Feb 27, 2025
Calibration of the OpenRAMAN DIY Raman spectrometer
- 1Arcadia Science
- Arcadia Science
Protocol Citation: Sunanda Sharma, Ben Braverman 2025. Calibration of the OpenRAMAN DIY Raman spectrometer. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmemj6g3p/v1
Manuscript citation:
Braverman B, Mets DG, Sharma S. (2025). DIY Raman spectroscopy for biological research. https://doi.org/10.57844/arcadia-cd7e-443b
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 16, 2024
Last Modified: February 27, 2025
Protocol Integer ID: 105860
Keywords: OpenRAMAN, calibration, DIY spectroscopy, Raman spectroscopy
Disclaimer
This is one of many approaches to calibration that you can take, and we found it useful for the types of biological and chemical investigations we're interested in.
Abstract
The OpenRAMAN system, created by Luc Boussemaere, is an open-source, low-cost, modular Raman spectrometer. In this protocol, we share a calibration procedure for two configurations of the system, which includes optional steps of assessing system performance using common materials used for Raman analysis. We also share an iPython notebook for data processing and sample data collected on the system. This protocol aims to ensure reproducibility of results, elucidate system limitations and capabilities, and allow for effective comparison to published literature or results from other instruments on solids and liquids.
Image Attribution
Arcadia Science
Materials
- Built and functional OpenRAMAN system (solid or liquid cuvette configuration)
- Acetonitrile: HPLC grade, VWR
- Ethanol: USP Spec, KOPTEC
Safety warnings
Use appropriate laser safety glasses.
Before start
To follow this protocol, you should already have an OpenRAMAN system built and functional. If you don't, check out the guide here: https://www.open-raman.org/build/starter-edition/.
Hardware setup
Hardware setup
To follow this protocol, you should already have a functional OpenRAMAN system built. Below is a labeled diagram of the system used for this protocol, which is a "Starter Edition."
We put our system in a black corrugated box with two layers of black fabric to prevent background room lighting from hitting the detector. When possible, we typically turn off overhead lights and reduce vibration to the table.
Select the appropriate tab for your cuvette configuration, liquid (standard) or solid. The liquid cuvette is primarily used for liquid samples (e.g., isopropanol) and high-concentration solutions (e.g., salts). The solid cuvette is primarily used for solid samples, including crystals and powders. The path is slightly different for each configuration, so we calibrate each separately.
Liquid cuvette5 steps
Materials
Materials
Assemble your materials: a neon bulb connected to power and acetonitrile in a disposable culture tube.
The exact materials we're using are listed below:
- Neon bulb: An NE-2H lamp with a 1000 Ω resistor soldered to an extension cord with a switch. Ensure all wires are safely insulated.
- Acetonitrile: HPLC grade, VWR
- Ethanol: USP spec, KOPTEC
- Parafilm or other cover for tube
Neon acquisition
Neon acquisition
Place the neon bulb into the liquid cuvette directly in front of the lens. You should be able to see neon light coming through.
Take a live measurement and move the bulb around until you get a spectrum like the one below. Make sure 'Enable Baseline Removal (Schulze et al. Algorithm)' and 'Enable Median Filtering' are both OFF and use the following settings:
| A | B | |
| Exposure | 1000 ms | |
| Gain | 0.0 | |
| ROI | 100 px | |
| Num avg | 5 |
You can manually save your file as a ".csv" or ".spc" by selecting the save icon in the upper left corner of the toolbar. We recommend saving files as ".csv" because ".spc" files can only be viewed on the Spectrum Analyzer software.
A screenshot of the Spectrum Analyzer software displaying the spectrum of neon from a liquid cuvette.
Acetonitrile Acquisition
Acetonitrile Acquisition
Pipette 2 mL of acetonitrile into a disposable glass culture tube and cover it with a piece of Parafilm or similar.
Acetonitrile in tube in the liquid cuvette attachment.
Make sure "Enable Baseline Removal (Schulze et al. Algorithm)" and "Enable Median Filtering" are both OFF and use the following settings:
| A | B | |
| Exposure | 1000ms | |
| Gain | 0.0 | |
| ROI | 100px | |
| Num Avg | 5 |
Capture a spectrum using the play button.
If you select "Automatically log acquired spectra on disk," your data will be saved automatically. You can save data as either a ".csv" or ".spc." We recommended saving files as ".csv" because ".spc" files can only be viewed on the Spectrum Analyzer software.
File path has been removed in this image.
A screenshot of the Spectrum Analyzer software displaying the spectrum of acetonitrile from a liquid cuvette. Configuration settings are shown on the right.
Dark spectrum acquisition
Dark spectrum acquisition
A "dark spectrum" is just a measurement with the laser off. This spectrum gives you information about background noise in your environment. You can do this without a sample in the cuvette.
Turn the laser off and acquire a dark spectrum to record the background environment.
Make sure "Enable Baseline Removal (Schulze et al. Algorithm)" and "Enable Median Filtering" are both OFF and use the following settings:
| A | B | |
| Exposure | 1000 ms | |
| Gain | 0.0 | |
| ROI | 100 px | |
| Num avg | 5 |
Acquisition parameters for the dark spectrum.
Capture a spectrum using the play button. Note that you can change the exposure to match the typical acquisition settings you'll use for your samples.
If you select "Automatically log acquired spectra on disk," your data will be saved automatically. You can save data as either a ".csv" or ".spc" file. We recommend saving files as ".csv" because ".spc" files can only be viewed on the Spectrum Analyzer software.
File path has been removed in this image.
A screenshot of the Spectrum Analyzer software displaying a dark spectrum. Configuration settings are shown on the right, but make sure to uncheck "Enable median filtering."
Data processing
Data processing
All the data collected with the liquid cuvette can be processed using a Python calibration notebook available on GitHub. The references quoted above are used to create the calibration equations. This notebook has four general steps:
1. Use the neon spectrum to create a linear equation to convert from pixel # to wavelength (nm). Atomic emission sources are great for this since they have known absolute wavelength positions. They also have well-defined, sharp peaks that aren't affected by environmental factors like temperature and pressure.
2. Convert wavelength to wavenumber (cm-1). The equation is: Raman_shift_in_cm-1 = (10^7)((1/532) - (1/wavelength_in_nm).
3. Test conversion and apply an additional adjustment based on the acetonitrile spectrum. Acetonitrile is a known, commonly referenced material with strong peaks across a wide range. This additional step helps catch systematic errors that might occur in the previous two steps, acting as a check. The acetonitrile spectrum, unlike neon, is from Raman scattering and can help correct for small variations in laser behavior and track laser power over time.
Protocol references
2. Strong lines of Neon ( NE ). Available at: https://physics.nist.gov/PhysRefData/Handbook/Tables/neontable2.htm (Accessed: 25 September 2024).
3. Shimanouchi, T., Tables of Molecular Vibrational Frequencies Consolidated Volume I, National Bureau of Standards, 1972, 1-160.
4. Calcite R150020 - RRUFF. Available at: https://rruff.info/R150020 (Accessed: 25 September 2024).
5. Okuno, M. (2021) ‘Hyper‐Raman spectroscopy of alcohols excited at 532 nm: Methanol, ethanol, 1‐propanol, and 2‐propanol’, Journal of Raman Spectroscopy, 52(4), pp. 849–856. doi:10.1002/jrs.6066.