Training LabRAM Spectometer and Software

Ref: Doc.TM-LabLS/En

HORIBA Jobin Yvon

Training “LabRAM Spectrometer and LABSPEC Software”

Gwénaëlle Le Bourdon – HORIBA Jobin Yvon SAS

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The LabRAM – Description, functioning and optical adjustment P.2

1. Description of the Spectrometer – different parts and optical path

2. Alignment : frequency calibration, verification of the laser alignement,

change of the exciting wavelength

3. Optimisation of the options available: choice of grating, choice of

objective, slit and confocal hole

LabSpec – how to obtain and save spectra P.34

1. Acquisition modes

2. Multi-point analysis

3. Raman mapping

4. Kinetics measurements

5. Depth profiling

6. Configuration menu (acquiqition parameters storage)

7. Saving options : formats, autosave mode

LabSpec – Modes of visualisation and treatment of spectres

1. Visualisation options (spectra, mappings, depth profiles)

2. Spectra treatments : baseline, smoothing, peak fitting, normalisation

3. Mapping treatment

4. Modelling

Use with samples

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1st PART

The LabRAM

Description of the spectrometer

Adjustments and optical alignment

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LabRAM Description:

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Initialize the confocal hole by closingthe hole and opening it to the previousvalue.

Set the confocal hole to the maximumvalue of aperture.

Close the confocal hole aperture

Set the value of the confocalhole aperture

The different parts of the LabRAM :

Laser :

- internal : HeNe 17mW. Wave length: 632,817 nm

- external

Notch Filter :

1 Notch FIlter for each exciting wave length

Density Filters : To decrease the laser power

Microscope :

Illumination : 2 modes : transmission and reflection

Objectives : X10, X50, X100 (standard)

Confocal Hole: linked to spatial resolution

By clicking here, select one of the 6 neutral filterswith the optical densities 0.3, 0.6, 1, 2, 3 or 4.filter [---] = no attenuation (P

0), [D0.3] = P

0/2, [D0.6]

= P0/4, [D1] = P

0/10, [D2] = P

0/100, [D3] = P

0/1000

and [D4] = P0/10000

I = I0 x 10-D

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Slit entrance : linked to spectral resolution

Spectrometer :

2 gratings

Movement controlled by Sin Bar

Detector : CCD

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Optical chamber :

Upper part of LabRAM

Spectrograph Lower part of LabRAM

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Optical Path and stem options:

STEM ACTION ROLE POSITION

1 Moves the beam splitters BS12

Allows to choose between a Raman recording or a visualization of the laser sample

Pushed : visualization with the camera Pulled: Raman measurement

2 Moves the block : Lenses L3, L4, L5, L6 and hole H2

Allows to choose between « line » mode and « point » mode

Pushed : « point » mode Pulled: “line” mode

3 Moves the mirror M10

Allows to choose between the analysis of a signal from the microscope or from the fiber optics entrance

Pushed : « point or line » mode Pulled: fiber optics analysis

4 Moves the two gratings 1800 g/mm and 600 or 300 g/mm

Choice between the two gratings

Pushed : 1800 g/mm grating Pulled: 600 or 300 g/mm grating

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This should be carried out when there is a significant loss of signal (a test to verify the

confocality by using a silicon sample will reveal any misalignment see P. 29-30) or when

the spot laser shows problems with the centering or focus.

Aim : to ensure that the laser is perfectly centred on the confocal hole image.

In order to do this, use the internal diode which follows the return path of the ‘Raman’

beam.

Laser Alignment

With the Labram, you have the possibility to see the image of the confocal hole projected on the sample when you switch on the laser diode for alignment. The laser diode for alignment is placed inside the spectrograph, and, when the grating is turned at an appropriate angle, the diode beam exits from the entrance slit of the spectrograph and illuminates the confocal hole. If you put a flat reflective sample under the microscope (like silicon or even a glass surface) you can see the projection of the confocal hole on it.

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Aligning the laser on the internal diode: 1. Turn on the internal diode:

2. Select the grating 1800 g/mm

3. Move the grating to the “diode reference position”

4. Position the camera for visualisation

5. Using the 100X objective – focus on the silicon sample

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6. To locate the centre of the confocal hole easily, close to 100 µm.

