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Home / Applications / Raman Imaging of Samples with Uneven or Rough Surfaces using Confocal Microscopy with Surface Scan Imaging (SSI)

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Raman Imaging of Samples with Uneven or Rough Surfaces using Confocal Microscopy with Surface Scan Imaging (SSI)

By Carlos Morillo

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April 25, 2024

Introduction

NRS-4500 Confocal Raman MicroscopeA confocal Raman microspectrometer is an excellent choice for analysis of materials with high spatial resolution – in the order of submicrons. This makes it possible to characterize and image small components such as APIs or bulking agents and foreign materials down to 1 μm, as well as extremely thin layers. However, to achieve high spatial resolution it is necessary to use objective lenses with high magnification. This results in a depth-of-field that is extremely narrow; any slight deviation in the focus at the measurement surface greatly affects to the intensity of the measured spectrum. Imaging measurement of a sample with an uneven or a sloping surface may result in poor measurement unless the objective lens is refocused at each measurement point and can lead to extremely long measurement times.

To make extremely fast measurement in a short time JASCO has developed Surface Scan Imaging (SSI). SSI is used to assess the surface shape of the sample as height information of the stage prior to measurement and Raman measurement is made while adjusting the stage Z axis according to this surface shape information. Using SSI, a sample with a very uneven surface can now be measured to give good Raman spectra with good S/N at all data points.

Keywords

260-AN-0017, Raman, Surface Scan Imaging, SSI, Confocal Raman Imaging

Results

Comparison of SSI with Conventional Autofocus Techniques

Conventionally, Raman imaging measurement of a sample with surface roughness was performed with autofocus (AF) was carried out at each measurement point (figure 1-a), or XYZ 3D imaging measurement was carried out (figure 1-b).

SSI (figure 1-c) calculates the sample height (irregularity) information from a multifocus* image and performs imaging measurement while scanning the stage based on this information. Although it also depends on measurement conditions and sample shape, the time required for Raman imaging measurement is greatly shortened compared with the conventional method.

* The multifocus image (all-focus image) is an image obtained when the stage is scanned in the Z direction to acquire multiple observation images with different focal points, and each layer that is in focus is extracted and a ‘single in-focus image’ is synthesized for the entire field of view. This multifocus image allow the details the entire surface shape of the sample to be identified prior to measurement.

Uneven surface sample measurement.
Figure 1. Uneven surface sample measurement.

Raman Imaging Measurement of a Pharmaceutical Tablet using SSI

Imaging analysis of the uneven part of the surface of a tablet (concave and convex difference: approximately 170 μm) was measured using SSI. The measurement parameters are as follows:

Measurement Conditions
InstrumentNRS-4500 Laser Raman Spectrometer
Excitation Wavelength532 nm
Objective Lens20x (long working distance type)
Interval10 µm
Measurement Points54 x 28 points
Exposure Time0.5 second
Measurement results of uneven surface sample
Figure 2. Measurement results.

When measurement is performed following the surface shape by using SSI, a clear Raman chemical image can be obtained because the focus match at each point. By overlapping the Raman chemical image on the 3D surface shape image, the quantitative result and shape information of concave-convex part of the sample can be checked simultaneously.

Conclusion

Using SSI, Raman imaging measurement of a sample with surface unevenness and inclination can be performed easily and quickly. In addition to the tablet measurement shown here, SSI make it easier to analyze a wide range of samples with surface roughness including rock and concrete, DLC coating, resins, foods, medicines, fertilizer granules etc.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

Featured Products:

  • NRS-5000 Series Confocal Raman Microscope

  • NRS-4100 Confocal Raman Microscopes

About the Author

Dr. Carlos Morillo received his Post Doc at Advanced Industrial Science & Technology in Fukuoka and was a Research Scientist at Kyushu University in Japan where he lived for several years. Carlos received his Doctor of Engineering from Kyushu University and his Masters and BS from Simon Bolivar University in Caracas Venezuela. He is an Applications Scientist at JASCO.

JASCO Application Note

Raman Imaging of Samples with Uneven or Rough Surfaces using Confocal Microscopy with Surface Scan Imaging (SSI)

Introduction

NRS-4500 Confocal Raman MicroscopeA confocal Raman microspectrometer is an excellent choice for analysis of materials with high spatial resolution – in the order of submicrons. This makes it possible to characterize and image small components such as APIs or bulking agents and foreign materials down to 1 μm, as well as extremely thin layers. However, to achieve high spatial resolution it is necessary to use objective lenses with high magnification. This results in a depth-of-field that is extremely narrow; any slight deviation in the focus at the measurement surface greatly affects to the intensity of the measured spectrum. Imaging measurement of a sample with an uneven or a sloping surface may result in poor measurement unless the objective lens is refocused at each measurement point and can lead to extremely long measurement times.

To make extremely fast measurement in a short time JASCO has developed Surface Scan Imaging (SSI). SSI is used to assess the surface shape of the sample as height information of the stage prior to measurement and Raman measurement is made while adjusting the stage Z axis according to this surface shape information. Using SSI, a sample with a very uneven surface can now be measured to give good Raman spectra with good S/N at all data points.

Results

Comparison of SSI with Conventional Autofocus Techniques

Conventionally, Raman imaging measurement of a sample with surface roughness was performed with autofocus (AF) was carried out at each measurement point (figure 1-a), or XYZ 3D imaging measurement was carried out (figure 1-b).

SSI (figure 1-c) calculates the sample height (irregularity) information from a multifocus* image and performs imaging measurement while scanning the stage based on this information. Although it also depends on measurement conditions and sample shape, the time required for Raman imaging measurement is greatly shortened compared with the conventional method.

* The multifocus image (all-focus image) is an image obtained when the stage is scanned in the Z direction to acquire multiple observation images with different focal points, and each layer that is in focus is extracted and a ‘single in-focus image’ is synthesized for the entire field of view. This multifocus image allow the details the entire surface shape of the sample to be identified prior to measurement.

Uneven surface sample measurement.
Figure 1. Uneven surface sample measurement.

Raman Imaging Measurement of a Pharmaceutical Tablet using SSI

Imaging analysis of the uneven part of the surface of a tablet (concave and convex difference: approximately 170 μm) was measured using SSI. The measurement parameters are as follows:

Measurement Conditions
InstrumentNRS-4500 Laser Raman Spectrometer
Excitation Wavelength532 nm
Objective Lens20x (long working distance type)
Interval10 µm
Measurement Points54 x 28 points
Exposure Time0.5 second
Measurement results of uneven surface sample
Figure 2. Measurement results.

When measurement is performed following the surface shape by using SSI, a clear Raman chemical image can be obtained because the focus match at each point. By overlapping the Raman chemical image on the 3D surface shape image, the quantitative result and shape information of concave-convex part of the sample can be checked simultaneously.

Conclusion

Using SSI, Raman imaging measurement of a sample with surface unevenness and inclination can be performed easily and quickly. In addition to the tablet measurement shown here, SSI make it easier to analyze a wide range of samples with surface roughness including rock and concrete, DLC coating, resins, foods, medicines, fertilizer granules etc.

Keywords

260-AN-0017, Raman, Surface Scan Imaging, SSI, Confocal Raman Imaging

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

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