Biology Imaging

Welcome to the Leica

DM 5500 Microscope tutorial

• How to start up the system (p.3) • How to use the stand DMI 5500 (p.4)• Which soft to use? (p.12)LAS (for color images)• How to use the stand from the soft (p.13)• The Differential Interference Contrast (DIC) (p.15)• How to acquire a Bright Field image and its different adjustments (p.16)• How to focus easier (p.22)• The Mark & Find positions option (1) (p.23)• The Multi-step acquisition mode ( Mark & Find (2), Mosaic) (p.24)• How to create a shading reference (p.30)• How to see, to save your images and their information (p.31)• How to annotate your image (p.32)LAS AF (for black and white images)• Create your experiment (p.34)• How to acquire a fluorescence image and its different adjustements

(p.35)• How to acquire several channels and overlay them (p.38)• The Relative Focus Correction (p.40)• How to acquire a Z-stack (p.41)• The Mark & Find positions option (p.45)• The Tile scan option (p.46)• How to shut down the system (p.47)

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Biology Imaging

Leica

DM 5500 tutorial

Before using the Leica DM 5500

You need to reserve the

system =http://svint

ranet.epfl.ch /index.php?o ption=com_vi ew&task=vie w&id=52

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Biology Imaging

System Start up

• Turn on the Fluorescence lamp

• Turn on the Microscope button

You have to turn on the light only if it is cold (approximately 30 minutes after its last use)

• Turn on the PC

• Introduce your Username• Introduce your password

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Biology Imaging

Microscope (left side)

• Intensity of halogen or fluorescence lamp (+/-) (INT)

• Condenser Aperture for the contrast (+/-) (AP)

• Field diaphragm for the Köhler adjustment (+/-) (FD)

• Toggle between reflected and transmitted light

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Biology Imaging

Microscope (front part)• On the downer front part of the

microscope, you’ll find a tactile screen

• On its left side, you will see some tabs, to open the one you need to setup more options, you have to touch it

You only need to use two tabs:• The Eyes-piece-camera position

selection• The stage position

For the other options (Settings summary/Illumination/Magnification

), you can change them from the software

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Biology Imaging

Tactile screen (step1)Eyes-pieces/camera• When you’ve touched and opened the

tab of eyes-piece -

camera position selection,

you can change between eyes-pieces and camera

position (or a fifty-fifty mode)

You will also find both of the shutter.• For fluorescence• For brightfield

To open or close them, you have to touch the shutter button, and you will see

• The LCD off window allow you to switch off the tactile screen. To switch on again, just touch it

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Biology Imaging

Tactile screen (step2)About the stage

• When you’ve touched and opened the tab of stage controller,

You’ll see two news tabs:

• The first one named Focus

and care about the Z-focus-Axis

• The second one named

Stage

and care about the XY-Stage position:you will see the X, Y positions in mm

and you can change the step of X/Y stage controller between fast

and precise

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Biology Imaging

Tactile screen (step3)About the stageUnder the window FOCUS, you can

setup the Z Axis Stage

• By choosing a fine

or coarse focus wheel step

• By doing go down and up the stage in some positions

• Neared focus position

( Z=0)you can modify it with the set button and delete it with the clear button

• a down position, to put your sample or your immersion oil

In both cases, you have to click on the GO TO

button to activate your chosen position.

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Biology Imaging

Microscope (upper part) Cameras

• On the right up side of the microscope, you will find The camera button selection.

Indeed this microscope has two cameras:

• A color camera

(the upper one)• A black and white camera

( the right one)

When you’re opened your soft, you have to choose the right camera:

• With the LAS soft

select the color camera

(pull the camera button)• With the LAS AF

select the black and white one

(push the camera button)

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Biology Imaging

Microscope (right side)

• Magnifying lenses

( 1x/1.25/1.6x), to change the lens, you have to turn this wheel

Choose your objective

manually:

• HCX PL FLUOTAR

5x/0.15• HC PL FLUOTAR

10x/0.30• HC PL FLUOTAR

20x/0.50• PL FLUOTAR/ OIL 40x/1.00-0.5• HCX PL APO/ OIL

63x/1.40-0.60

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Biology Imaging

The JoystickNext to the microscope, you will find a

Joystick.This Joystick allows you to manage

the stage:

Turning

the upper button, you will manage the Y-axis direction

Turning the downer button, you will manage the X-axis direction

Turning the backwards button

you will manage the Z-axis direction

On each side of the joystick you will find two small black buttons which allows you to choose Between a coarse

or fine

moving in Z direction

OrBetween a fast

or precise X-Y moving direction

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Biology Imaging

Which soft to use?

When you arrive on your computer session, you will see that this computer has two softs, you have to open the right one.