8. The diode spot determines the point which is used to align the spot laser.

There are two possible types of misalignment :

A centring problem:

A focusing problem:

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Should there be a significant disalignment of the laser, it is necessary to adjust the path by

using the mirrors as follows:

<Reference Laser 633nm>

You can adjust intensity by this mirror. This mirror is very sensitive. 1,At first,you have to search maximum intensity by Z axis.

2, : Confirmation of intensity. 3,Touch that mirror,and search maximum intensity value. 4,When you took a good value,you should check laser focus after.

5,When you got bad focus,you can adjust by this mirror. 6,When you adjusted this mirror,you should check intensity again.

A new confocality test has to be realised after the alignment (see P 29-30).

<Others wavelength Laser> You can adjust intensity by this mirror. This mirror is very sensitive. 1,At first,you have to search maximum intensity by Z axis.

2, : Confirmation of intensity. 3,Touch that mirror,and search maximum intensity value. 4,When you took a good value,you should check laser focus after.

5,When you got bad focus,you can adjust by this mirror. 6,When you adjusted this mirror,you should check intensity again.

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The gratings are driven with a sinus

arm that is linear in wavelength. In

fact the formula for the dispersion of

a grating is given by:

λ = [2⋅cos(ϑ0)⋅sin(α)]/n⋅N , where N

is the number of grating grooves/mm;

n is the order and the angles ϑ0 and

α are represented in the picture. As

[2⋅cos(ϑ0)]/n⋅N is a constant, λ is

proportional to sin(α).

The easiest way to test if your Labram is perfectly calibrated in frequency is to run a

silicon sample. You should find the Si ν1 line at 520.7 cm-1.

If not, the « zero order » position of the grating has to be checked.

The coefficient ZERO is the mechanical position used as a reference for a value of

0nm. This corresponds to a number of steps of the motor between the switch of the

mechanical reference and the position 0 nm.

Verifying the calibration frequency : zero order position of the gratings

At the angle α=0, the “zero order” of the grating must be centered on the detector. If it

is not the case, a constant shift in all the spectrum is observed (and thus on the silicon

band).

A small shift of the “zero order” can be produced by temperature changes. A shift of 6

pixels each 10 degrees of temperature change should be considered as normal.

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Adjusting the “zero” coefficient of the grating : This is to be carried out for each grating. 1. Select the grating to be calibrated. Move the grating to the zero order position by clicking on:

2. Select the following values for the confocal hole and slit entrance : Hole = 400µm Slit = 150 µm Remove all samples and turn on the reflecting white light. Let the white light enter the spectrometer by putting the camera beamspiltter. Change the units of measurement to nm.

3. Use the icon : to save a spectre and change the acquisition time of the intensity of the light to obtain a signal around several thousands of counts. 4. Press STOP. Use the red cursor to determine the position of the band. It should be at 0 nm, at about +/- 1 pixel. 5. If the band is not at 0 nm, open the calibration window, by clicking on: . Before changing anything, note the values ZERO et KOEFF.

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In the second menu « Mfluo », change the value of ZERO :

Zero value [+] : Spectrum is going to minus direction.

Zero value [-] : Spectrum is going to plus direction.

Now adjust the ZERO and watch the band position move, adjust until the band is within

+/- 1 pixel of 0 nm

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6. Now change the UNITS to cm-1 and move the spectrograph to the position at which you should monitor the Si Raman band (520.7cm-1) at the centre of the CCD (the central pixel corresponds to the pixel for which one the frequency position is the same than the spectrograph window value). 7. Insert your standard silicon sample and focus the laser in the normal way. Using the

spectrum adjustment icon again , you should now be able to see the Silicon Raman line. Press STOP. 8. Again use the RED cursor to measure the position of the band. It should within +/- 1pixel of 520.7cm-1. 9. Once you are satisfied with the calibration, close the calibration window and ensure that you save the changes when prompted.

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The Notch Filter is used to reject the laser and filter

the Rayleigh diffusion.

