Training LabRAM Spectometer and Software

HORIBA Jobin Yvon Training “LabRAM Spectrometer and LABSPEC Software”

Gwénaëlle Le Bourdon – HORIBA Jobin Yvon SAS

Ref: Doc.TM-LabLS/En

1

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

- LABRAM TRAINING

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

1 Ref: Doc.TM-LabLS/En

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2

1st PART

The LabRAM

Description of the spectrometer Adjustments and optical alignment

2 Ref: Doc.TM-LabLS/En

3

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LabRAM Description:

3 Ref: Doc.TM-LabLS/En

4 The different parts of the LabRAM :

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

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- external

Notch Filter : 1 Notch FIlter for each exciting wave length

Density Filters : To decrease the laser power I = I0 x 10-D

By clicking her e, select one of the 6 neutral filters with the optical densities 0.3, 0.6, 1, 2, 3 or 4. filter [---] = no attenuation (P0 ), [D0.3] = P0 /2, [D0.6] = P 0 /4, [D1] = P 0 /10, [D2] = P 0 /100, [D3] = P 0 /1000 and [D4] = P0 /10000 Microscope : Illumination : 2 modes : transmission and reflection Objectives : X10, X50, X100 (standard)

Confocal Hole: linked to spatial resolution

Close the confocal hole aperture

Initialize the confocal hole by closing the hole and opening it to the previous value.

Set the value of the confocal hole aperture

Set the confocal hole to the maximum value of aperture. 4 Ref: Doc.TM-LabLS/En

5 Slit entrance : linked to spectral resolution

Spectrometer :

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2 gratings Movement controlled by Sin Bar

Detector : CCD

5 Ref: Doc.TM-LabLS/En

6 Optical chamber :

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Upper part of LabRAM

Spectrograph Lower part of LabRAM

6 Ref: Doc.TM-LabLS/En

7 Optical Path and stem options:

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1

2

3 4

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 : Allows to choose Pushed : « point » mode between « line » mode Lenses L3, L4, L5, L6 Pulled: “line” mode and « point » mode and hole H2

3

Moves the mirror M10

4

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

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

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

7 Ref: Doc.TM-LabLS/En

8

Laser Alignment

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.

- LABRAM TRAINING

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.

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.

8 Ref: Doc.TM-LabLS/En

9 Aligning the laser on the internal diode:

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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

9 Ref: Doc.TM-LabLS/En

10

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 :

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A centring problem:

A focusing problem:

10 Ref: Doc.TM-LabLS/En

11 Should there be a significant disalignment of the laser, it is necessary to adjust the path by using the mirrors as follows: You can adjust intensity by this mirror. This mirror is very sensitive.

- LABRAM TRAINING

1,At first,you have to search maximum intensity by Z axis. : Confirmation of intensity. 2, 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.

You can adjust intensity by this mirror. This mirror is very sensitive. 1,At first,you have to search maximum intensity by Z axis. : Confirmation of intensity. 2, 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). 11 Ref: Doc.TM-LabLS/En

12

Verifying the calibration frequency : zero order position of the gratings

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.

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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 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. 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. Ref: Doc.TM-LabLS/En

12

13 Adjusting the “zero” coefficient of the grating : This is to be carried out for each grating.

- LABRAM TRAINING

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.

.

13 Ref: Doc.TM-LabLS/En

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14

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

14 Ref: Doc.TM-LabLS/En

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15

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 line. Press STOP.

again , you should now be able to see the Silicon Raman

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.

15 Ref: Doc.TM-LabLS/En

16

Notch Filter The Notch Filter is used to reject the laser and filter

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the Rayleigh diffusion.

EASY

OPTICAL DENSITY 7 6

POSSIBLE RAMAN 5 4

NO RAMAN

3 2

α

1

(degree) 0 15

10

5

0

T (%) 100

80

60

40

RAMAN (cm-1)

20

0 0

50

100

150

16 Ref: Doc.TM-LabLS/En

17 Characteristics of Notch Filters :

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 2

Edgewidth and cut-off definition edgewidth 1 0 -1

3

-2

3

-500

4 4

5 5

80 Intensity (%)

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1

0

Wavenumber ( 1)

60 40

50 %

20 0 -500

0 Wavenumber (cm-1)

17 Ref: Doc.TM-LabLS/En

18 Aligning the Notch Filter.

Notch filter

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Spacer

Spacer Number Diameter (mm) Angle of the notch (degrees)

1 4 9,84

2 5 8,69

3 6 7,58

4 7 6,59

5 8 5,69

6 9 4,86

7 10 4,05

8 11 3,3

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 :

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. 18 Ref: Doc.TM-LabLS/En

19

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

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

- LABRAM TRAINING

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.

