Leica SP2 Manual de usuario

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Manual for Leica SP2 Confocal Microscope

Enter you name, the date, the time, and the account number in the user log book.

Things to check before start-up.

•Make sure that your sample slides are clean and sealed. Use Windex and cotton balls or Kimwipes to clean

coverslips. Fixed samples need to be sealed with nail polish.

•Check the objectives. The 10x and 20x objectives are DRY objectives, and should NEVER have oil on them!

There are 40x and 63x OIL immersion objectives, and a 63x WATER immersion objective also on this

microscope. If oil immersion objectives have oil on them, wipe the lens gently only with lens paper. With

the microscope controllerON, you see the information of the objective in position on the reading panel

at the front of the microscope. The order of

the objective lenses in the lens turret is:10X, 20X,

40X Oil, 63X Oil, and 63X Water.

•The 63X objective has aperture collar adjustment

rings that control the size of aperture opening. It

should be wide open for most imaging condition.

If your sample looks dim or the whole view field

is not illuminated evenly when you look through

the eyepieces, turn the collar to open the aperture.

•The field diaphragm and aperture diaphragm in

the excitation path of the mercury lamp are

controlled by round, black dials on the left side

toward the back of the microscope, and control

the fluorescence illumination from the mercury

lamp. For all imaging with the mercury light

source, these should be set wide open by turning all the way counter-clockwise.

Setting up for bright-field and fluorescence

viewing.

Basic Operation of Leica DM IRE2 Microsocpe.

Turn on the microscope controller .

Turn on the mercury arc lamp.

Bright-field viewing:

Place your sample slide with coverslip (preferably #1.5

thickness) facing down to the objective on the stage.

For bright-field viewing, turn on the microscope light by turning the wheel

toward you and turn the VIS-SCAN switch to VIS position (Note:

switch back to Scan position for confocal scanning, see below)

Start with low magnification objectives (10X or 20X) first to find and focus

onto your specimen. The information of the selected objective and their z-

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position can be read on the readout panel at the front of the scope. You

can place the desired objective in position by turning the lens turret by

hand or by pressing the objective change button on the left side of the

microscope.

Pressing upper objective change button once rotates in next higher

objective into place and pressing lower change button rotates in next lower

objective into place.

However, pressing these buttons does not change between dry and oil

objectives. To do this, first press buttons  simultaneously,

and when the readout panel reads “change objective I (or D by

pressing again)”, then push the objective change button .

To focus, use coarse movement button (upper one for bring lens

up to a safety distance, “0” position and lower one for lowering

lens down) and fine focus knob (9) for focusing. The coarseness of

focusing movement (“S0” (fine) to “S3” (coarse)) can be set by

pressing “STEP” button.

For using oil or water immersion lens, lower the objective

turret slightly, expose the lens by turning the turret half

way, and place a drop of oil or water on the objective (be

careful not to touch the lens directly with application

tools!). Place the slide over the objective and raise the

objective until the oil just spreads out as it contacts the

slide. Use find focus knob to gradually bring your sample

into focus. Check your slide occasionally to make sure that

you are not pushing up the slide with the objective. This

can damage the objective lens and your sample!

Please DO NOT press “LEARN” and “CHANGE”

buttons.

Fluorescent viewing:

You can check your sample to see if your fluorescence labeling

works by using the mercury lamp illumination and the

appropriate filter set. The microscope has filter sets for viewing

DAPI (A), Green fluorescence (I3) and red fluorescence (N2.1).

Press the arrow horizontal button  on the front of the scope to

choose the filter set. “Scan” position has no filter set in place and

is used for laser confocal scanning. The fluorescent light can be

blocked (closed) or emitted by pressing Shutter  button. The

“closed” light should be off for fluorescent viewing.

Turn the VIS-SCAN switch to Scan position to block the

transmitted bright-field light, if necessary.

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Setting up for Confocal Imaging.

Turn on the Scanner/Laser He/Ne switch  (the

scanner should be on for about 1 min before starting

the LCS program).

Turn on the PC/Stand switch  and turn on the

computer. Log onto using your User ID and

password or TCS User.

Start the LCS program by double-

clicking the LCS icon.

