HORIBA Jobin Yvon
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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
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1
stPART
The LabRAM
Description of the spectrometer
Adjustments and optical alignment
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LabRAM Description:-L
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Initialize the confocal hole by closing the hole and opening it to the previous value.
Close the confocal hole 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 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 (P 0), [D0.3] = P0/2, [D0.6] = P 0/4, [D1] = P0/10, [D2] = P0/100, [D3] = P0/1000 and [D4] = P 0/10000 I = I0 x 10-D
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Slit entrance : linked to spectral resolution
Spectrometer :
2 gratings
Movement controlled by Sin Bar
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Optical chamber : Upper part of LabRAM Spectrograph Lower part of LabRAM-L
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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
Choice between the two gratings Pushed : 1800 g/mm grating 1 2 3 4
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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
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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:
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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.
<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).
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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.
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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 5 10 15 OPTICAL DENSITYα
(degree) T (%) RAMAN (cm-1) POSSIBLE RAMAN EASY NO RAMAN-L
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Characteristics of Notch Filters :
1 0 -1 -2 -500 0 Wavenumber ( 1) 80 60 40 20 0 Inte nsity (% ) -500 0 Wavenumber (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 8 Diameter (mm) 4 5 6 7 8 9 10 11 Angle 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
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1. Removable mirror2. 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 –
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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 –
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Influence of the grating :
10000 8000 6000 4000 2000 Intens ity (a.u.)
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 grooves TM light polarized normal to the grooves
939.4 929.9 923.5 908.8 707.9 695.2 953.7 966.1 12000 10000 8000 6000 4000 2000 Intensity ( a .u.) 700 750 800 850 900 950 1000 Wavenumber (cm-1) ⎯⎯ 1800 l/mm ⎯⎯ 600 l/mm
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250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 Pine excitation at 633 nm Pine excitation at 780 nm IN T [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 .
0 10 20 30 40 50 60 200 300 400 500 600 700 800 900 1000 1100 Wavelength, nm . Quantum efficiency, % . 633 - 787 nm 780 - 1030 nm
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αNA = 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 : 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 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 10x 0 10 20 30 40 50 60 70 80 90 100 0 0,2 0,4 0,6 0,8 1 Numerical Aperture ² Intensity (%) 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 sampleThe principle of confocality
Advantages :
(1) small increase in the lateral resolution (2) large improvement in the axial resolution
Hardware Section –
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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)
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Device bar Take aspectrum : Confirmation of intensity. : Take in spectrum. (1 sec)
Device bar
Take a
spectrum : Confirmation of intensity. : Take in spectrum. (1 sec)
Check
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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
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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 –
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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.
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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’
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Second Part
LabSpec
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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 gratings Do not for get to initialize the spectrograph after a grating change
Move the spectrograph to the zero order position (0 nm)
Enter here the spectrograph position 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
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60000 50000 40000 30000 20000 10000 0 Intens ity (a.u. ) 500 1000 1500 2000 2500 3000 3500 Wavenumber (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
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8000 6000 a.u. ) 3000 2500 2000 1500 1000 500 Int ensi ty (a .u.) 0 500 1000 1500 2000 Wavenumber (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
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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
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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
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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’
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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
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‘Configuration’ Window-L
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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,
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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.
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Yo u can choose a name
You can choose to use
the day, the month and the year as the name
Yo u can choose a name
You can choose to increment
the name by number, hour and minutes of 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.
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
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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).
Baseline Correction
To save the baseline Automatic calculation Saves as a spectrumClears 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.
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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.
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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
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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’.
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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.
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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 Length Y (µm) 400 500 600 Length X (µm) Images : ≈ 4500 pixels (pas de ≈ 5 µm) 2h30 acquisition time TEM 1340 1335 10 5 200 150 100 50 10 µm x1000 40 20 intensity Backg
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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.