increase this factor

• For live imaging, we don’t need to set the pixel size to reach the best XY resolution and the scan speed should be 400Hz

Live

• Click on the “Live” button to begin the acquisition

Look up table


• Click on the “Quick LUT” button to toggle between the different LUT (False color, B&W, saturation) and choose the saturation LUT.

Gain / offset

• Select the one called the saturation LUT which displays your signal between black and red, saturated pixel are in blue and pixels with a value of 0 are in green

• Set the Gain in order to increase the dynamic of your image (few pixels in blue). You can also play with the laser power and the detection range to enlarge the dynamic range (or decrease it if the signal is too high)

• Keep the laser power as low as possible to avoid the photobleaching effect • If you increase too much the gain, the noise will increase at the same time • Now reduce (it’s a negative value) the offset. This will set the low signals to 0. You should by this

way remove only the noise or the background (nonspecific labeling, autofluorescence). If you reduce too much the offset, you will begin to remove INFORMATION…


Image format (pixel number is linked to the pixel size!!!), scan speed and average

For live imaging (low quality but faster) • Image format: 512x512 (we don’t need resolution for live imaging) • Speed: 400Hz (to avoid photobleaching)

For the final acquisition: • Image format: depends of your objective and your zoom factor (we cannot give you a number!)

o Increasing it will lead to a better resolution (stop when the pixel size is bigger than the half of the resolution) and to an increase of the photobleaching process

o Decreasing it will lead to a less defined image but is faster to acquire and reduce the photobleaching • Scan speed: 200 Hz

o Decreasing it will lead to reduce the noise and to an increase of the photobleaching process o Increasing it will lead to the opposite phenomena

• Average line number (or frame but not needed): 2 to 4 o Increasing it will lead to reduce the noise and to an increase of the photobleaching process o Decreasing it will lead to the opposite phenomena

• Click on “START”: your image is displayed on the right panel. Change the LUT if you want to see your image in color (see page Look up table)

Advices: Let’s take an example: the pixel size should be equal to 100nm of the 40X or 63X with high NA but 200nm for the 20X with low NA to each the best resolution

• If the best resolution is not needed, you can increase the pixel size (i.e. decrease the format) • The pixel size is directly linked to the image size. If you apply a zoom to scan a smaller area, the

image size will decrease and then you have to change the image format

• A format of 2048x2048 or 4096x4096 pixels is not MANDATORY, except if it’s needed! If the pixel size reaches the resolution, don’t increase the number of pixels


D. Second channel: AF488 We will follow these steps:

• Deactivate the first channel and laser without changing their parameters • Follow the previous steps one by one for the second fluorophore

• Deactivate the 1st PMT • Switch the shutter of the 405nm laser OFF • Don’t change anything else on this 1st channel

a. Excitation parameters

• Select the WLL laser: click on the right part of the laser panel as indicated


WLL

• Switch the laser ON with its own shutter • Activate the 1st laser line: click on the checkbox • Set the wavelength: move the laser line to the desired wavelength (or double-click on the

wavelength value) • Set the laser power (10 to 20% at the beginning) with the black arrow on the right of the laser line


b. Detection parameters

• Activate the channel • Select the fluorophore, the detection range • Set the PMT Gain/Offset in the live mode (with the saturation LUT, 512x512 pixels, speed 400Hz) • Set the false color of the channel

c. Image parameters • Set the image format (number of pixels), the scan speed, the number of averages, the zoom

factor • Acquire your second channel: click on “Start”


E. Third channel: AF568 We will follow these steps:

• Deactivate the second channel and laser without changing their parameters • Follow the previous steps one by one for the third fluorophore

• Activate the 2nd laser line: click on the #2 small square on the top of the laser panel, click on the checkbox

• Set the wavelength, set the laser power, activate the channel, select the fluorophore, set the detection range, set the PMT Gain/Offset in the live mode, set the false color of the channel