Notch Filter

100

80

60

40

20

0

0 50 100 150

0

1

2

3

4

5

6

7

0 51015

OPTICAL DENSITY

α (degree)

T (%)

RAMAN (cm-1)

POSSIBLE RAMAN

EASY

NO RAMAN

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Characteristics of Notch Filters :

1

0

-1

-2

-500 0Wavenumber( 1)80

60

40

20

0

Inte

nsit

y (%

)

-500 0Wavenumber (cm-1)

3

4

5

1 : filter references 2 : transmission of the filter in function of the angle 3 : optical density in function of the angle 4 : cut-off position in function of the angle 5 : spectral edge width in function of the angle

Edgewidth and cut-off definition

edgewidth

50 %

1

2

3

4

5

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Aligning the Notch Filter.

NB. Each Notch Filter must be used with the adapted spacer.

You can see the position of the cut-off of the notch filter looking at the white lamp in transmission with a x10 objective. If the spectrograph is positioned on the exciting line you can record a spectrum like :

Spacer Number 1 2 3 4 5 6 7 8Diameter (mm) 4 5 6 7 8 9 10 11Angle of the notch (degrees)

9,84 8,69 7,58 6,59 5,69 4,86 4,05 3,3

Spacer

Notch filter

The cut-off of the notch is often asymmetrical, to achieve a lower edge of transmission. The lowest wave number that can be measured reliably is around 100-120 cm-1.

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1. Removable mirror

2. Changing the Notch Filter (remember to choose a suitable spacer)

3. Software : enter the new wavelength value.

4. Choose a suitable grating (see p.20)

5. Position the spectrometer in the centre of the spectral window.

Hardware Section – Practical Advise Sheet n°1 : Changing the excitation wave length

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The spectral resolution depends on :

- the grating

- the slit entrance

- the excitation wavelength

- the spectometers’ focal distance

- the number of pixels of the CCD

Parameters which can be optimised by the user:

- the grating

- the excitation wavelength

- the size of the slit entrance (in most cases a slit of 100 µm is used)

Depending on the grating selected:

- the resolution differs

- the observed spectral range will differ

Hardware Section – Practical Advice Sheet N°2 : Choosing a suitable grating

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Influence of the grating :

10000

8000

6000

4000

2000

Inte

nsity

(a.u

.)

500 1000 1500 2000 2500

Wavenumber (cm-1)

Gratings at fixed positions (1700 cm-1)

⎯⎯ 1800 l/mm⎯⎯ 600 l/mm

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To conclude:

Choice of grating depends on the application and aims of the measurement.

NB : other grating characteristics :

- The gratings are appropriate to a certain wavelength, meaning they have a reflection

maximum in certain spectral ranges.

The reflection of a grating is hence subject to the wavelength (1) and the light polarisation

(2)

TE light polarized parallel to the groovesTM light polarized normal to the grooves

939

.4

929

.9

923

.5

908

.8

707

.9

695

.2

966

.1

953

.7

12000

10000

8000

6000

4000

2000

Inte

nsity

(a.u

.)

700 750 800 850 900 950 1000

Wavenumber (cm-1)

⎯⎯ 1800 l/mm ⎯⎯ 600 l/mm

Different gratings – same excitation wavelength

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250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 30000

2000400060008000

100001200014000160001800020000220002400026000280003000032000

Pine excitation at 633 nm Pine excitation at 780 nm

INT

[a.u

.]

Wellenzahl [cm-1]

IMPORTANT when working in the Near InfraRed!

- choice of grating (the grating 1800tr/mm is not suitable : optimised in visible and limited

mechanically) 950 tr/mm

- effect of the detectors’ response

Typical spectral response at 193 K .

0102030405060

200 300 400 500 600 700 800 900 1000 1100

Wavelength, nm .

Qua

ntum

eff

icie

ncy,

% .

