Leica confocal microscope Manual

Leica Confocal Manual

Neuroscience Imaging Core

Rightmire Hall

Ohio State University

Director: Tony Brown

060 Rightmire Hall

292-1205

[email protected]

Facility Manager: Paula Monsma

062 Rightmire Hall

292-3025

[email protected]

This manual prepared by Paula Monsma.

Leica Confocal Manual .................................................... 1

Getting Started ................................................................. 4

Start the Leica Confocal Software ................................... 4

Left Monitor ...................................................................... 5

Information Window ......................................................... 6

Beam Path Setting Window ............................................. 8

Format Button ................................................................ 10

Mode Button .................................................................. 10

Obj button ...................................................................... 11

MicCtrl Button ................................................................ 11

Pinh Button .................................................................... 12

Speed Button ................................................................. 12

Right Monitor ................................................................. 13

The Viewer Window ................................................... 13

Viewer Toolbar ............................................................... 14

Collecting an image ....................................................... 15

The Beam Path Settings Window ............................... 15

Take the first image .................................................... 16

Create an overlay of the two channels ....................... 17

Optimize the gain and offset of the two channels ....... 17

Averaging ....................................................................... 19

Averaging taking a single image ................................ 19

Crosstalk ........................................................................ 20

Check for Crosstalk .................................................... 20

Adjust beam path settings for crosstalk ...................... 22

Change the detection slot ........................................... 23

Set up a sequential scan ................................................ 25

Optimize the settings for each channel ...................... 25

Save the sequential setting ........................................ 26

Load the sequential scan settings .............................. 27

Acquire an image using the sequential scan settings . 28

Zoom Functions ............................................................. 30

Zooming on a specific area ........................................ 30

Centered Zoom .......................................................... 31

DIC................................................................................. 32

Take a DIC image ...................................................... 32

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Saving the Data ............................................................. 33

Saving the experiment ................................................ 33

Saving the overlay image ........................................... 35

Experiments ................................................................... 36

Create a new experiment ........................................... 36

Open a saved experiment .......................................... 37

Renaming images ...................................................... 38

Browsing all the images in an experiment .................. 38

Adding a scale bar ......................................................... 39

Taking a Z series ........................................................... 41

Set the Start and End Points ...................................... 41

Set the number of sections ......................................... 42

Collect the series ........................................................ 43

Viewing the Z-series ................................................... 44

Create a 3D Projection and Animation ........................... 45

Select the Projection Type ......................................... 46

Preview the Projection ................................................ 46

Export an Animation ...................................................... 48

Archiving Data - Create a data disk ............................... 49

The Nero Express Window ......................................... 50

Logging off of the computer ........................................... 53

Shutting Down ............................................................... 54

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Getting Started

1.

Turn on the nitrogen tank valve

2. Turn on the mercury bulb

(top)

3. Turn on the microscope

(bottom)

4.Turn on the fans for the lasers

(red switches)

5. Turn on the three lasers (keys)

7. Log in as a user

Start the Leica Confocal Software

Double click on the Leica Confocal Software icon.

4

6. Turn on the computer

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Left Monitor

Pull down menus and buttons-

Many of the functions here can be done somewhere else in the software. The most important button here is the

“SAVE” button.

Experiments Window –

Images are sent here as files.

Images can be saved, renamed, deleted and exported from this window.

Blank area for opened windows to be placed so that they don’t cover up other information.

Information Window – All of the settings for the current image are available here.

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Information Window

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Menu buttons – Each opens a new set of tools. Generally, these are used in a left to right order.

Z Series controls

Tools for acquiring images.

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Beam Path Setting Window

Laser Line Settings-

Indicates which laser lines are active and at what power.

Window-Saved beam path settings. Save your own settings here.

Transmitted light

PMT – make active to take DIC images.

Dichroic

(Beam Splitter)

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Select the detection slots for each PMT

Seq –Opens Sequential Scan Settings

Save – Saves the current Laser line and detection slot settings.