• Set the image format (number of pixels),set the scan speed, set the number of averages, set the zoom factor

• Acquire your second channel: click on “Start”


F. Simultaneous three channels imaging

• Activate all laser lines: open the 405nm shutter and activate all the lines of the WLL • Activate all the three PMTs • Click on “START”: the three images appear in the right panel. You can double-click on these images

to enlarge them, unclick their number on the right part of the right panel in order to display or not one channel or to display or not the merge image (as indicated above)


V. Sequential acquisition of 3 channels

Here we will show how to be sure that there is no crosstalk between channels or cross-excitation (one laser exciting two dyes at the same time).

We will show how to avoid the crosstalk by sequential acquisition.

A. Check the possible problems: switch the different lasers OFF 405nm

• Switch the two laser lines of the WLL OFF (the sample is only excited @405nm) • Keep the three channels activated • You need to see a signal in the DAPI channel but not in the other channels • In this example, the AF488 dyes is not excited @405nm BUT DAPI is detected on the green channel


499nm

• Switch the 405nm OFF and the 594nm laser line OFF • Keep the three channels activated • You need to see a signal in the AF488 channel but not in the other channels • In this example, everything is correct: AF488 is not detected in the red channel and AF568 is not

excited by the 499nm laser

594nm

• Switch the 405nm OFF and the 499nm laser line OFF • Keep the three channels activated • Nothing happens except in the red channel: OK!


B. Sequential acquisition Here we will show how to set a sequential acquisition by creating two sequences: one for DAPI + AF568 and one for AF488 alone. For each sequence, we will select the corresponding lasers and channels.

• Click on the “Seq” button: another panel appears • Click on “+” to create two sequences • Select “Between frames”: this means that the switch

between both sequences will only appear between frames and not between lines. This avoid problems of crosstalk

• Switch the 405nm and 594nm ON and the 499nm laser line OFF • Keep the DAPI and AF568 channels activated • Inactivate the AF488 channel


• Select the second sequence • Switch the 405nm and 594nm OFF and the 499nm

laser line ON • Inactivate the DAPI and AF568 channels • Activate the AF488 channel

• Click on “Start” to acquire both sequences • Click on “Capture image” if you want to acquire only the selected sequence • Click on live: only the selected sequence will appear (except if you select “Between lines”)


VI. Z scan The interest of confocal imaging is to do Z-stacks. To do so, you have to:

- Define the lower and upper limits in Z - Define the Z step size

• Choose the Z-galvo (the sample is moving and not the

objective: faster and more precise) • Click on the “Live” button • Click on the Cube • Using the mouse wheel, move to the top of the

sample • Click on the “Begin” arrowhead (turned from black to

red) • Using the mouse wheel, move to the bottom of the

sample • Click on the “end” arrowhead (turned from black to

red) • Click on “Stop” • Set the “Z step size” to the desired value (not less

than 0.5µm for the high NA objectives or click on system optimized)

• Click on “Start”


VII. Go further

A. Zoom factor

If you need to zoom in your image, you can use a zoom factor. We suggest you to use integer numbers (1-2-3, etc.), this will allow you to show and compare images in your future article more easily (how to compare the localization of a protein into two images obtained with a zoom of 1.7 and 2.6?

Pinhole

White Light Laser

AF568 excited at 576nm

AF568 excited at 568nm

Z scan

• Select “Zoom in” and set a zoom factor of the desired value (4 in this example) • You can see directly that the image size is automatically calculated • You have to CHANGE the “image format” in order to reach the best resolution • This means that previously, with the 40X, the image format was 2048x2048. As the image is now 4

times smaller, we need to divide the number of pixels by 4: 512x512 is now the best choice for the image format


