Skip to content
Skip to content
  • News
  • Events
  • eBooks
  • Blog
  • Careers
  • Contact
  • News
  • Events
  • eBooks
  • Blog
  • Careers
  • Contact
KNOWLEDGEBASE
  • About Us
  • Products

    Fluorescence Spectrometers

    • FLS1000 Photoluminescence Spectrometer
    • FS5 Spectrofluorometer
    • LifeSpec II Lifetime Spectrometer
    • Mini-tau Lifetime Spectrometer

    Raman Microscopes

    • RM5 Raman Microscope
    • RMS1000 Multimodal Confocal Microscope

    Transient Absorption

    • LP980 Transient Absorption Spectrometer

    FTIR Spectrometers

    • IR5 FTIR Spectrometer

    Lasers and LEDs

    • Pulsed Lasers
    • Gas Lasers
    • Customisation Options
    View All Products
  • Techniques
  • Applications
KNOWLEDGEBASE
Edit Content
  • About Us
  • Products
  • Techniques
  • Applications
  • Knowledgebase
  • eBooks
  • News
  • Events
  • Blog
  • Careers
  • Contact Us

RESOURCES

Spatial Resolution in Raman Spectroscopy

  • July 8, 2021

KEY POINTS

  • Lateral resolution in a Raman microscope is determined by the excitation wavelength and the numerical aperture (NA) of the objective lens used.
  • Shorter wavelength lasers can be used to achieve higher spatial resolution.
  • Higher NA objectives, especially immersion types, improve resolution
Edit Content

Spatial resolution is vital in Raman microscopy for discriminating different structures in a sample.

The better the spatial resolution, the more detailed information can be gained. For example, differentiating different components in a single cell or detecting defects in graphene materials.

Lateral Resolution

There are two main factors that determine the lateral (XY plane) resolution in a Raman microscope: the excitation wavelength (λ) and,

the numerical aperture (NA) of the objective lens used. There a several definitions of lateral spatial resolution but the most commonly used is:

Spatial resolution equation

For example, using a 405 nm laser and an objective with a NA of 0.9 the theoretical spatial resolution achievable is 275 nm. However it should be stressed that this is a theoretical ideal. It is rarely achieved in practice due to sample dependent scattering properties. A practical Raman spatial resolution is on the order of 1 µm.

Since the spatial resolution is proportional to laser excitation wavelength, shorter wavelength lasers can be be used to achieve higher spatial resolution. Raman Microscopes are often equipped with multiple laser wavelengths, enabling the user to find the best compromise between required spatial resolution and other constraints such as fluorescence from the sample.

The numerical aperture is determined by the type of objective lens used. Standard air objectives have a maximum NA of 0.9 but for applications where spatial resolution is critical, immersion objectives can be used to obtain a higher NA. Here, 1 an immersion fluid with a higher refractive index than air (1) is placed between the front lens of the objective and a coverslip/sample where in normal objectives air would be. The two most commonly used fluids are water with a refractive index of 1.3, and oil which has a refractive index of approximately 1.5 (dependent on oil type). Water immersion objective have particular use in confocal Raman microscopy for the study of live cells in cell media, whilst oil objectives can be useful for depth studies.

The highest lateral spatial resolution can only be achieved using a confocal Raman microscope. Confocal Raman microscopes utilise a pinhole that is used to reject out of focus light from detection. Figure 1 highlights the improvement in spatial resolution of polystyrene beads by narrowing the pinhole diameter of the Edinburgh Instruments RM5 Raman Microscope.

Figure 1: polystyrene beads mapped on the RM5 Raman Microscope with varying pinhole diameters A) 2 mm B) 100 µm C) 50 µm D) 25 µm.

Axial Resolution

The axial spatial resolution (Z axis) is proportional to λ/NA2 and like the lateral resolution it improves with decreasing wavelength and higher NA objectives. However, the main parameter that controls the axial resolution is the confocal pinhole diameter, the narrower the pinhole the better the axial resolution.  At the narrowest pinhole diameters a depth resolutions on the order of 1 µm can be achieved in ideal samples. More information about the relationship between pinhole diameter and resolution can be found in our Spectral School tutorial on the role of the pinhole in Raman microscopy.

