Page 101 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 77

Figure 3.7c shows adipocytes containing well-preserved lipid drop-
lets following paraformaldehyde (PF) fixation with OsO postfixation
4
and critical-point-drying (CPD).
There are several advantages to this sample preparation over for-
malin-fixation, water-rinsing and air-drying: (a) Formalin in PBS con-
tains methanol, which permeates the plasma membrane and results
in a faster fixation compared with PF that does not contain methanol.
However, methanol extracts intracellular lipids, which is inappropri-
ate for adipocyte fixation. (b) The OsO postfixative preserves lipids,
4
however, does not itself absorb in the mid-IR range where most biomol-
−1
ecules absorb, except for a peak at 960 cm . (c) The three-dimensional
structure of the adipocyte is retained, since the sample is dried without
surface-tension effects, through CPD, and the localization of intracel-
lular lipid droplets of the adipocyte is persevered. The disadvantage
of this fixation protocol is that the mode of action by which OsO
4
preserves lipids is through complexation-reaction within the double
bonds of lipid hydrocarbon chains or complexation and cross-
linking between unsaturated hydrocarbon chains (Fig. 3.7e). Thus, the
v (=C-H) signal from unsaturated hydrocarbons is present in the
s
lipid-deposit spectrum of the spectrum of the formalin-fixed, water-
rinsed, air-dried adipocyte [Fig. 3.7d(ii)], but is not observed in the
lipid-droplet spectrum of the PF-OsO -CPD adipocyte [Fig. 3.7d(iii)].
4
Additionally, both methods of fixation (formalin-water rinse-air dried
and PF-OsO -CPD) result in a decrease in peak resolution of the
4
v (CH) and v (CH) modes and v (CH) and v (CH) modes.
as 2 as 3 s 2 s 3
The PF-OsO -CPD protocol outlined above was used to preserve
4
samples of prostate cancer cells (PC-3 cell line; prostate cancer cells
derived from bone metastases) that were co-cultured with adipocytes
43
preloaded with deuterated palmitic acid (D -PA). This specimen
31
was used in an FTIR tracing experiment to determine whether PC-3
cells could uptake the fatty acids stored within adipocytes. Figure 3.8a
shows an optical image of a PF-OsO -CPD-fixed adipocyte surrounded
4
by PC-3 cells and stroma cells. In this figure the adipocytes are visu-
alised as large dark bodies (designated with Adp in Fig. 3.8a), whereas
PC-3 cells (1 to 4) are lighter in appearance and possess lamellipodia-
pointed processes. The dark stain results from the binding of OsO to
4
the lipids. The boxed area was mapped using FTIR microspectros-
copy and the v (CD) signal intensity distribution is shown in
as 2+3
Fig. 3.5b. As expected, there was localization of the v (CD) signal
as 2+3
with high intensity to the adipocyte; however, it was also found that
this signal illuminated the PC-3 cells (Fig. 3.8b). Since, the only source
of v (CD) signal in the PC-3 cells is through incorporation of D -
as 2+3 31
PA released by the adipocytes, this data unequivocally demonstrates
the translocation of D -PA between these cell types without cell iso-
31
lation or external labeling. Appropriate fixation was necessary in this
experiment, since delocalization/bleeding of lipid molecules from
adipocytes in the adipocyte—PC-3 cell coculture system could result