• If you work with color camera,

you have to open

the LAS

soft

• If you work with black and white camera,

you

have to open the LAS AF soft

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Biology Imaging

LAS Soft

Microscope on the soft(MIC1)

Under Acquire and Mic1,

• you can see the objective

that you’re using

• You can setup the aperture

and field diaphragm

• You can setup the intensity

of the transmitted light and use the shutter

• You can select the eye piece

or camera

position

• For Acquiring your image after all settings done

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Biology Imaging

Microscope on the soft, step 2 (MIC2)

Under

Acquire and Mic2, you can first

• setup the focus wheel(fine or coarse)

• setup the X/Y stage manager

(fast/precise)

You can also Mark some positions

(6) that you could find later.

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Biology Imaging

Differential interference contrast (DIC)

Under Acquire and MIC1, select DIC contrast

• To change and choose the right contrast , turn the DIC prism wheel

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Biology Imaging

Acquire a transmitted light image• Under Acquire and Camera,

you can adjust• the exposure time • The contrast (gamma)

As you’re working with a color camera, you have to apply a white balance

before

acquire a picture (see next slide)

If you have no other option to setup

You can click on Acquire an image

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Biology Imaging

White Balance

As you’re acquiring a Bright filed picture

with a color camera, you

need to do a white balance

You have to• select an area of your live image

which has to be white

• Create around this area a selection with the left button of your mouse and Click on

White

balance

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Biology Imaging

Histogram and saturation options

Under Acquire and Camera and Histogram,

• You can see the grey scale

• You can tick Show Over/Under Exposure, and you will see the over exposure in red and the under exposure in blue, you shouldn’t have some red dots on your picture

• You can also reduce the grey scale

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Biology Imaging

Input option

Under Acquire and camera and input option, you can choose

• if you want a greyscale

picture

• The bit depth (8/12 bits)

• The right format (2088x1550 HQ)

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Biology Imaging

The binningUnder Acquire and Camera and

Input option,

• You can choose a binning, it will increase the camera sensitivity, reduce the exposure time

But you will lose

some resolution

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Biology Imaging

Scale Bar

• Under Camera and scale bar,

You can choose to show and change the scale bar, if you click on show

Be careful, with this option the scale bar won’t stay on your picture. if you really want a scale bar

on

your image you have to do it under process

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Biology Imaging

How to focus easier

Under Acquire and Camera and Region of interest,

• If you select zoom focus, it will zoom the region you want so you can easily find the right focus. When it’s done click on off, and the right focus will appear on all the picture

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Biology Imaging

Mark and Find options (1)Under

Acquire and Mic2,

You can Mark

some positions

(maximum 6) that you could find later.

• To mark a position

you have to click on the dark tab, to see all positions and select one of them and click on

store

When you’ve clicked on store, your position will be saved.

You will see the coordinates of your chosen position next to its name and on the right part of your screen, a circle surround the number of the position

When all positions you want have been marked, to find them you have to

Select the position

you want to find andClick on

GoToOrDobble

clicked

on the right part of thescreen on the right number surroundedposition.To clear position you have to select it and click

on CLEAR

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Biology Imaging

Multi step options, step1As the DM 5500 owns a

motorized stage, under multi-step you will find some applications linked to the stage, the two most important:

• A Mark and Find option

• A multi-step option, which allows you to create a mosaic

picture with several single images

To accede to the options you have to select

the MULTI STEP acquisition mode

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Biology Imaging

Multi step options, step 2When you’re opened the multi-step

acquisition mode, the “S”

tab will appear and open,

under Methode

choose the Mark and Find

options

• To mark a position, you have to choose the position you want and click on Create Do that for each position you want to mark

• To see your saved positions, Click on them

• To delete your positions clickon Clear. If you want to remove

only one position, select it and click on remove

To acquire

each position

which was marked, click on

Acquire Multi-Step

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Biology Imaging

Multi step options, step3(mosaic mode)With the Multi step

option, you can scan a “large”

part

of

your sample in several images which be put together,

By default, you will use the bi-directional scan

option

Then you have to select the start point

of your

mosaic and click on create

Afterwards you have to choose the end point

and click on

expand

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Biology Imaging

Multi step options, step4 (mosaic mode)You can now see your mosaic.

• if you’re area is too small, you can zoom it• To remove it, you have to click on clear

Before acquiring our picture, under option you can

• Save or load configurations

• Choose to create

a Mosaic image

or/and sub- images

• Choose what you want to see during the acquisition (Mosaic or singles images)

• Change the reduction factor

of your mosaic

When all your settings have been doneyou can click on Acquire MultiStep

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Biology Imaging

Multi step options, step5(mosaic mode)During acquisition you can see the advance of your mosaic

When your acquisition is finished,it will open under Browse, you can again

• Choose to see single images

ora Multi Step image

• Change the factor reduction

of the Multi Step image

If you want to attenuate boarders betweenSingle images, you have to apply a shading reference

(see next slide)

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Biology Imaging

Create a shading referenceYou will find the shading reference

under Acquire, camera and processing.