Close – Closes the Beam Path Settings window

Many different options for formatting the image.

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Format Button

Choose the resolution of the image to be collected. The default is

512 x 512.

Mode Button

Determines the type of scan that will occur. Default is xyz. Modes with “t” are timed.

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Obj button

Displays the objective currently in use. Also, can change the objective in use by clicking on the desired objective.

MicCtrl Button

To easily change the microscope settings between scanning and looking at your sample thru the eyepieces, select the “MicCtrl” button.

Choose “Scan” if you want acquire images with the confocal software.

Choose “Visual” if you want to look through the eyepieces of the microscope. If you get an error message when you try to acquire an image in the software check this se tting to make sure that “Scan” is selected.

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Pinh Button

Displays the current pinhole setting. The default for each objective is

1 Airy Disk. Increasing the pinhole will increase the signal, but will also reduce the confocality of the image.

Speed Button

Displays the current scan speed, which is equivalent to exposure.

Default is 400 Hz. Increasing the speed will decrease photobleaching and brightness of image, as well as the time it takes to acquire the image.

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Right Monitor

The Viewer Window

13

Viewer Toolbar- Buttons that will facilitate taking and displaying an image.

Viewer Window – The acquired images will be displayed here.

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Viewer Toolbar

Single, Tiled, Gall. and Ovl –Ways of determining the type of view that you would like in the viewer window.

Ch 1 thru Ch 4 –

Selecting/Deselecting will determine which channels are displayed in the viewer window.

Q LUT and Lut – Buttons for optimizing the gain and offset, as well as adjusting the display of the image.

Buttons for acquiring images. Many buttons are duplications of what is under the Acquire menu.

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Collecting an image

Select the MicCtrl button. Set it to scan.

The Beam Path Settings Window

Select the “Beam” button. The Beam Path Settings window will open.

To select excitation and detection settings, double click on one of the options under the Leica directory in the Beam Path Settings window

(blue arrow). The settings for FITC-TRITC are selected here.

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Take the first image

Select “Single Scan” to take one scan of your image.

The first scan on the right monitor. This image will be flipped on a horizontal axis as compared to what is seen through the eyepieces of the microscope.

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Create an overlay of the two channels

Choose the “Ovl” button to create a merged image of the two channels.

Optimize the gain and offset of the two channels

Select the “Q LUT” button. This will create an image where pixels of maximum value are displayed as blue and pixels with a value of zero are displayed as green. Pixels with values in the mid ranges are displayed as reds and whites. Adjusting this will change the way the data is collected by the detectors. This is not simply changing the way the image looks on the screen.

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Select “Continuous” to start a continuous scan. Select each channel, and using the control panel, use the smart gain knob and adjust the gain to show maximum values and use the smart offset to adjust the offset to show minimum values. Select the other channel and adjust the gain and offset for that channel.

This image has been adjusted, so that there are some maximum pixels and some minimum pixels, but nothing is saturated. To change the display back to colored image, simply click on Q LUT twice more.

The gain and offset will need to be checked and adjusted each time a setting is changed.

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Averaging

Averaging taking a single image

Select “Aver” Select single scan. Choose the number of frames you would like to average. Averaging two to four frames is most common.

Select “Single Scan”. An image will be created in the Experiments window.

The single scan is saved as an image in the Experiments window.

The small red check on the file icon indicates that this is the image that is currently being shown on the right monitor.

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Crosstalk

Check for Crosstalk

Reduce the 488 laser line to zero. Take a scan. Check the gain and offset.

Because there is no signal in the FITC channel, we would say that there isn’t crosstalk in this direction.

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Check for crosstalk from the other laser line

Reduce the 543 laser line to zero. Set the 488 laser line to its previous setting. Take a single scan.

Because we see a signal in the TRITC channel, this shows crosstalk.

Since we see signal in this channel it can mean that the 488 laser line is also exciting TRITC (excitation crosstalk) or that the emission curve for FITC is so large and the signal so strong that the detection slot for

TRITC is actually detecting FITC.