On the main menu, select the Personal that contains your setting for acquisition parameters you have created or

Company for a default setting.

To start the laser(s);

(1) Turn on only the lasers you will use. They do not

need more than a minute or so to warm up and

stabilize, so it is not necessary to turn them on

until you know you need them.

(2) Argon (for 458, 476, 488, and 514 nm laser): Turn

on the red Laser Ar/ArKr switch  and turn the

Level Ar/ArKr knob  to MIN. Then, turn the

left Ar/ArKr key  to “ON”, then push to

“START” and release. Adjust the laser power with

LEVEL knob (usually 9 o’clock position is

sufficient).

(3) Melles Griot yellow DPSS (561 nm) laser: push

the green button .

(4) He/Ne (633 nm) laser: turn on the right He/Ne 633

key.

(5) Diode UV laser 405 nm: Turn on the red power button and turn the key  to Iposition.

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LCS Imaging program setting

Upon LCS program start, the basic image acquisition menu will appear. The “Acquire” button should appear

pressed.

Click “Beam” button and select the appropriate detector setup for the

fluorescent labels you used on your sample from the drop-down list .

This will set up the emission detector band-width, activate the proper

detector (PMTs), dichroic beamsplitter, and excitation laser wavelength

and power. You can adjust the range and position of detector bandwidth

and the laser power level as necessary. 

You also can change the color scheme of individual images into any color

or grey scale by clicking the pseudo-color selector  associated with

PMTs on the window screen (Note: the color information will be saved

with image files when saved).

Press Mode button to select Scan Mode (default is XYZ).

Press Format, Speed, and Bit buttons to select these parameters (defaults are 512x512 format, 400Hz scan

speed, and 8 bit image).

Detector

bandwidth

and range

Laser power level

Dichroic

beamsplitter

setting

Detector selector &

pseudo-color selector

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Press Z-scan button and select z-Wide in order to use Z POS knob to

move the objective for focusing (Default is z-Galvo, which is used with

Galvo stage adaptor).

Click MicCtrl button to switch to “Visual” mode, in which you can

examine your sample visually at the microscope. Switch “MicCtrl” to

“Scan”mode for laser scanning (it will automatically shut off visual

output through the eyepieces).

Press “Continuous” button to start scanning. An image or images, depending on the number of active

detectors, will appear on the right monitor.

Control Panel

Optimize the images by adjusting parameters including;

•The z-position (focusing) within the specimen (Z-POS knob).

•Zoom factor (ZOOM knob ): Default is 1. Increasing zoom will magnify the image but also will bleach

the sample faster!

•PMT and Smart Offset knobs : Turn PMT knobs to increase the signal intensity of each fluorescent and

transmission image channel. To decrease background signal, click the mouse over the image channel to

select it and turn Offset knob counterclockwise. By activating Q LUT button , a full dynamic range of the

PMT can be obtained. In this mode, pixels saturating the PMT will appear blue and pixels which are black (0

value) appear green. Set the PMT gain so that the brightest pixels are just under the saturation and set the

PMT offset such that the darkest pixels are just above the zero value.

•Laser power: adjust with dragging the laser power bar on the menu window if necessary.

Click the Continuous button again to stop scanning.

You can save the current parameter setting with Save button for later use.

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Working with Images.

Single button–

puts up full screen

of one fluorescent

channel.

Ch1, 2, 3, 4

buttons– activate

the channel on the

screen.

Tiled button–

views up to 3

channels

simultaneously.

Ovl button –

overlays (merge)

multiple channels

into one.

Lut button- open

color look up

table.

Acquiring Single Optical Section Image.

Make sure to switch ”MicCtrl” to Scan mode.

Press “Single Scan” button to acquire an image by a single laser scanning at a scanning speed (normally

400Hz). The resulting image may display background noise. Image averaging is a process to decrease this noise

and improve the signal-to-noise ratio.

To use averaging, click either “Aver”(frame averaging) or “Li.A”. (line averaging) button and select the

number of frames (or lines) to be averaged. Then, click “Single Scan”button.