You can first choose one already- existed shading reference

, clicking here

To create

a shading reference, you have to

• Find an empty area of your sample or better a new empty slide

• Setup illumination until our picture becomes totally white

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Biology Imaging

Create a shading reference• Go under Acquire, camera and

processing and click on Create a shading reference

(the downer

selection you can do)

• Name it

If the illumination setup doesn’t be alright, the soft will tell you

• Finally click on Apply to live

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Biology Imaging

See your images and their information

• Under Browse and file information, you can see the information linked to the image

• Under Browse and File directory, you can see in which file is saved your image

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Biology Imaging

How to annotate your image• Under Process and Annotate,

you can choose to note some information

about the image

• The name , description , date and time

• The scale bar

• Lines, measurements (ex. distance line)

• To save

the picture with the information, you have to select Merge.

Be careful, if you do it, you can’t go back. So if you need the original picture, copy it before you annotate it

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Biology Imaging

LAS AF soft

When you’re opened the LAS AF soft, during the configuration time, you will see this window appears:

If you want to use the motorized stage

to acquire some

tile scan

or marking some positions, you have to initialized the stage

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Biology Imaging

How to acquire a fluorescence image: Create your experiment

Under Acquire and Experiment

When the software is open, experiment name

is create, you can change it

You can also open

some saved experiment

And if you want more than one experiment, you can create them with the New

button

When you experiment is finished you have to save it

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Biology Imaging

How acquire a fluorescence image?(step1)Under Light Path Settings,

you

have to set up your channel

• Choose the name• Choose the color

• Select the FLUO contrast method

• Choose the filter

Under load/save single settings, you can save

or

load

this settings

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Biology Imaging

How acquire a fluorescence image?(step2)

You have to click

on Live

to see your live image

Under Acquire and Acquisition, you can adjust

• the exposure time

• Change the intensity

For acquiring

your image after all settings done, you can click on Single image

; Capture image;

Start

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Biology Imaging

Bit depth

Under Configurations, and camera

You can choose between a

8

or

12

bits

depth image

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Biology Imaging

HistogramIf you click on this button,You will see• The histogram

of your image

For seeing over or under exposure

you have to go on the right screen.

Normally your image has to be in color, then you have to click on this button to change into Over-under exposure mode

(Over-exposure would be in blue and under exposure in green)

or Greyscale

mode

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Biology Imaging

How acquire several channels?Under the Light Path Settings, • you can add

or delete

some new channels, clicking on the plus or minus

For each channel you have to• Select the channel

Under the Light Path Settings,• Choose a name, a color, the FLUO

contrast method and the filter

Under Acquire and Acquisition,• Setup the exposure time and the

intensity

You can also

save

or load

some configuration

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Biology Imaging

How to overlay several channels?On your right screen, on the right

side you will see each channel you’re acquiring.

If you want to see only one channel, you have to select the channel

which interested you

If you want to

see all your channels, select all your channels

Finally if you want to see

an overlay of channels, select channels you

want to overlay and select the overlay mode

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Biology Imaging

Relative Focus Correction• The Focus changing option, permit

you to acquire a several channels image , when several channels have different focus point between them

• To open the focus changing option , click on this button

Relative Focus Correction (RFC)

Then you have to• Select the channel• Find the focus• Click on Store Z PositionFor each channel you want

Finally you have to click on apply Relative Correction Focus

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Biology Imaging

How to acquire a Z-stack (step1)

First, under Acquire and setup, you have to select the Lambda then Z

Z-movement

Under Acquire and Acquisition, you have to

• open the Z-stack mode

• Go to the Z-stack dialogue

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Biology Imaging

How to acquire a Z-stack (step2)

• Activate live mode

• 1) Find the first section by moving the z position until the objective is positioned on the top of your sample

• 2) select Begin

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Biology Imaging

How to acquire a Z-Stack ?(step3)• Continue in Live Mode

• 3) Draw and setup the last section by moving the z position until the objective is positioned below to your sample = move the red plane into the z-stack dialogue

• 4) Select End

• 5) Stop live mode

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Biology Imaging

How Acquire a Z-stack? (step4)

• 6) Introduce the step size

into the calculator

• Do enter

Or select the system optimized mode

• 7) Select

Start

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Biology Imaging

How to mark some positionsWith the mark and find option,

you can choose to save the position of several points on your sample and it will be able to find them later

You have to • Select the Mark and Find

option

• Select a new position

• Choose

on the sample the position

you want and click on this button

You can check the X-Y position

Then you have to click on Start

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Biology Imaging

Tile scan optionWith the tile scan option, you can scan a

“large”

part

of your sample in several images which be put together

You have to• Open the tile scan

option

• Select the start point

of the scanning on your sample and click on this button

• Select the end point

of the scanning on your sample and click on this button

On the tile scan dialogue, you can see, which surface will be scan and also in how many tiles it will be done

If you want to delete

your mosaic to create a new one, you have to stop

the Live mode

You can also see the positions of your point

Then you have to click on

Start

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Biology Imaging

How to shut down the system

• If you was using oil/glycerol objective, clean

the objective with ethanol/water

Before shutting down the system, you have to look on the reservation web-page if there is somebody using this microscope after you. In this case you haven’t to shut it down but you have to log off your session

If nobody comes after you, you have to• Exit the software• Shut down the PC• Turn off the microscope• Turn off the light

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