[Spectroscopy Ninja] Spectroscopy Ninja
© 2016: Dr. Friedrich Menges
[SpectraGryph] Spectragryph
- optical spectroscopy software -
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Functions of the Acquire ribbon
=> Click onto a button for descriptions

This ribbon works together with the acquisition tab, which is for spectrometer control and live data acquisition. Here you can connect to a variety of spectrometer types, set acquisition parameters, acquisition and measurement modes, execute x axis calibration and define live post-processing steps.
All parameters and settings can be changed on the fly, even during continuous acquisiton, and effects will be immediately visible.

New Acquisition view

Create a new Acquisition view as the tab for spectral data acquisition from spectrometers. It is possible to connect to several spectrometers by creating several acquisition tabs. The acquired spectra can be handled and processed in all ways just like "normal" spectra by using all the normal functions available. The acquisition tab behaves like a normal Spectra tab, with live data acquisition added.
The initial steps for data acquisition:
- have your spectrometer plugged into the computer
- select spectrometer device type from [Device Type] list
- connect with [Connect] button
- acquire a live spectrum with [Acquire] button

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Spectrometer & Acquisition control

As a first step, always select the device type from the "Device type" list. Currently, there are: Andor cams, OceanOptics and Stellarnet spectrometer modules and USB webcams. The dropdown list below shows the available devices to select from, if there are more than one devices attached. Only after successful selction of a device type does it make sense to actually connect to a device with the [Connect] button. The connector image will change after successful connection to a "plugged" state. The desired exposure time can be set anytime (in milliseconds) and will apply to the next spectrum acquisition.

To acquire live spectra, use the "Acquire" button. This button shows a "down" state only during acquisition, thus it is always visible if there is an acquisition ongoing. Several acquisition modes are possible:
- single shot: grab a single spectrum from the spectrometer, replace the last spectrum in the plot
- continuous: continuing live update of acquired spectra, as fast as exposure time and data transfer allow
- additive: grab a single spectrum from the spectrometer and add it as a new spectrum to the plot
- burst: continuous spectra acquisition, adding them as new spectra to the plot. Process will slow down with present spectra number going up
- loop: continuous spectra acquisition in preset time intervall (use the entry field on the right). To be stopped by pressing [Acquire] button again
Spectrometer settings :
This gives access to multiple parameters at once and allows to save them together as "Spectragryph acquisition settings" file (*.sgas). This file can be loaded again and applied to the currently connected spectrometer.
The upper part give access to parameters that are common to all devices, like exposure tome, loop time, acquisition mode and averaging. On the right, there are the calibration coefficients and designated axis types used for x axis calibration. This part is disabled for spectrometers that have their calibration onboard. The lower part of the window gives access to device-specific settings.
After entering or loading parameters, they can be put to use by clicking the [Apply] button below, or changes can be ignored by closing the window again with the [Close] button below.

Andor settings
- detector temperature
- readout mode with single track positions
- shutter open mode
- shutter signal type
- opening/ closing time

OceanOptics settings
- correct detector nonlinearity
- electrical dark correction
- strobe lamp trigger
- external trigger mode

Stellarnet settings
- temperatur compensation
- show black pixels
- x timing resolution

USB webcam settings
access to webcam settings is provided by a MS Windows dialogue, that may look different between OSes. Exposure time is often not accessible. At least you can prevent clipping by controlling "Brightness" parameter.
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Data control

The selected measurement mode defines the y axis type of the measured live spectrum, with one or more auxiliary spectra needed, depending on the chosen mode. As auxiliary spectrum, there can be dark, reference and Blank spectrum, while each of those can be set, removed and viewed anytime. As soon as these are assigned, they are kept behind the scenes for further use. To update any of them, set them again with a newly measured live spectrum. The dark and the Blank spectrum type are optional, so their use has to be activated by clicking onto the respective button. Reference spectrum is mandatory for each y axis type except intensity, so it is used automatically, when necessary.

