System operate without software running 5) Perform calibration (ToolsCalibrationQuick

System start 1)      Turn on computer on gantry2)      Turn on Raman microscope using main switch located at the side3)      Turn on laser sources (select laser source according to sample under investigation, 830 nm offers higher power and better spectral resolution but sensitivity in CH region is significantly reduced due to drop in CCD (charge-coupled device) sensitivity) à 830nm diode laser.

4)      Start WiRE software (icon on desktop) – laser will not operate without software running5)      Perform calibration (ToolsCalibrationQuick calibration)System operationSelect correct optics for desired excitation wavelength (830nm edge Leica for 830nm)-          Sample alignmentCalibration can be checked using internal Silicon reference (MeasurementNew measurement830 nm with Si ref) à RunDisplay peak wavenumber using the icon below the menu bar à if peak position is not correct it can be shifted using ToolsCalibrationOffsetUse green plastic standard (Allen key holder) and record spectrum in static mode (centre at 1500 cm-1) to get reference spectrum for comparisonPlace sample on sample stage, select icon to view sample under video (adjustment of brightness, aperture and symbol with “f” might be necessary) à White light spot should be visible on sample surfaceManually move stage for coarse position and focus adjustment à close sample chamber door gently to avoid sample misalignment Fine adjustment can be realised using motorised stage controller à Always press one of the four outer buttons first to select speed of movement (5x: fast, 100x: slow) à Trackball moves sample stage horizontally (e.g. in x-y-plane) focussing ring around trackball serves for vertical movement (z direction)Actual coordinates are displayed in top right corner of programme window/use “set origin” and “go origin” for easy position recovery after movement.When accessing/changing sample always press button “door release” before opening sample chamber door. If not doing so interlock will be engaged à to reset interlock go to Toolsinterlock and hit “Reset”   –          MeasurementIf desired sample position is reached, measurement parameters can be selected using MeasurementewSpectral acquisition.

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It is important to select the parameters here as changes using icons below the menu bar sometimes do NOT work.There are 2 options selecting the desired spectral range1)      If only small range is required “static” can be selected à enter position of central wavelength and accessible range is determined automatically à Attention: minimum wavenumber must be larger than at least 50cm-1 to avoid exposure of CCD to direct laser wavelength2)      If extended range (or small signals between intense peaks) should be investigated, select “Extended” à default range is 100-3200cm-1 but can be customised by changing start and ending wavenumber, minimum integration time in this case is 10 seconds. For signal adjustment, real time scanning using 1 s integration time and 1 accumulation is recommended; laser power has to be adjusted accordingly to get sufficient signal intensity à Select “static” range to contain prominent Raman band to optimise its intensity à use icon “Cycling” for continuous data readout à optimise signal (or better signal to noise ratio) by carefully tuning the z-position (slow movement speed (100x or 50x) is recommended)For spectral recording measurement parameters can be changed using Measurementsetup measurement (all parameters selected previously using Measurementewspectral acquisition are maintained)As detector is quite sensitive, automatic removal of cosmic rays is recommended (tick box where selecting integration time and number of acquisitions) à this requires a minimum of 3 accumulationWhen using high laser power and/or long integration times detector might become saturated à this is indicated by the red line in the actual spectrum view changing from solid to dashedIf measurement is completed save spectrum in *.wxd file format (FileSave as) à this file format maintains all file associated informationFor import in Excel additionally save file in *.

txt (ASCII) formatSaved spectrum will be displayed in active window à select new position on sample and hit “Run” icon à new measurement is started with same parameters as previous acquisitions à this allows for easy spectral comparison (alternatively, saved spectrum can be removed from display)-          Special featuresSoftware offers also possibility for automated mapping and depth scans (select appropriate option in Measurementew) “Focus track” option might be useful when mapping non-flat sample à system optimises focus position at each sampled position before recording Raman spectrumSpectrum MUST be saved in *.wxd format (otherwise information will be lost) à data can be converted into ASCII compatible format using “batch converter” (shortcut on desktop)If e.g. doing spectral mapping resulting *.txt file will contain four columns (x position, y position, wavenumber, intensity) à data can be rearranged using Excel macro.System shutdown1)      Quit WiRE software 2)      Shut down computer3)      Turn off 830nm laser source 4)      Turn off Raman microscope using main switch located at the side