Sequential Scanning Mode

This confocal microscopy can detect up to four fluorophores simultaneously as long as all the excitation and

emission spectra of fluorophores are well separated. Imaging of samples stained with different dyes thus simply

requires manipulation of laser power and adjustment of the detector bandwidth. However, often excitation of

one fluorophore may cause emission into the range of another (bleed-through) or can be induced by neighboring

laser lines (cross-talk), which produce false signals. To avoid these, the sample can be scanned sequentially by

collecting one fluorophore signal at any given time.

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To scan sequentially:

•Choose the beam setting parameter

for your fluorophores (i.e. GFP and

DsRED) by selecting laser lines,

dichroic mirror, and PMTs.

•Set up a condition for one fluorophore by only activating and adjusting laser level and PMTs. Save this

parameter set up as your own setting (i.e. “GFP-seq”) .

•Repeat the same process for the other fluorophore and save the condition as another setting (i.e. “DsRED-

seq”).

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•Click the Seq button in the beam window. Add the fluorophore acquisition setting one at a time by

selecting them in the beam window and then click Add button in the Sequential scan settings window.

Keep the scan mode at “between lines”

Do not close the settings window.

You can save these sequential

acquisition settings by Save  button.

•Start the acquisition by clicking Single

Scan or Series button depending on

your imaging condition.

Acquiring Z-series images.

Z-series allows obtaining optical sections through the certain volume of your sample that can be used for

making 3D images.

To set the z range, press “Continuous”button (it will be highlighted with “Stop”), turn Z-POS knob in a

direction to focus to a specific z position of your sample and click Begin button (it will appear depressed).

Turn Z-POS in opposite direction to reach a desired position and click End button .

Click Stop button to stop scanning.

Click Series button to check the thickness of the z-series and determine the number of optical sections.

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Click Sect button  to select the predetermined number of optical section, or to select Others …

It brings up a z-configuration window to determine the number of sections and spacing (step size) between

sections. Press OK.

Choose the number of averaging using “Aver”button and click “Series”button to begin Z-series stack

imaging.

Acquiring Time Series images.

Time series allows time-lapse

imaging of live samples to

study the changes and

dynamics of your object of

interest. The movie can be

saved as multiple Tiff files and

AVI files for playing in movie

programs.

To set obtain time-series images,

select scan Mode as xyt (or

other modes containing‘t’). It will

activate “Time” button .

Click “Time” button to open the

time-lapse setting dialogue and set

the parameters such as the time

interval between frames and the

number of frames. Hit Enter key

on keyboard, which will calculate

the complete time. Click “Apply”.

(Option) Click on Aver or Li.A. button to set the number of averaging per frame. Note: If your object of

interest changes or move faster than scanning speed, averaging will generate distorted or ghost images. To

increase the signal, use slower scan speed.

Click “Series” button to start the time series imaging.

Set time

interval

Set the number

of frames

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Saving Images.

This is a very important for your effort and data. Computer crash or power outage will result in loss of

unsaved data! SAVE FREQUENTLY!!

To save the imaging data files, click “Save” button at the top of the window. Select Ddrive and users folder.

Create your folder, rename the Experiment# and save.

Since the storage memory in the computer will be filled up quickly and this lack of hard drive space prevents

image acquisition, all user files will be deleted after two weeks. Be sure to copy your files to a flash memory or

CD/DVD disks.

Shut-down Procedure

Please enter your time of use in the log book and note any problems and suggestions during your time.

1. Clean the oil from the objectives only with lens paper (not Kimwipe). Clean the microscope and stage.

2. Close the LCS program.

3. Copy your data files to a flash drive or CD. Users are responsible for their own files, as there is no

automatic backup at present.

4. Log off the computer (not turning off the computer) and turn off the microscope control box.

5. If the next user won’t be ready within 1 hour: Turn off the mercury lamp.

6. If you are the last user of the day:

a. Turn off the mercury lamp.

b. Minimize the power of the Ar for >3min before turning it off.

c. For the Ar laser, turn the key to “off” and after 10 min turn off the red button switch.

d. Turn the Melles Griot DPSS laser off by pushing the OFF button.

e. For the He/Ne, turn the key to off position.

f. Exit windows and shut down the power for PC stand, Scanner/Laser HeNe, and Laser Ar/Kr with the

red buttons.

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