633 - 787 nm

780 - 1030 nm

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α

NA = n sin (α)

Numeric aperture of an objective : N.A. = n. sin(θm)

θm being the half open aperture

and n the refraction indice

Lateral Resolution Optical characteristics of the main objectives used:

Type of objective 10

X

50 X

LWD

50 X

MPlan

100 X

LWD

100 X

MPlan

Half aperture

max (θm)

33°.4 48°.6 53°.1 64°.2

NA=n.sin (θm) 0.25 0.55 0.75 0.8 0.9

W. D. (mm) 7 8.1 0.38 3.2 0.21

Spot diameter

632.8 nm

3.1 1.4 1.03 0.96 0.86

Hardware Section – Practical Advice Sheet n°3 : choosing the best objective

Maximum diameter of the luminated spot is

limited by diffraction phenomena’s:

T = 1,22 x ( λ / NA )

eg : for a x100 objective of NA: 0,9

T = 1,22 x ( 632,81 / 0,9 )= 858 nm or 0,86 µm

This resolution can be limited by the confocal

hole.

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Axial resolution – field depth

The depth probed depends on the numeric aperture (NA) of the objective :

High aperture : small volume studied

Low aperture : large volume studied

The choice of objective will determine the intensity of the Raman spectra. Depending on

the sample type (opaque or transparent), the same objective will not have the same

behaviour.

1 Opaque sample

When there is almost no penetration of the laser in the sample, the Raman spectrum is

obtained mainly from the surface and its intensity is proportional to the collected flux. It

will be better to use a microscope objective with a high numerical aperture (x100,

NA=0.9) so that the solid angle (NA = n⋅sin(α)) is bigger and you have a maximum

Raman signal.

The following drawing compares a x100 objective with a macro objective, which supports

this argument.

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Silicon line intensity = f(NA²)

obj 100x

obj 50x

obj 10x0

10

20

30

40

50

60

70

80

90

100

0 0,2 0,4 0,6 0,8 1

Numerical Aperture ²

Inte

nsity

(%)

2- Transparent sample

If you have an homogenous sample, it will be better to use a microscope objective with a

big depth of focus (for example a x 10) so that it will collect the signal from a bigger

volume with a macro objective supports this argument.

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Rejected Beams

Confocal Hole

Multilayered sample

The principle of confocality

Advantages :

(1) small increase in the lateral resolution

(2) large improvement in the axial resolution

Hardware Section – Practical Advice Sheet N°3 : Confocality

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Relationship between the aperture of the confocal hole (µm) and the signal intensity (%)

This is also a test to verify the laser alignment.

Relationship between Depth (µm) and

Intensity (a.u.) For 6 Confocal Hole Apertures from 100 to 1100 µm

Relationship between Confocal Hole Aperture (µm) and Axial Resolution (µm)

(Full Width at Half Maximum)

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Device bar

Take a spectrum : Confirmation of intensity. : Take in spectrum. (1 sec)

Device bar

Take a spectrum : Confirmation of intensity. : Take in spectrum. (1 sec)

Check

Choose a 1000um spectrum. : Select this icon.(Multiply Const)

Calculate the ratio of 1000um to 200um.

Use of a quick confocality test to check laser alignment

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Laser He-Ne (632.817 nm) :

- The optimum confocality value = 60% with a confocal hole at 200 µm/

confocal hole aperture at 100µm

- The laser is required to be adjusted if confocality becomes <40 %

Diode laser 785 nm :

- Optimum confocality value = 35-40 % with a confocal hole at 200 µm/

confocal hole aperture at 1000µm

- The laser is required to be adjusted if confocality becomes <20 %

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With the Labram you have the possibility of working with macro-samples, gases and liquids. For that you have to mount the accessory that is shown in the drawing below which replaces one of the microscope objectives.

This macro-sampling device uses a 40 mm focal length lens, but other focal lengths are available. You can even mount a microscope objective at the place of the 40 mm lens. The advantage is that your laser beam exits horizontally and not vertically. An accessory with a spherical mirror for double laser pass is also available for transparent material (see below).

Harware Section – Practical Advice Sheet N°4 : Taking measurements in Macro

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Check the following :

- Wavelength (nm/cm-1) - Camera pull handle - Z axis (Focus) - Try reinitialization. (Hole/Slit/Wavelength) - Is there some obstacle on light axis? (Paper) - Checking of Power supply switches.

Troubleshooting : ‘I can’t acquire my spectrum’

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Check :

• That the internal diode hasn’t been left on

• That the white light is switched off

• That the light in the room is switched off

Check :

• That there is no density filter on the laser beam

• That the objective is clean

If necessary, clean the obhective with a mix 5% ether / alcohol. Soak the lens and place it

in an ultra sound bath.

