Time did not allow us to answer all of the many and varied questions that came in during the webinar, and during my travels rolling out the new system, I’ve received lots of additional questions. Therefore, I have taken the opportunity to answer many of these here. If you have any further questions that aren’t answered here, please don’t hesitate to contact me directly.
The overall particle size range for a morphological analysis is from 0.5 µm to 1300 µm (or larger for some applications). For MDRS/Chemical identification measurements, although the spot size on the instrument is 2 µm, for some samples it is possible to measure Raman spectra from particles smaller than this. The instrument can return to and center the laser on particles as small as 1 µm. However, the signal-to-noise ratio of the Raman spectrum will depend upon how strong the Raman scattering is from the sample, compared to the Raman scattering from the sample substrate.
Laser diffraction is a volume-based technique that assumes a perfect sphere particle shape, whereas static image analysis is a number-based technique that measures the largest face of the dispersed particles. Both techniques can mathematically convert to the other type of result for comparison. You would expect comparable results between the techniques when measuring perfect spheres but you would expect differences between the results for more irregularly-shaped particles. However, the techniques are often used in parallel and you would expect similar trends when comparing between results for different samples. You can find out more here
Raman spectroscopy provides chemical information mainly from molecular vibrations. It is particularly well-suited to a wide range of organic materials and inorganic materials. Unsuitable samples include metals, black or very dark samples, and samples which fluoresce at the wavelength of the laser light used (785 nm).
The Sharp Edge algorithm finds regions of interest in each frame and then uses a combination of automated intensity thresholding and edge detection to find the particles. Sharp Edge is a new feature for the Morphologi 4 platform and is an alternative to thresholding, which separates particle images from the background based on a specified greyscale intensity value. The Morphologi 4 software is not backward compatible so it cannot be used with Morphologi G3.
Yes, you can use the Morphologi 4-ID as a Morphologi 4 for imaging only, and then when you need the extra capability of chemical identification enabled by the Morphologi 4-ID, you can apply that as well.
The Morphologi 4-ID has a class 1 laser safety rating. It is possible to perform Morphologi measurements with the door open, but the door must be closed for the Raman measurement.
Morphological classifications for agglomerates can be set up based on any combination of size or shape parameters. Typically, we use a combination of size and circularity or convexity. The Morphologi 4-ID takes a Raman spectrum from a spot approx. 2 µm in diameter at the center of mass of the particle. For samples with agglomerates up to 10’s of µm, it may be possible to see contributions in the Raman spectra from more than one component. As always, this will depend upon the relative Raman scattering from the different components. Such agglomerates will give intermediate correlation scores for the individual components.
The Morphologi 4 and Morphologi 4-ID both come with an integrated dry powder dispersion unit and a holder for standard microscope slides. There are also optional wet cell accessories for measuring particles in suspension and filter holders for measuring particles captured on membrane filters. In addition, there are specifically-designed glass filters for use with Raman Spectroscopy. See the Morphologi 4 range brochure for more information.
For successful image analysis, a good separation between particles is required. The dilution, if required, will depend on the particle size range.
The Raman part of the MDRS analysis is the time-consuming step of the measurement, but the flexible approach enables the user to optimize the chemical identification according to the requirements of the application. The following options are possible:
if required and depending on the total number, 100% of the imaged particles can be targeted
a subset of particles selected objectively by the software can be targeted
only particles meeting predefined morphological classes can be targeted (or a subset of the morphological class selected objectively by the software)
the user can manually select particles of interest from their images
Typical measurements for the morphological analysis take between 15 and 30 minutes depending on the size range and the number of particles that need to be measured. The chemical measurement time depends upon two variables: the time needed to collect a suitable Raman spectrum, and the number of particles required. Typically, Raman acquisition times vary between 2-30 seconds but this will depend on the Raman scattering efficiency of the sample. A 1,000 particle measurement with a 10 second acquisition time would typically take around 5 hours for both the morphological and chemical analysis. Longer measurements can be left to run unattended overnight.
The instrument does not come with any libraries as standard. It is recommended practice that for chemical classification applications, the user constructs a library from known reference samples using the Morphologi 4-ID and thereby optimizes that library for their specific application. However, chemical identification of unknown samples can be achieved by directly exporting spectra to KnowItAll from BioRad, or by exporting spectra in a compatible format for use with other third-party libraries.
The Morphologi 4-ID uses a 50x lens to acquire Raman spectra with the optimum signal-to-noise ratio. The laser spot size at this magnification is approx. 2µm and is automatically placed at the center of mass of the particle. Therefore, chemical identification methods for particles >2µm assume that the particles are chemically homogeneous.
The optional pre-processing applies a Savitzky-Golay filter to the raw signal using the derivative order and filter width selected by the user. Correlation scores are then calculated from the pre-processed spectra according to well established mathematical methods:
You can find out more here.
The Morphologi 4-ID currently only has a 785 nm laser. This laser provides the optimum compromise between signal strength and sample fluorescence for a wide range of organic materials.
No mapping capability is provided. Currently, automated Morphologi 4-ID measurements support single point spectra from the center of mass of each selected particle. Manual targeting by the Raman spectrometer is possible in the manual microscope interface, allowing point spectra and position to be recorded individually. Please contact me if this is a feature that is of specific interest to you.
Discriminating between different APIs is one of the key strengths of the MDRS technique, provided there is sufficient contrast between the particles and their background to be able to measure them during the image analysis part of the measurement and provided the APIs of interest are good Raman scatterers. Watch this webinar for some examples.
Yes we have MDRS users who have studied minerals, cement, volcanic ash deposits and tsunami deposits, plus some applications for forensics such as looking at minerals within soils and analyzing samples from different geological regions. Here are some links to an application notes and some webinars which discuss these applications in greater detail:
Component-specific characterization of cements using Morphologi ID
Forensic Analyses by Morphologically-Directed Raman Spectroscopy
Forensic soil analysis explained
The Morphologi 4-ID has been developed with the analysis of protein-based samples in mind. A number of recent advances will help with this type of application, including Sharp Edge particle separation for low contrast samples, a fully enclosed system and increased laser power.
Depending on the particle size range, this should be possible. If these particles are suspected to be present in relatively low concentrations, it may be more efficient to filter the samples prior to analysis.
Depending on the age of the system, Morphologi G3 systems will be supported for up to 7 years from when the End of Manufacture notice is released.
Previous webinar Q&As:
- A basic introduction to Electrophoretic Light Scattering (ELS) for Zeta potential measurements – Q&A
- A basic introduction to Dynamic Light Scattering (DLS) for particle size analysis – Q&A
- NanoSight Sample Assistant – Q&A
- Basic Principles of Nanoparticle Tracking Analysis – Q&A
- DLS in 30 Minutes – Q&A