Hot stage polarizing light microscopy
The three major lipids, CE, phospholipid (PL) and Cx found in atherosclerotic lesion
can be easily identified by HSPLM. CE in liquid crystal phase appears as Maltese crosses
and the transition temperatures from liquid crystal phase to isotropic liquid phase are in
the 25oC to 55oC range. Birefringence remaining after 55-60oC
represents Cx with the appearance of typical plate-like morphology and acute edge angles
of 79 degrees, and phospholipids with the morphology of myelin figures or Maltese crosses
(see Figure). Briefly, the aortic sample for physical microscopy was placed on the
microscopy stage with a heating stage control, and viewed using crossed polarizer. The
sample was heated at the rate of 1-2oC/min to 60oC, the temperature
at which all CEs become isotropic. The remaining birefringence was Cx and PL. Then the
same sample was cooled to 10oC with dry ice to observe all lesion lipids in
crystalline phase. Two sets of photomicrographs were taken at 10oC and 60oC.
Set No.2 was used exclusively for subsequent DIA. In set No.2, the camera exposure time
was set constant for both temperature settings.
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