B. Pinhole effect Sometimes, you could be forced to use a non-unitary pinhole size:

• Your signal is really too weak and you don’t need a high resolved image in Z • You need to acquire a signal (calcium flux, FRET) which can occur everywhere in your sample. Then

acquiring thicker planes is requested and the resolution in Z is not a problem

• Increase the pinhole size and click on “Live” • The effect is clear: we collect more signal, the channels are saturated • Change now the laser power and the gain/offset parameters to avoid saturation

• Click on “Start”: you can see clearly that the image looks like a widefield image (blurred due to out-of-focus photons)


C. White Light Laser: interest??? The WLL allows you to set precisely the excitation wavelength in order to:

• Excite a fluorophore with the better efficiency • Excite less efficiently this fluorophore to avoid crosstalk or cross-excitation

AF568 excited @ 594nm

AF568 excited @ 568nm

• Less signal than @594nm because the maximum excitation peak is 594nm


D. Transmission image (pseudo brightfield) A pseudo-brightfield image can be acquired. This mode of imaging uses lasers (but it’s not linked to fluorescence!!!) and not the white lamp. The detection is performed by a PMT located above the condenser. The signal is proportional to:

- the transparency of your sample (for fixed cells, the contrast is really weak and you will probably not see lots of details)

- the laser power you use for the other channel(s)

• Activate the “PMT Trans” channel • Click on “Live” • Change the PMT Trans gain • The resulting image and more interestingly the merge of the different channels is displayed


VIII. Saving files and apply previous settings

The Leica format for files is .lif • A .lif file can be opened by ImageJ/FiJi • A .lif file is called an “Experiment” • An experiment contains one or multiple images

When you click on “start”, a temporary Experiment is created. To save it:

• Right click on your image and rename it • Right click on the experiment file, rename it, and save

it in the PICSL server (everything is managed by right clicks)

• For each new acquisition, rename the image and click on “Save All”

If you need to reproduce a previous experiment with exactly the same settings, you have to:

• Open a previous experiment from your PICSL file • Select the image you want to reproduce. The system

will keep: o Lasers (power, wavelength) o Channel configuration o Sequences o Image format o Etc.

BUT o Many parameters have to be adjusted every

day for every sample: you HAVE TO set the gain/offset/power/pixel size, etc.

• Click on apply • Never use the settings from someone of your lab if

you don’t know precisely what he did!

Experiment

Images


IX. Switch OFF procedure

A. Between users

• Log off from your Windows session • Fill the notebook • Clean the objective(s) • If the next user will come in at least 2

hours, switch the fluorescence lamp OFF

B. If you are the last user of the day

• Switch the fluorescence lamp OFF • Fill the notebook with the lamp hours /

your name / the date / comments • NEVER press the small red button, this is

to reset the lamp hours • Shut down the computer

When the computer is completely OFF: • Turn the key to the OFF position • Switch the three green switches OFF

(Laser power, Scanner power, PC + microscope)


I. IntroductionA. BookingB. LocationC. General view

II. Startup procedureA. General (general switch box, see page 5)B. Fluorescence lamp (see page 5)

III. Sample positioningIV. Simultaneous acquisition of 3 channelsA. Introduction to acquisitiona. Software window

B. QUIDELINES to followa. Excitation parametersb. Detection parametersc. Image parameters

C. First channel: DAPIa. Excitation parametersb. Detection parametersc. Image parametersPinholeScan direction, zoom factor and scan speedLiveLook up tableImage format (pixel number is linked to the pixel size!!!), scan speed and average

D. Second channel: AF488a. Excitation parametersWLL

b. Detection parametersc. Image parameters

E. Third channel: AF568F. Simultaneous three channels imaging

V. Sequential acquisition of 3 channelsA. Check the possible problems: switch the different lasers OFF405nm499nm594nm

B. Sequential acquisition

VI. Z scanVII. Go furtherA. Zoom factorB. Pinhole effectC. White Light Laser: interest???AF568 excited @ 594nmAF568 excited @ 568nm

D. Transmission image (pseudo brightfield)

VIII. Saving files and apply previous settingsIX. Switch OFF procedure

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