• Aligning the laser on a silicon sample (check the spot is centred and there is no

major focusing problems)

Troubleshooting : ‘My spectrum look odd’

Troubleshooting : ‘My spectrum have a weak intensity’

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Second Part

LabSpec

Acquiring and saving a spectrum

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5 Practical Advice Sheets for acquiring :

- a spectrum in point mode

- spectra at multiple points in the sample

- a Raman mapping

- kinetics

- an in-depth profile

Precautions before recording spectrum :

Be sure that the laser power will not harm the sample ! Use density filters to decrease the

laser power in case the sample is sensitive to laser heating.

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1. Choosing the grating

Select the grating from the LabSpec software (LabRAM : a message will appear asking to

choose the correct stem position for the grating)

Choose the grating with the use of stem 4.

In case the grating has not been used before, check its zero order.

2. Focusing the laser

Using either the video monitor or the video image function within LabSpec

3. Positioning the spectrograph

Centre the grating at the desired position

Practical Advice Sheet N°1 : recording a spectrum / point mode

Click here to select one of the available spectrograph gratingsDo not for get to initialize the spectrograph after a grating change

Move the spectrograph to thezero order position (0 nm)

Enter here the spectrographposition value

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4. Define the size of the slit (100µm) and that of the confocal hole

5. Define the parameters ‘exposure time’ and ‘number of accumulations’

3 possible acquisition modes

Type of acquisition Parameters to define Note

Simple window

Adjustment

- Acquisition time

- Central position of the spectral

zone

The previous

spectrum is

automatically erased

Multi-window

Accumulation Mode

- Acquisition mode

- Number of accumulations

- Complete spectral mode

(Multi-window)

Scanning Mode

Continuous (Kiefer

Scanning mode)

- Acquisition time

- Kiefer Scan parameters

(Spectral zone and number of

accumulations)

Number of accumulations

Exposure time in seconds

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60000

50000

40000

30000

20000

10000

0

Intensity (a.u.)

500 1000 1500 2000 2500 3000 3500Wavenumber (cm-1)

Principle of the different acquisition modes :

Generally to get the whole Raman spectrum, multiple spectral windows are required. This is achieved by moving the grating position in the spectrometer in some manner so as to move the specific part of the spectral range which illuminates the CCD. The first option is to use discreet spectral windows, which are glued automatically. (see multi window acquisition option).

Multi-Window Mode

Kiefer Scan Mode The method used by the new Kiefer scanning works in a different way and consists of shifting the spectrum step by step so that each individual spectral element is detected several times by the detector, rather than as a discreet spectral window. The software and hardware can scan the spectrometer through a defined spectral region, off-setting each subsequent acquisition viewed by the CCD detector to a certain amount. This can be by a large number of pixel steps or even a sub-pixel value, depending upon the required effect. (i) Mode (I) - Larger Pixel overlap values : In mode (I), a larger number of pixels is chosen as the offset, and an averaging effect is produced. For this method the operational principle is that a datapoint ( in cm-1 ) is seen by a number of different pixels on the CCD detector, and the average signal for this datapoint is calculated.

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8000

6000

4000

2000

0

Intensity (a.u.)

428 430 432 434 436 438Wavenumber (cm-1)

3000

2500

2000

1500

1000

500

Intensity (a.u.)

0 500 1000 1500 2000Wavenumber (cm-1)

(i) Mode (II) - Sub pixel offset. The second way of using the Kiefer CREST scan is to use very small overlaps in the spectrum, which provides an enhanced band definition. Here a shift in position, entered in the ‘sub Pixel’ value box, will move the grating position by an amount less than one pixel on the detector. - A Sub Pixel. By selecting a sub-pixel value (a value of 1 is for full integer values and hence deactivates this mode), the step selected will increase the number of data points obtained for the spectrum. Hence, a 2 subpixel value will give twice the number of data points, 3 subpixel value three times and so on. If you consider a usual acquisition, you have 1024 pixels (and 1024 data points). Using the subpixel arrangement of a factor of 2, will give 2048 data points. Whilst this operation does not affect the actual spectral resolution which remains defined by the spectrometer focal length, grating and entrance slit, it does provide a better band definition for Raman bands and can hence provide a better basis for analysis of band shape and position for a given instrument.

