Fixed cell imaging protocol

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Raman spectroscopic imaging has achieved remarkable results in the label-free imaging of fixed cells. This has enabled studies of drug carrier uptake, cellular response to pharmaceuticals, and characterization of different micro environments. However, in order to obtain such insights, it is important to optimize the Raman experiment. Here we detail some of the important experimental considerations for fixed cell imaging and provide an example protocol for the acquisition of high quality Raman spectral images of fixed cells.

Choice of cell type

For 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

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 and not be affected by the substrate
  • Raman substrate background signal
  • Substrate transparency (for monitoring cell growth prior to imaging using conventional widefield microscopy)
  • Substrate costs

Choice of fixative

Different fixatives operate by different mechanisms and thus cause varying degrees of changes to the cell biochemistry and Raman spectra (for more details see Fixatives).

Choice of imaging environment

Different imaging environments (e.g., air, DBPS, hydrogel etc) preserve the cellular morphology to varying degrees as well as heat transfer from the focused laser (for more details see Imaging Environments).

Choice of laser wavelength

The choice of laser excitation wavelength needs to be optimized for different biological imaging applications. In general for imaging fixed cells a visible excitation such as 532 nm represent a good choice. The following guidelines dictates the choice of laser wavelength for cell imaging.
​Shorter wavelengths (vis)
  • Increased Raman signal / faster imaging
  • Better resolution / confocal sectioning capability
  • Increased cell autofluorescence interference
  • Increased risk of photodamage (in particular of cytochrome/mitocondria)
​Longer wavelengths (NIR)
  • Decreased Raman signal / slower imaging
  • Poorer resolution / less confocal sectioning capability
  • Decreased cell autofluoresence interference
  • Decreased risk of photodamage

Choice of microscope objective

The microscope objective characterized by its magnification and numerical aperture represents one of the most important aspects for performing imaging. In general a low magnification objective can be used to obtain a wider field overview of the sample while high NA objectives gives better sectionaing capability and signal intensity.
Low magnification (e.g., 10-40x ) and low NA
  • Relatively low signal (low NA) / slow Raman imaging
  • Lower spatial resolution
  • Can often result in more background signal and out-of-focus Raman light.
  • Enables widefield overview of tissues
  • Less sensitive to sample gradients
High magnification (e.g., 40-100x )and high NA
  • Increased Raman signal / faster imaging
  • Higher spatial resolution
  • Often low background signal and good confocal sectioning capability
  • Can be very sensitive to sample gradients

Choice of pinhole for confocal imaging (fiber coupled to spectrometer)

The fiber coupling on the confocal microscope controls the confocal capability of the microscope and must be chosen accordingly with consideration to the laser excitation. For instance a 785 nm laser with a 50um pinhole often results in
Large pinhole (e.g., 50-100um fiber)
  • Relatively high signal / fast Raman imaging
  • Lower spatial resolution
  • Results in more background signal and out-of-focus Raman light.
  • Poorer confocal sectioning capability
  • Less sensitive to sample gradients
Small pinhole (e.g., 25-50um fiber)
  • Relatively low signal / slow Raman imaging
  • High spatial resolution
  • Result in less background signal
  • Excellent confocal sectioning capability
  • Very sensitive to sample gradients

Example of a fixed cell imaging protocol

Substrate: CaF2 or MgF2
Fixative: Formalin
Imaging Environment: Hydrated (DPBS)
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: 1 second (dependent on laser wavelength and power)
Spatial Resolution: 0.5 um

Procedure:
  1. ​Seed cells on selected substrate and allow to attach.
  2. Apply treatments to cell as required.
  3. Fix cells in 4% (v/v) paraformaldehyde for 15 minutes at room temperature.
  4. Remove fixative, rinse cells in DPBS, and store at 4C in DPBS until required for imaging.
  5. Turn on Raman confocal microscopy system and laser.
  6. Perform microscope calibration (see Microscope Calibration).
  7. ​Place sample in glass petri dish and fill to 3/4 with DPBS for imaging.
  8. Place sample underneath microscope objective and, using brightfield imaging, bring the cells into focus.
  9. Focus objective onto the centre of selected cell and switch to Raman acquisition mode.
  10. Define imaging area.
  11. ​Optimise Raman signal acquisition by adjusting microscope focus (Raman and brightfield focus planes will be different).
  12. Perform low quality scan of imaging area (e.g. 0.05s acquisition time, 1 um resolution) to ensure it is defined correctly.
  13. Once imaging area is correctly defined, perform high quality scan (e.g. 1 s acquisition time, 0.5 um resolution).
  14. Save data and record image acquisition parameters for data processing. See Data Processing for next steps.