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Alexa 546

Spectra

488 FITC 543 TRITC

Looking at the spectra for both channels, you can see that the

488 laser line will excite both fluorophores, FITC more than

Alexa 546, so there is possibility of excitation crosstalk. When we look at the

488 FITC 543 TRITC detection slots (shaded areas) for both channels, we see that the tail of the emission spectra for FITC is clearly detected in

Alexa

488

Spectra the TRITC channel. So, if we excite with just the 488 laser at a power low enough that it doesn’t excite the TRITC channel, we will still see data in the TRITC channel, because the FITC emission curve is being detected in the TRITC channel.

Adjust beam path settings for crosstalk

Reduce the 488 laser line to 7% to see if that helps. The 488 laser line is very powerful and is generally used at a low level. Take a scan, adjust the gain and offset.

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There is still a large amount of crosstalk.

Change the detection slot

Using the slider, change the detection slot for the TRITC channel, so that the tail of the FITC emission is no longer being detected. Take a scan.

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The signal in the TRITC channel is reduced, but there is still some crosstalk.

Because the emission tail of the FITC is so large and there is so much overlap into the TRITC channel, this would be a good situation to use sequential scan.

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Set up a sequential scan

A sequential scan will take a scan with the settings for one channel and then the settings for another channel. It’s a good way to reduce crosstalk between channels.

Optimize the settings for each channel

Here, we are optimizing the settings for the FITC channel. We widened the detection slot and set the laser setting at reasonable level.

Take a scan. Optimize the gain and offset.

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Save the sequential setting

Select “Save” (blue arrow) in the Beam Path Settings window. The

“Name of Parameter Setting” (yellow arrow) dialog will open. Name the setting (here Seq Green Convallaria) and click “OK”. The setting will be saved under “User” (red arrow) in the Beam Path Settings window.

Here we are optimizing the settings for the TRITC channel.

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The resulting image.

Save this setting as for the other channel.

Load the sequential scan settings

Select “Seq” (blue arrow) in the Beam Path Settings window. The

Sequential Scan Settings window will open. Select one of the channel settings that you saved (red arrow and highlighted) and click

“Add” (yellow arrow) in the Sequential Scan Settings window. Then select the settings for the other channel and click “Add”.

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Here the settings for both channels have been added. Now choose a mode.

Acquire an image using the sequential scan settings

With the “Sequential Scan Settings” window open, select “Single

Scan” or “Continuous”. The resulting image will be collected using the sequential scan settings.

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The resulting image. Check and adjust the gain and offset.

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Zoom Functions

Zooming on a specific area

To choose an area to zoom in on, select “Z. In”. Go to your image and draw a box around the desired area. Collect an image using either Single Scan or Continuous scan. This is not simply zooming in on the already acquired image, it is scanning smaller area with the same resolution.

Resulting zoomed image.

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The amount of zoom is indicated in the information box.

Centered Zoom

Select the “Zoom” button. It will tell you what the current zoom is, as well as allow you to choose a specific zoom. This will zoom in on the center of the image. This is handy if you have several areas of interest in one larger image because you can use it to reduce the zoom to 1.00, so that you can see your original image again.

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DIC

Take a DIC image

To take a DIC image, make sure that the the objective prism and the condenser prism are correct for the objective you are using, the transmitted light detector selection knob is set to scan, and that the analyzer is not in the light path. Then check the “Active” box for the transmitted light PMT.

DIC Image Overlay Image

Take an image, adjust the gain and offset.

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Saving the Data

Saving the experiment

The program places all the images that you acquire in temporary memory until you save them. The images that are automatically sent to the experiment are the individual channels. If you have created an overlay of both channels, you will need to specifically save that. If the program crashes, upon restart it will notify you that there are temporary files and it will ask if you want to save them. If you exit out of the program purposely and choose not to save your images, they are not recoverable.

To save the experiment

Select “Save” at the top of the screen.