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How to use the Kiefer Scanning Modes?

indicates the Kiefer scanning modes, by clicking on this, the window for the Kiefer Scan will open:

• ‘start point’ is the starting point for the spectrum you wish to have (in cm-1 or in nm) • ‘finish point’ is the end point for the spectrum you wish to have (in cm-1 or nm) • ‘accumulation number’ activates the first mode of the Kiefer scan used to generate extended spectral regions and for spectral averaging, Mode (I) The value represents how often a spectral data element will be detected (any value can be entered) ie. the larger the number the greater the averaging. • ‘sub-pixel factor’ defines the linearized data point (maximum is 6). This is used to generate the higher definition mode. Again the greater the value, the greater the definition, Mode(ii).

It can be considered that there are three cases for using the Kiefer scanning: - reduction of spectroscopic phenomenon ( on a wide spectral domain): 1) just enter the “start point” and “finish point” values, 2) select the “accumulation number” required 3) and set “sub-pixel factor” to 1. 4) Press “start” and the software drives the spectrograph, acquires the data and linearizes the spectrum.

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The selection of the “ accumulation number” depends on the required quality of the end spectrum. The higher the number, the better the quality. Integration time : for instance, if integration time T for classic acquisition is selected and N accumulation for the Kiefer Scanning is also selected, the integration time should be changed to T/N before starting the Kiefer Scanning.

- Improving the definition of the line shape ( on a short spectral domain) : 1) just enter the “start point” and “finish point” values, 2) set the “accumulation number” to 1 3) and select the “sub-pixel factor” required 4) Press “start”. For the same reason as above, change the integration time to a lower value.

- A combination of both modes is possible but this is not really recommended as this method will take time, especially if you require a complete Raman spectrum with a definition four times better!

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LabRAM equipped with a motorised XY table

1. Focusing the laser on the sample

2. Recording the video image on the sample surface

Stop the acquisition of the continuous video

3. Define the measurement points with the cursor

4. Define the acquisition parameters :

- grating

- acquisition time and number of accumulations

- spectral zone « multi-window »

- confocal hole aperture and slit entrance

5. To start an acquisition use the icon ‘spectral image’

Practical Advice Sheet N°2 : recording spectra in several points in the sample (Multi-points analysis)

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LabRAM equipped with a motorised XY stage

Initially, determine the best measurement conditions (acquisition time and number of

accumulations, confocal hole and slit apertures, density filter, spectral range) by

acquiring some few spectra in different areas of the sample. This allows one to optimize

the parameters and to prevent from detector saturation.

1. Displaying the sample with white light

Verify that the objective selected in LabSpec corresponds to the objective used.

Recording the video image

2. Open the window ‘Acquisition Options’

Select ‘table’ in the sub-menu ‘X-scanning’ and ‘Cursor’ in ‘scanning area’. Define the value in ‘Refresh Time’ :which determines the period of time between the two updates displaying the spectrum recorded on screen. In case of a long mapping increase this time (eg. 3600 s) in order not to lose time with the use of display updates. Close the ‘Acquisition Options’ window.

Practical Advice Sheet N°3 : recording Raman Maps

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3. Choosing the cursor type to define the zone analysed

- a- sloping line

- b- horizontal line

- c- rectangle

- d- elipse

- e- polygon

3. Selecting the parameters of the measuring zone

Open the ‘Acquisition Data Parameters’ window

- Click on ‘X’ and ‘Y’ to define either the number of measurement points or the steps

between two points (µm), then the software calculates automatically the second

parameters.

4. Define the acquisition parameters:

- grating

- acquisition time and number of acquisitions

- ‘multi-window’ spectral zone

- Apertures of confocal hole and slit entrance

5. To start an acquisition, use the icon ‘spectral image’

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This option enables you to take a series of spectra or spectral images as a function of time. Follow the same procedure as the one to record point by point spectra or spectral images. But you also have to choose:

• In the ‘Data size’ window (select the ‘time’ parameter, there are two types of boxes, the first is the number of measurements and the second is the time interval between them). • Please refer to the «Index section» • Be careful, because the acquisition time is included in the interval of time between two measurements.

