Tissue Imaging

​Raman microscopy can be used for imaging both tissue sections and bulk tissues. This has enabled characterisation of native tissues and tissue engineered systems. It is important to optimise a number of parameters to ensure high quality spectral information. Here we detail some of the important considerations for imaging of both tissue sections and bulk tissues and provide example protocols for the acquisition of high quality Raman spectral images of tissues.
References:
  1. Ali, S. M., Bonnier, F., Tfayli, A., Lambkin, H., Flynn, K., McDonagh, V., Healy, C., Lee, T. C., Lyng, F. M., Byrne, H. J., Journal of Biomedical Optics, 18, 6, 2013.

Tissue type

​For Raman tissue imaging applications, the choice of an appropriate tissue is essential. For most imaging techniques, tissues are excised, fixed, and then paraffin embedded. However, for Raman spectroscopy, paraffin embedding can cause significant artefacts, requiring either physical or digital dewaxing. Neither procedure is perfect, and so if possible, it is best to obtain frozen or fixed frozen tissue. Key considerations include: 
  • Select a tissue type that is not very autofluorescent (e.g. lung, liver, oral cavity tissue is very autofluorescent)
  • If imaging single cells, select a cell type that can be sparsely seeded
  • Consider tissue size - larger tissues take longer to image, while smaller cells might not enable certain types of analysis
FFPE (Formalin-Fixed Paraffin-Embedded vs. Fresh Frozen)
​FFPE (Formalin-Fixed Paraffin-Embedded)
This is the most common type of tissue preparation, used extensively in clinical tissue banks.
  • Archival samples readily available
  • Long-term storage at room temperatures
  • Preserves tissue morphology
  • Microtome sectioning
  • Denatures proteins (not suitable for immunohistochemistry)
  • Requires physical or digital dewaxing for Raman spectroscopy studies
Fresh Frozen
Though not as common as FFPE tissue, fresh frozen tissue is available in many clinical tissue banks and presents several advantages for Raman spectroscopic imaging.
  • Less commonly available
  • Medium-term storage at -80C
  • Preserves antigen function
  • Cryostat sectioning
  • Fixation in alcohol post-sectioning
  • Suitable for Raman microscopy
Bulk tissue vs. Tissue sections
Bulk Tissue
​Bulk tissue is typically what is available in clinical tissue banks and is often preferable for Raman spectroscopy as:
  1. It will have undergone fewer processing steps
  2. It can still be suitably sectioned for Raman spectroscopic imaging if required
However, as confocal Raman spectroscopic imaging is sensitive to sample topography, imaging of bulk tissues should be performed on flat samples (i.e. sectioning of top layers to reveal a smooth, flat surface) either with a cryostat or vibrotome.
​Tissue Sections
Tissue sections can be easier to work with, but it is important that the sections are thick enough to provide sufficient Raman signal which again depends on the laser excitation and microscope objective. We would typically recommend a section thickness of 10 um or more if possible. Further, due to the limited sample thickness, choice of substrate is of particular importance to maximise Raman signal and limit any interference presented by the substrate signal.

Choice of substrate

Different substrates have different implications for Raman spectroscopic imaging (for more details see Substrates), the key factors to consider are:
  • Biocompatibility - cells seeded on the substrate need to attach prior to imaging
  • Raman background signal
  • Transparency (for monitoring cell growth prior to imaging)
  • Cost

Choice of fixative

Different fixatives operate by different mechanisms and thus cause varying degrees and types of biochemical and hence Raman spectral distortion (for more details see Fixatives).

Choice of imaging environment

Different imaging environments preserve the cellular morphology to varying degrees (for more details see Imaging Environments).

Choice of laser wavelength

The choice of laser excitation largely depends on autofluorescence of tissue. Further the thickness of tissue needs to be considered in this as aspect as well as NIR excitation offer less confocal capability and can for very thin tissues collect interferences from the substrate below
Shorter wavelengths (vis)
  • Increased Raman signal
  • Increased tissue autofluorescence
  • Increased risk of photodamage
​Longer wavelengths (NIR)
  • Decreased Raman signal
  • Less tissue autofluoresence
  • Less risk of photodamage

Bulk tissue ​imaging protocol in air or hydrated environment

Histological Tissue Slice ​Imaging Protocol
Substrate: CaF2 or MgF2 (for thin samples), Cheaper alternative (e.g. aluminium) for thick samples
Imaging Environment: Air/DPBS
Objective: 63x water immersion/50x
Laser Wavelength: 785 nm
Laser Power: 80 mW (dependent on laser wavelength and spectral acquisition time)
Spectral Acquisition Time: 1 second (dependent on laser wavelength and power)
Spatial Resolution: 1 um+ (dependent on microscope stage capability and objective magnification)

Procedure: bulk tissue sectioning
  1. ​Place bulk tissue onto sample holder for cryo-sectioning in cryo-chamber.
  2. Apply droplets of water to cover sample and allow to freeze, fixing tissue in place.
  3. Section bulk tissue to achieve a smooth, flat surface
  4. Remove tissue and transfer to selected substrate for Raman spectroscopic imaging.
Procedure: Raman imaging
  1. Turn on Raman confocal microscopy system and laser.
  2. Perform microscope calibration (see Microscope Calibration).
  3. ​Place tissue on substrate and place under objective (for imaging in air) or place tissue and substrate in glass petri dish and fill to 3/4 with water/DPBS (for water immersion imaging).
  4. Place tissue underneath microscope objective and, using brightfield imaging, bring the tissue surface into focus.
  5. Focus objective onto the centre of selected tissue region and switch to Raman acquisition mode.
  6. Define imaging area.
  7. ​Optimise Raman signal acquisition by adjusting microscope focus (Raman and brightfield focus planes will be different).
  8. Perform low quality scan of imaging area (e.g. 0.05s acquisition time, 1 um resolution) to ensure the entire tissue is in focus.
  9. Once imaging area is correctly defined, perform high quality scan (e.g. 1 s acquisition time, 1 um resolution).
  10. Save data and record image acquisition parameters for data processing. ​See Data Processing for next steps.
Substrate: CaF2 or MgF2 (for thin samples)
​
Imaging Environment: Air
Objective: 50x
Laser Wavelength: 785 nm
Laser Power: 30 mW (dependent on laser wavelength and spectral acquisition time)
Spectral Acquisition Time: 1 second (dependent on laser wavelength and power)
Spatial Resolution: 1 um+ (dependent on microscope stage capability and objective magnification)

Procedure: Raman imaging
  1. Turn on Raman confocal microscopy system and laser.
  2. Perform microscope calibration (see Microscope Calibration).
  3. Place tissue section (on substrate)  underneath microscope objective and, using brightfield imaging, bring tissue section into focus.
  4. Focus objective onto the centre of selected tissue section region and switch to Raman acquisition mode.
  5. Define imaging area.
  6. Optimise Raman signal acquisition by adjusting microscope focus (Raman and brightfield focus planes will be different).
  7. Perform low quality scan of imaging area (e.g. 0.05s acquisition time, 4 um resolution) to ensure it is defined correctly.
  8. Once imaging area is correctly defined, perform high quality scan (e.g. 0.5 s acquisition time, 1 um resolution).
  9. Save data and record image acquisition parameters for data processing. See Data Processing for next steps.