Leica TCS SP5 X Manual de usuario

Cell Imaging Unit – UIC

LEICA TCS SP5 MICROSCOPE MANUAL

TABLE OF CONTENTS

ANATOMY OF THE LEICA SP5 CONFOCAL MICROSCOPE 3

INITIALISATION PROCEDURE 4

SP5 MICROSCOPE 5

1. RIGHT SIDE OF MICROSCOPE (DETAILED VIEW) 5

2. FRONT SIDE OF MICROSCOPE (DETAILED VIEW) 6

3. LEFT SIDE OF MICROSCOPE (DETAILED VIEW) 6

4. CONTROL PANEL BUTTONS 7

STARTING THE LAS SOFTWARE 8

1. CONFIGURE FOR ACQUISITION 8

1.1

THE LEFT PANEL ALLOWS YOU TO CONFIGURE THE IMAGE

ACQUISITION PARAMETERS

1.2

THE RIGHT PANEL ALLOWS YOU TO LOAD A PRE-MADE

CONFIGURATION OR TO CREATE YOUR OWN ONE

1.3

CREATE A SEQUENTIAL ACQUISITION 10

2. ACQUIRE IMAGES 11

3. ACQUIRING A Z-STACk 12

4. SAVE AND EXPORT YOUR IMAGES 13

SHUTDOWN PROCEDURE 14

ANATOMY OF THE LEICA SP5 CONFOCAL MICROSCOPE

1. Research inverted microscope 1. Transmitted light source

2. Scanhead 2. Stage

3. TCS Workstation 3. CLSM port

4. Microscope control unit 4. z -Drive

5. Laser and power supply 5. Display

6. Computer table 6. Controller

7. Microscope table

7. x-Drive

10. Control panel 8. y-Drive

11. Control monitor 9. z-Drive

12. Image Monitor 10. Eyepieces

13. Supply control 11. Condenser head

12. field diaphragm

INITIALISATION PROCEDURE

1. Turn on the “PC”, “SCANNER” and “FAN” switch.

1. Turn the LASER key on

2. For fluorescence samples, turn on the Fluorescence light power source (check

first if the lamp is not hot), otherwise leave it off and proceed

3. Turn on the microscope

4. Log on into your personal/group account

5. Start the “Leica Confocal Software -LAS” available both as a Desktop

shortcut and the Windows start menu.

SP5 MICROSCOPE

The microscope is fully automated which means that you have now button in stead of

knobs. It you know what you do in a conventional microscope, it takes 5 minutes to

understand what the buttons are for.

1. RIGHT SIDE OF MICROSCOPE (DETAILED VIEW)

Displays the following information:

1. Microscope stage up movement (DO NOT USE).

2. When used with 7 is sets the maximum and minimum threshold (DO NOT

USE).

3. Microscope stage down movement (DO NOT USE).

4. Speed of knob 7

5. Change objective to high magnification.

6. Change objective to low magnification.

7. Focus knob.

2. FRONT SIDE OF MICROSCOPE (DETAILED VIEW)

Displays the following information:

1. Change cube for blue excitation filter cube – FITC, GFP

2. Change cube for green excitation filter cube – RFP, CY3

3. Use if for cutting some “umpolarized light” from the laser (might be

interesting for good DIC)

4. Empty position

5. Empty position

6. Empty position

7. Fluorescence shutter

3. LEFT SIDE OF MICROSCOPE (DETAILED VIEW)

Displays the following information:

1. Change from transmitted light to transmitted light detector (not confocal

detector)

2. Enables switching from oil to dry objectives and vice-versa, minimizing the

risk of having oil in dry objectives

3. Move one filter cube position

4. Change from confocal mode to visualization mode

5. Change aperture diaphragm just for florescence

6. Change field diaphragm just for transmitted light

7. Change light intensity in both fluorescence and transmitted light, depending

on which mode you are

8. Toggles between transmitted light and fluorescence

4.CONTROL PANEL BUTTONS

Displays the following information:

1. Smart gain

2. Smart offset

3. Rotation

4. Pinhole

5. Zoom

6. Z position

THE LAS SOFTWARE

1. Double-click on the LAS AF icon to start the program.

2. In the window coming up check that MACHINE is selected under

CONFIGURATION and START.

3. In the software window, go the CONFIGURATION tab, LASER and turn on

the lasers you need

4. Set the percent power on the Argon laser (25-30% is recommended).

1. CONFIGURE FOR ACQUISITION

It’s time now to configure the software for a confocal acquisition:

1.1THE LEFT PANEL ALLOWS YOU TO CONFIGURE THE

IMAGE ACQUISITION PARAMETERS:

•Acquisition type. Standard mode is XYZ.

•Image size in pixel. Standard size is 512x512 and acceptable for

publication, although in some instances 1024x1024 may be desirable

for publication. Lower and higher resolutions are available in the drop-

down menu, but beware, the higher the resolution the larger the file.

The pixel size and image size are shown.

•Acquisition speed in Hz de 10-1000 Hz. Standard speed is 400Hz.

Faster scanning speeds may be required for imaging live cells or

moving specimens. For even faster scanning, you can activate

bidirectional scanning (“Bidirectional X”). This doubles the scanning

speed, but requires “Phase” adjustment.

•Pinhole size. The default setting of 1 Airy Unit (AU) is the

recommended setting. (The pinhole setting of 1 Airy Unit will display

a different physical pinhole aperture size on the control panel for each

objective). For faint specimens, increasing the pinhole size may help,

but the images you are capturing may no longer be truly “confocal”

images.

•Zoom level. Refer to notes on “Zooming and selecting a Region of

Interest”. By zooming in the pixel size will decrease. For high

resolution imaging optimal pixel size according to the Nyquist

criterion should be ensured. Note that the pixel size can also be

decreased by enhancing the format.

•Level of averaging used to reduce the background noise. It’s

unnecessary to use an average of more than 8x. To determine whether

you should average your image more times, try capturing with more

averages and see whether the image is improved. Line averaging is

faster and therefore recommended for live cells. For fixed specimens, it

doesn’t really matter whether you use line averaging or frame

averaging or a combination, although frame averaging may be better

suited for some applications to allow for a brief recovery from photo-

bleaching.

1.2 THE RIGHT PANEL ALLOWS YOU TO LOAD A PRE-MADE

CONFIGURATION OR TO CREATE YOUR OWN ONE

1.2.1 If you want to use a pre-made configuration, just use the drop

down menu « Load/Save single settings »:

1.2.2 If you want to create your own configuration, follow this step-by-

step:

•Activate the laser lines you need (« fluo » and/or « Visible »).

•Tune their output power (usually between 15 and 50 % depending on the line

and the sample that is used).

•Activate the PMTs (detectors) that you need (check « Active »). Use first the

PMTs located just under the spectral band you want to detect.

•Choose the bandwidth according to your staining for each PMT.

1.3 CREATE A SEQUENTIAL ACQUISITION:

This mode allows you to avoid « crosstalk » problems by acquiring each channel one

after the other.

Click on the « sequential » button then create a configuration only one

channel with only one excitation wavelength.

Add a new sequence by clicking on the « + » button, the preceding sequence being

used as a template for the new one. Create the configuration for the next channel. You

can add as many sequences you want. To delete a sequence, click on the « - » button,

and the last one created will be deleted.

Click here to

select a pre-made

configuration

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