How to measure the auxiliary spectra:
- dark spectrum: light source off, shutter closed, so that no light falls onto the detector, measurement mode: intensity
- reference spectrum: light source on, full light (100% level) falling onto the detector, measurement mode: intensity
- blank spectrum: with blank sample present (like pure solvent or buffer in sample container), with the final measurement mode selected
Each one should be measured again after changing exposure time. Reference should be measured again after any changes in the excitation light intensity.

Here are the calculations that happen for the measurement modes, starting with the initially acquired raw spectrum:

  • intensity/counts: Live = Raw - Dark - Blank

  • transmittance: Live = (Raw - Dark) / (Reference - Dark) - Blank

  • reflectance: Live = (Raw - Dark) / (Reference - Dark) - Blank

  • absorbance: Live = - log10 ( (Raw - Dark) / (Reference - Dark) ) - Blank

Remember: Dark and Blank are used optionally and therefore can be acitvated/deactivated, while reference is mandatory, therefore transmittance/ reflectance/ absorbance can't be measured without a previously measured reference spectrum. Not all combinations might make sense for all spectroscopy types, you have to know what suits you best. For example, while it might be well advised to subtract both a dark and a blank spectrum in intensity mode for measuring fluorescence intensity, this would make no sense for an absorbance measurement. There, you would subtract the Blank spectrum in absorbance mode. And so on...

  If "averaging" is checked, the defined number of raw spectra gets averaged, before being shown as live spectrum.
X axis calibration:
For devices that don't have their calibration on board and give out pixels as x axis data values, an x axis calibration has to be executed. For this, first a light source or sample with known peak positions is measured. Then the measured positions get mapped onto the known positions with polynomial regression. The polynomial coefficients are then ready for use, until the calibration gets invalidated by changes in optical setup or whatever. It is possible to calibrate for
- absorbance: by using a sample with known peaks, like a Holmium or Didymium filter. Target: wavelength scale
- fluorescence: by using a light source with known emission peaks, like a pen lamp source (Hg, Hg/Ar, Xe, Ne). Target: wavelength scale
- Raman shift: by using a sample with known Raman lines, as described in ASTM E1840 standard guide. Target: Raman shift scale

After measuring an appropriate spectrum, open the calibration dialog from the "calibr. x axis" button's drop down menu. In the upper field, select the spectrum to be used. Change the peak finding threshold, if necessary. Select the desired x axis type after calibration on the right side. Below are the fields for the calibration coefficients, that result from the calibration process.
In the table below, the x axis positions of the found peaks from the selected spectrum are displayed. You can either enter the known positions into the "desired position" column manually, or select from a number of precompiled positions that are shown as dropdown list. The content of this dropdown list depends on the calibration source chosen above. Only the "custom" option allows to manually enter position values. Check the values to be used during calculation.
Finally, select the polynomial order for the regression calculation and press the "Calculate" button. The calculated calibration coefficients will show up in the respective fields above. To keep the coefficients and the set target x axis type, use the "Apply" button below.
The peak data for the selectable calibration sources is in a file called "calibration_lines.csv" in the program folder, this file's content can be changed to adapt or enhance the range of available calibration peak data.

Turn the calibration-induced axis transformation on/off with the "calibr. x axis" button. This can be done on the fly while measuring.

HINT: To better find the peaks to be used during calibration, you might turn on "Peak labels" before starting the calibration procedure.

ANOTHER HINT: The calibration coefficients are saved and loaded together with other settings from the "Settings" dialogue.

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Live post-processing

All the parameters and settings described above affect the acquired raw data before being shown in the plot. Therefore, they get active with the next acquired spectrum. The post-processing options however affect the displayed live spectrum, so the differences while turning them on and off are directly visible.

Each button has a dropdown menu to access it's settings. It is wise to inspect the currently set parameters before activating the respective option. Activation just happens by clicking the button down, another click will deactivate again. It is possible to use any function in any combination, from only one to all six of them, as there are:
- cut off spectrum part
- remove spikes
- advanced baselining
- advanced smoothing
- Raman transformation (as alternative to directly transforming pixels to Raman by calibration)
- calculate derivative

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© 2001-2017: Friedrich Menges. Last Change: 19 September, 2017
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