To start an acquisition, use the icon ‘spectral image’

Practical Advice Sheet N°4 : recording Kinetics

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• Focus the laser on the sample,

• Take a video image and freeze it with the icon,

• Choose the cursor,

• Select the time of exposure,

• Select the

• In the window « Data size », select the Z scanning parameters. - Please refer to the «Index section» • Press the icon to start the acquisition

Practical Advice Sheet N°5 : Recording a profile in depth

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‘Configuration’ Window

Saving the measuring options

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• Save one of the displayed spectra, by activating the ‘spectrum’ window, select the desired spectrum by pressing the «radio» button, then choose one of the following methods:

• Click on the following icon: then enter the file name, its location and format.

• From the « file »Main Menu, select the « Save As » option, then enter the file name, its location and format. Before saving, it would be useful to complete the information list about the spectrum (operator, laser power, etc...) by pressing the icon NB. Some parameters (hole, slit, spectro, grating, time, accum, date) are automatically updated. LabSpec file formats. The following is a list of the various file formats that LabSpec supports to save the acquired spectra or images: • Dilor ASCII format (*. ms0): This format is used for single spectrum. • Extended Tiff - (*.tsf): It is a specific format for single spectrum and LabSpec software. - (*.tvf): It is a specific format for spectral image. • Standard Tiff (*.tiff): Standard TIFF format for image. • Text format (*.txt): This is an ASCII mode format which uses two columns: wavelength/Wavenumber and intensity, without header. Spectra Calc format (*.spc): This format is used by ‘SpectraCalc’ and ‘Grams’ processing softwares.

Important comments:

- It is possible to save several spectra one after the other by using MULTI, (do not forget

to de-select after use) icon:

- when saving as txt, remember to activate Axe+Text

- Saving images: The « save » procedure always saves the content of the active window. To save an image, activate the window that contains all the spectra. If the « Spectrum: Point » window is activated, only this spectrum will be saved, and not the whole spectral image.

Saving spectrum

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Saving in ASCII Format

This module permits to save all the spectra (or the spectra of a Raman image) of a window as ASCII files (the maximum spectra saved in one shot is 64)

Destination path : indicate the address of the directory where the spectra will be saved. Conversion option : Different conversion options are available: to remove the converted objects of the LabSpec window., to split the activated image to spectra that will be saved in ASCII format, to saved the ASCII file in two columns or two lines to write the frequency values and/or the position values. To activate the saving of the spectra press the button : SAVE ALL

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You can choose a name

You can choose to usethe day, the month and the year as the name

You can choose a name

You can choose to incrementthe name by number, hour and minutesof the acquisition.

- You can choose the directory where the spectra will be recorded:

MAXIMUM 8 CHARACTERS

- You can choose the file name of the recorded spectra:

MAXIMUM 8 CHARACTERS. - You can choose the format of the files:

Auto Save

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Two options are available: You can choose to work with the « Auto Save » and « Auto Repeat » options: For this the delay between two acquisitions (eg: 10 seconds) must be chosen. Then after clicking on the icon the software takes an acquisition and repeats every 10 seconds, saving the spectra automatically. However it is possible to work only with the « Auto Save » option: - When the icon is selected, the software takes an acquisition, stops and records it. - When the icon is selected (which is used for spectral imaging, time scanning...) the software takes a spectral image and at the end of the acquisition will be recorded. MAKE SURE TO CHOOSE THE « TSF » FORMAT FOR THE SPECTRAL IMAGES.

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Third Part

LabSpec

Processing Spectra and Raman Mappings

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The main procedure is to build up a baseline point by point, to best fit the spectrum and then subtract it from the spectrum. If you have to subtract the baseline from a spectrum, activate this spectrum. If you have to subtract the baseline an image, activate the ‘spectral image’ window.

• Verify that the option Ins/Del is active (in the ‘operations’ box)

• Select the type for interpolation: it can be linear or polynomial (in the box ‘type’). For the polynomial interpolation, you can also choose the degree of the polynom (in the ‘degree’ box).