19 Ref: Doc.TM-LabLS/En

20

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

The spectral resolution depends on : - the grating - the slit entrance

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- 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

20 Ref: Doc.TM-LabLS/En

21

- LABRAM TRAINING

Influence of the grating :

Gratings at fixed positions (1700 cm-1) 10000

⎯⎯ 1800 l/mm ⎯⎯ 600 l/mm

Intensity (a.u.)

8000

6000

4000

2000

500

1000

1500

2000

2500

Wavenumber (cm-1)

21 Ref: Doc.TM-LabLS/En

707.9

22

12000

Different gratings – same excitation wavelength 10000

953.7 939.4

966.1

923.5 908.8

4000

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⎯⎯ 1800 l/mm ⎯⎯ 600 l/mm

929.9

6000 695.2

Intensity (a.u.)

8000

2000

700

750

800

850

900

950

1000

Wavenumber (cm-1)

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 grooves TM light polarized normal to the grooves

22 Ref: Doc.TM-LabLS/En

23 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

Quantum efficiency, % .

60

633 - 787 nm

50 40 30

780 - 1030 nm

20 10 0 200

300

400

500

600

700

800

900

1000 1100

Wavelength, nm .

32000 30000

Pine excitation at 633 nm Pine excitation at 780 nm

28000 26000 24000 22000

INT [a.u.]

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Typical spectral response at 193 K .

20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 250

500

750

1000 1250 1500 1750 2000 2250 2500 2750 3000 -1

Wellenzahl [cm ]

23 Ref: Doc.TM-LabLS/En

24

Hardware Section – Practical Advice Sheet n°3 : choosing the best objective Numeric aperture of an objective : N.A. = n. sin(θm) θm being the half open aperture and n the refraction indice

- LABRAM TRAINING

Maximum diameter of the luminated spot is

NA = n sin (α)

limited by diffraction phenomena’s: T = 1,22 x ( λ / NA )

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

α

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

This resolution can be limited by the confocal hole.

Lateral Resolution Optical characteristics of the main objectives used: Type of objective

10

50 X

50 X

100 X

100 X

X

LWD

MPlan

LWD

MPlan

33°.4

48°.6

53°.1

64°.2

Half aperture max (θm) 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

3.1

1.4

1.03

0.96

0.86

632.8 nm

24 Ref: Doc.TM-LabLS/En

25 Axial resolution – field depth The depth probed depends on the numeric aperture (NA) of the objective : B High aperture : small volume studied B 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

- LABRAM TRAINING

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.

25 Ref: Doc.TM-LabLS/En

26

Silicon line intensity = f(NA²)

100

obj 100x 90 80

Intensity (%)

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70

obj 50x

60 50 40 30 20 10

obj 10x

0 0

0,2

0,4

0,6

0,8

1

Numerical Aperture ²

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.

26 Ref: Doc.TM-LabLS/En

27

Hardware Section – Practical Advice Sheet N°3 : Confocality

The principle of confocality

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Confocal Hole

Rejected Beams

Multilayered sample

Advantages : (1) small increase in the lateral resolution (2) large improvement in the axial resolution

27 Ref: Doc.TM-LabLS/En

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

- LABRAM TRAINING

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)

28 Ref: Doc.TM-LabLS/En

29

Use of a quick confocality test to check laser alignment

Device bar

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Take a spectrum

: Confirmation of intensity.

: Take in spectrum. (1 sec)

: Confirmation of intensity.

: Take in spectrum. (1 sec)

Device bar

Take a spectrum

Check

Choose a 1000um spectrum. : Select this icon.(Multiply Const) Calculate the ratio of 1000um to 200um.

29 Ref: Doc.TM-LabLS/En

- LABRAM TRAINING

30

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