The video below shows an example of the axial information that can be obtained with a confocal Raman microscope. The 3D Raman map was reveals the different material layers of a transdermal drug delivery patch. The different colours show the identification and location of PET (red), PET/PIB (pink), PE (blue) plastic and the active pharmaceutical ingredient (green).

 

 

RELATED PRODUCTS

RMS1000

Multimodal Confocal Microscope

VIEW

RM5

Raman Microscope

VIEW

Contact our expert team today to find out more about how our products can improve your research

Contact Us
PrevPrevious
NextNext
Previous Role of the Pinhole in a Confocal Microscope Next What is Confocal Raman Microscopy?

RESOURCES

Tags:
  • Spectral School
  • RM5
  • RMS1000
  • Raman
Suggested Reading:

Improving Axial Resolution and Spectral Sensitivity in 3D Confocal Raman Imaging Using an Immersion Objective Lens

View more »

What is Confocal Raman Microscopy?

View more »

Role of the Pinhole in a Confocal Microscope

View more »
Laser Spot Size

Laser Spot Size in a Microscope

View more »
Share:

Keep up to date with the latest from Edinburgh Instruments

Join our mailing list and keep up with our latest videos, app notes and more!

LOCATION:
  • Edinburgh Instruments Ltd.
    2 Bain Square, Kirkton Campus, Livingston, EH54 7DQ.
  • sales@edinst.com
  • +44 1506 425 300
ABOUT:
  • About Us
  • Techniques
  • Applications
  • Knowledgebase
  • About Us
  • Techniques
  • Applications
  • Knowledgebase
PRODUCTS:
  • Fluorescence Spectrometers
  • Raman Microscopes
  • UV-Vis Spectrophotometers
  • Transient Absorption
  • FTIR Spectrometers
  • Lasers and LEDs
  • Customisation Options
  • Software
  • Upgrades
  • All Products
  • Fluorescence Spectrometers
  • Raman Microscopes
  • UV-Vis Spectrophotometers
  • Transient Absorption
  • FTIR Spectrometers
  • Lasers and LEDs
  • Customisation Options
  • Software
  • Upgrades
  • All Products
LEGALS:
  • News
  • Events
  • Blog
  • Careers
  • Contact Us
  • Terms and Conditions
  • Privacy Policy
  • News
  • Events
  • Blog
  • Careers
  • Contact Us
  • Terms and Conditions
  • Privacy Policy
SOCIALS:
Youtube Linkedin X-twitter Facebook
©2024 Edinburgh Instruments. Registered in England and Wales No: 962331. VAT No: GB 271 7379 37
Manage Consent
To provide the best experiences, we use technologies like cookies to store and/or access device information. Consenting to these technologies will allow us to process data such as browsing behaviour or unique IDs on this site. Not consenting or withdrawing consent, may adversely affect certain features and functions.
Functional Always active
The technical storage or access is strictly necessary for the legitimate purpose of enabling the use of a specific service explicitly requested by the subscriber or user, or for the sole purpose of carrying out the transmission of a communication over an electronic communications network.
Preferences
The technical storage or access is necessary for the legitimate purpose of storing preferences that are not requested by the subscriber or user.
Statistics
The technical storage or access that is used exclusively for statistical purposes. The technical storage or access that is used exclusively for anonymous statistical purposes. Without a subpoena, voluntary compliance on the part of your Internet Service Provider, or additional records from a third party, information stored or retrieved for this purpose alone cannot usually be used to identify you.
Marketing
The technical storage or access is required to create user profiles to send advertising, or to track the user on a website or across several websites for similar marketing purposes.
Manage options Manage services Manage {vendor_count} vendors Read more about these purposes
View preferences
{title} {title} {title}