Shortcut Icon on the desktop

Each lab will have a user folder on the D drive in the “users” folder, as well as a shortcut to that folder on the desktop. Save images only on the D drive. Here, McKinney lab is the user folder. To save, select

“McKinney lab’s Documents”.

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Rename the experiment (red arrow) and click “Save” (blue arrow).

The experiment will be saved as a folder. The folder will contain all of the images acquired in the experiment.

The software creates a file that tells what the names of the files are in the experiment folder. If you wish to change the name of your files and still be able to open those files in the Leica software, you must

rename them from within the Leica software. If you go into

Windows and rename the individual files there, when you try to reopen these images in the Leica software you will not be able to see them. A good way to eliminate this problem is to create a permanent archival copy of your raw data on CD or DVD. When you want to use the images, make a copy of the image and change that.

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Saving the overlay image

In order to save an image that looks like the Viewer window, right click on the images in the right monitor. Choose “Send to”, then

“Experiment”, and then “All(snapshot)”. This will send an image that when you open it in Photoshop, will have the individual channels as well as the overlay in one composite image.

The icon for the snapshot (arrow) in the experiment window.

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Experiments

Create a new experiment

This would be useful if you have several very different samples you want to image during the same session. You can rename the experiment so that it’s clear which set of images came from which experiment without having to rename each image that is taken.

Select “New” to create a new experiment. The new experiment will be created in the Experiment window.

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Open a saved experiment

Se lect “Open”. An open window will open. Browse until you find your experiment folder. Select it and open it. Here “4-4-05

MolProbesSlide” was selected.

When the experiment folder is open, only the .lei file is visible.

Double click it to open. This will open all the images in this folder in the Leica software.

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Renaming images

To rename an image, right click on the name of the file and select rename.

Browsing all the images in an experiment

Choose “Browse”. This will display all the images in the experiment in a thumbnail format.

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Adding a scale bar

Check “Scale” in the Display menu, under Settings.

A scale bar will be added to your images. To format the scale bar to a specific length, choose the “Scale” button (arrow). A Scale Bar dialog will open.

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Type in the length of the scale bar and choose apply.

The resulting scale bar. In order to save the scale bar, you must right click on the image and send it to the experiment as a snapshot.

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Taking a Z series

A Z series is a set of optical slices through the specimen, each at a different Z plane. It provides a more three-dimensional picture of the specimen.

Set the Start and End Points

Select the “Series” (red arrow) button. This will bring up the “Setting

Z/YPosition for Spatial Image Series” dialog. Start a Continuous scan. Using the control panel knob, change the Z position until you are at the top of the specimen. Select the “Begin” button (green arrow) to set this as the beginning of the Z series. Change the Z position in the opposite direction until you are at the bottom of the specimen and choose the “End” button (blue arrow) to set this as the end of the Z series.

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Set the number of sections

Here the beginning and end points have already been chosen. Select the “Sect” button to choose the number of sections or slices that will be taken. The software automatically chooses an “optimized” number of sections. To change the number of sections, select “Others” and the Z-configuration dialog will open.

Select the number of sections. A good rule of thumb is to double the optimized step size.

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Here, the step size has been changed to 0.24. This decreases the number of sections to 194.

Collect the series

Select the “Series” button to start the collection of the Z-series. The

“Setting Z/Y-Position for Spatial Image Series” will show the progress of the Z-series. The green line in the box is the begin point. The red line in the box is the end point. The yellow line in the box is the current position.

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Viewing the Z-series

These are all the images in the Z-series. The select this view, choose

“Gall” for the gallery view, “Single” to only have one image, both “Ch

1” and “Ch 2” since both channels are being captured and “Ovl” to have the overlay image.

The Z series file is named “Series” with a number, in this case

Series027.

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Create a 3D Projection and Animation

Select the “Process” menu. Choose “3D Visualization and

Animation”.

In 3D Visualization and Animation, choose “Projections with

Animation”.

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Select the Projection Type

There are six different choices of how the projection is created. Each of the windows displays a preview of what the projection will look like with that method. In this case, “Average Proj.” was chosen.

