Live cell imaging protocol

Raman spectroscopic imaging has achieved remarkable results in the label-free imaging of living cells. This has enabled studies of cell cycle dynamics, visualise changes during apoptosis, and perform time-course imaging. However, as with fixed cell imaging, in order to obtain such insights, it is important to optimise a number of parameters to ensure high quality spectral information.

Here we detail some of the important considerations for live cell imaging and provide an example protocol for the acquisition of high quality Raman spectral images of living cells.

Choice of Cell Line

or any cell imaging applications, the choice of an appropriate cell type is essential and it is no different for Raman spectroscopy. Key considerations include: 
  • Select a cell type that is adherent to the chosen substrate (or apply a substrate coating)
  • If imaging single cells, select a cell type that can be sparsely seeded
  • Consider cell size - larger cells take longer to image, while smaller cells might not enable certain types of analysis
  • Consider cell robustness to live imaging conditions

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 Imaging Enivronment

Imaging environment is critical for live cell Raman spectroscopic imaging owing to the long imaging times required. It is recommended to image the cells in a phenol red-free medium and, depending on the system setup you have, use a CO2-independent buffering cell medium (for more details see Imaging Environments).

Choice of Laser Wavelength

Due to the absorption profiles of different biomolecules, the laser wavelength needs to be optimised for different biological imaging applications. This is particularly important for live cell imaging, as UV and visible laser wavelengths can cause cell damage during imaging that distort any results obtained.
​Lower Wavelengths
  • Increased signal
  • Increased cell autofluorescence
  • Increased risk of photodamage
​Higher Wavelengths
  • Decreased signal
  • Decreased cell autofluoresence
  • Decreased risk of photodamage

Live Cell Imaging Protocol

Substrate: CaF2 or MgF2
Imaging Environment: Cell Medium (Leibovitz's L-15, Phenol red-free)
Objective: 63x/1.0NA Water-Immersion
Laser Wavelength: 532 nm
Laser Power: 35 mW (dependent on laser wavelength and spectral acquisition time)
Spectral Acquisition Time: 0.5 second (dependent on laser wavelength and power)
Spatial Resolution: 1 um (dependent on microscope stage capability and objective magnification)

Procedure:
  1. ​Seed cells on selected substrate and allow to attach.
  2. Apply treatments to cell as required.
  3. If cells not already in Raman imaging medium, exchange medium for Leibovitz's L-15 phenol red-free medium.
  4. Turn on Raman confocal microscopy system and laser
  5. Perform microscope calibration (see Microscope Calibration).
  6. ​Place sample in glass petri dish and fill to 3/4 with cell medium for imaging.
  7. Place sample underneath microscope objective and, using brightfield imaging, bring the cells into focus.
  8. Focus objective onto the centre of selected cell and switch to Raman acquisition mode.
  9. Define imaging area.
  10. ​Optimise Raman signal acquisition by adjusting microscope focus (Raman and brightfield focus planes will be different).
  11. Perform low quality scan of imaging area (e.g. 0.05s acquisition time, 1 um resolution) to ensure it is defined correctly.
  12. Once imaging area is correctly defined, perform high quality scan (e.g. 0.5 s acquisition time, 1 um resolution).
  13. Save data and record image acquisition parameters for data processing. See Data Processing for next steps.