• You can select with the mouse in the spectrum window (validate: left button, delete: right button) the points for the baseline computation Hint: (for the images, when you press the icon , a window that contains an average spectrum of the image is opened automatically).

• Activate the option « attachment », to fit at best the baseline to the spectrum. Hint: An automatic procedure can also be used for the computation of baseline: button « Auto »

• Press the button « SUB ». NB: The baseline can be saved as a spectrum: by selecting the ‘CONV’ button

Baseline Correction

To save the baseline

Automatic calculation

Saves as a spectrum

Clears the points of the baseline

Additional/Subtraction

Normalise the spectrum (maps)

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Main use for processing images:

Enter the weakest value for the spectra as ‘0’ and normalisation.

Correction

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2 main usess : - (1) to create a ‘profile’ file with several spectra separately saved and required to be treated as images - (2) to extract the profiles from a map. (1) Create an image with several spectra : use the ‘Add’ function after having activated the spectrum into which the profile is to be added.

(2) Extract a profile from a map, either by following the horizontal or vertical lines.

Profile

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Preparation : Remove spikes on the spectrum

NB: in the case of an image, this modification must be carried out for each spectrum. If a

spectrum has a spike, remove this spike in the window “Spectrum : Point” and Press

button “R” to insert the corrected spectrum in the Raman mapping.

Step 1 : If only one part of the spectrum is of interest, first extract this zone by using the

window: (if it is a spectrum or image)

Then, define the approximative positions of the bands

- by an Auto search (height parameter and neighbour to adjust)

- by using the following icon

Peak-fitting

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Step 2 : define the function for fitting/add a baseline

It is possible to choose a function independently for each band.

Step 3 : Define the parameters: number of iterations and deviations

Then start the peak fitting operation by clicking on

Then on

Step 4 : Visualise the reults by activating ‘Band sum’ and ‘Band shape’.

Then for a table click on

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NB : it is possible to save these results by using the SAVE function

NB. : it is possible to fix the value of a parameter

Step 5 : with images

It is possible to draw up a map for each of the predetermined parameters by pressing keys

« p », « a », « w », « g » or « s ».

« p » : frequency position of the band.

« a » : amplitude of the band.

« w » : width at half maximum.

« s » : surface of the band.

« g » : Gaussian/Lorentzian ratio.

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Examples of Raman mapping based on band Intensity/Frequency/Width (Thanks to Mr

Mermoux) :

15000

10000

5000

0

1200 1400

2.4

2.2

1333.0

1332.8

1332.6

1333.0

1332.8

1332.6

x1000

50

intensité

fréquence

largeur

200

300

400

500

600

Leng

th Y

(µm

)

400 500 600Length X (µm)

Images : ≈ 4500 pixels (pas de ≈ 5 µm) 2h30 acquisition time

Cathodoluminescence

TEM

1340

1335

1330

10

5

200

150

100

5010 µm

x1000

40

20

intensity Backg

frequency width

× 100 microscope objective Point spacing: 0.7 µm λexc.=514.5 nm

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It is the decomposition of each original spectrum of an image as a sum of « model »

spectra.

• Choose the spectra that will become « MODELS », which can be already saved on the

disk, or they can be particular spectra that have been extracted and saved from a spectral

image.

• If the spectrum is a particular and extracted from the image and save.

• Do the same with any other interesting spectra.

• Load all the selected spectra and put them overlapped in a window (for that use the

option « Behavior » in the menu « view » in the FORMAT menu).

• Activate the first spectrum selected and press the button ‘get’. Do the same for all the

selected spectra: a window is then created with all the « model spectra ».

• In the « single spectrum » window, there are:

- The « models » with relative intensity.

- The experimental spectrum in particular point of the image.

- The result of the fitting.

You will see in the mapping window additional pictures that correspond to each one of the

« model ».

You can overlap them to see the relative contribution of each of the spectra in a particular

point of the image (for that use the option « Behavior » in the menu « view » in the

FORMAT menu

You can save a whole spectral image with the « model » compounds choosing the « tvf »

format. When loading this image, the decomposition will automatically appear.

Modelling

Training LabRAM Spectometer and Software

Documents