Preview the Projection

Choose “Preview” (blue arrow) to create a preview in the Result window. Choose “Apply” (red arrow) to create a file in the

Experiments window that can be saved (red circle).

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A finished 3D Animation.

The projection will also be displayed in the Viewer window.

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Export an Animation

Right click on the 3D Animation file in the Experiments window. Here it is named 3DAnimAver_00. Choose “Export”.

A “Save As” window will open. Name the animation and choose from the “Save as type” menu. Here “Export-Animation-Avi (*.avi) has been chosen. Choose “Save”. The animation will be saved as an

AVI file.

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Archiving Data - Create a data disk

The computer has a CD/DVD writer as well as USB ports. Loading a

CD or DVD into the CD/DVD drive will cause burning software to open automatically. If it does not open automatically, click on Nero

SmartStart icon on the desktop.

Nero SmartStart icon

Choose “Data”. Choose “Make a Data CD” or “Make a Data DVD”, depending on the type of media you are using.

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The Nero Express Window

Open up your user folder. Select the items in the folder that you would like to save to the CD and drag them over to the CD project.

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The blue line indicates how much space the selected items consume.

Select “Next” to go to the next screen.

Select “Burn” to save the selected data to the CD.

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When the burn process is finished, a dialog will open. Click “OK”.

Then this window will open. Choose “Exit” to close the burning program.

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Logging off of the computer

After exiting out of the confocal software, log off of the computer.

Click on the “Start” button.

Choose “Log Off”.

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Shutting Down

7. Turn off the

nitrogen tank

4. Turn off the microscope

(bottom)

5. Turn off the mercury bulb

(top)

3.Turn off the lasers

(keys), but not the red buttons

6. At least 15 minutes

after #3! Turn off the fans for the lasers (red switches)

1. Shut down the

Leica software

2. Log out as a user

Alternative Shutdown Procedure:

1. Turn off the lasers (keys only) and the mercury bulb.

Wait 15 minutes before covering the microscope or turning off the fans for the laser (step 5).

2. Finish working with the software (save your data, burn a disk, etc). Shut down software. Log out as user.

3. Clean up microscope (clean off oil, leave 10x objective in place with the turret down).

4. Turn off the microscope.

5. Turn off the fans for the lasers (at least 15 minutes after

step 1!!).

6. Cover microscope.

7. Turn off the nitrogen tank. Shut off lights in the room.

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Troubleshooting

This section is intended to offer help in solving various problems. If y ou are still having problems with the confocal system, please don’t hesitate to contact Paula.

Hardware Legend Can’t Get All Values

If there is a problem with the way the microscope has initialized (or the microscope wasn’t on before the software was started), you will get this message – “Hardware legend can’t get all values.” (You will see this error message in other situations as well.) Click “OK”. The next screen will look like this.

DM IRE2 (SDK) is the microscope.

Make sure that the microscope is on and click “Initialize”.

You will see this message when the microscope is initialized and usable. Click “OK” and the software is ready to use.

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Device SCSI Controller V2 cannot find a Leica

Scanner on SCSI bus

If you see “Device SCSI Controller V2 cannot find a Leica Scanner on

SCSI bus”, the scan head has not completely booted up.

Click OK. Then you will see this message - “Hardware legend can’t get all the values”.

Click OK again. Then the software will load, but it will look like this and you won’t be able to take images.

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What to do:

1. Shut down the Leica software and wait a few minutes before restarting the software. Frequently, all the system needs is more time.

2. If step 1 doesn’t work, then the scan head might not have started up completely. In this case, turn off the lasers and the scan button (lower red switch), wait a few seconds and turn the scan button and the lasers back on.

Wait a few minutes and restart the software.

3. If neither step

1 or step 2 works, turn off the lasers and the scan button, as well as the microscope. Wait a few minutes and restart the lasers, microscope and scan button. Wait a few more minutes and restart the software.

4. If nothing works, do a complete shutdown of the system and then restart the whole system.

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