Capsule-Based Processing and Handling


Figure 3 : Rat ( Rattus norvegicus ) kidney tissue prepared using mPrep/s and mPrep/g capsules. (a) Rat kidney section stained with polychrome I, transmitted light microscopy. Image width about 100 µm. (b) Rat kidney TEM image from the same block as the light micrograph. The box indicates the region shown in Figure 3c. (c) Rat kidney TEM image at higher magnifi cation.


micrographs indicate good tissue fi xation and uniform staining. Figure 5 shows vampire bat brain sections stained using mPrep/g processing that exhibit uniform staining without precipitates. Figure 6 shows Dieff enbachia prepared using mPrep/s and mPrep/g capsule processing by technical college students learning electron microcopy. T is specimen also shows good fi xation and uniform staining.


Labor effi ciency and reagent


Figure 4 : Porcine tissues entirely prepared using mPrep/s and mPrep/g capsules. (a) Porcine heart TEM showing myofi brils in various orientations and mitochondria. (b) Porcine skin TEM.


was used for the Dieff enbachia specimen and a Hitachi H-7600 for the vampire bat brain.


Measuring reagent consumption and labor effi ciency . Reagent consumption for mPrep processing was compared to common vial and transfer pipette methods. T e reagent consumed for processing 12 tissue specimens with mPrep/s capsules was measured with the pipettor. Reagent that would have been used in standard vial processing was calculated assuming 1 ml per reagent exchange. T e reagent consumed for grid processing was not compared because mPrep/g capsules use 17.5 μ l per grid (two grids in one 35 μ l capsule), which is similar to typical droplet volumes of 25–100 μ l per grid. T e labor effi ciency for processing 12 specimens and 16 grids was calculated by counting the number of physical operations required for mPrep capsule processing and the number of physical operations that would be used for vial tissue processing and droplet grid staining, as enumerated in Tables 1 and 2 .


Results


Images of processed tissues . T ree mammalian tissues and resultant sections on grids were entirely prepared using mPrep capsule processing. Figure 3 shows rat kidney imaged with LM and TEM, and Figure 4 shows pig heart and skin tissue. T ese


34


consumption . T e method for pro- cessing tissue with mPrep/s capsules was described in the Materials and Methods section of this article. T e most commonly repeated steps were the 24 fl uid reagent exchanges from


fi xative to fi nal resin ( Table 1 ). As shown in Figure 1 , all these reagent exchanges were performed simultaneously by immersing the bottom ends of 12 mPrep/s capsules into reagent-containing reservoirs and operating the pipettor. Final embedding was achieved by simultaneously inserting all 12 resin-fi lled capsules into the silicone bench holder prior to transfer into the curing oven. Compared to vial processing, the number of manual operations was reduced 7-fold, from 324 to 49 operations with mPrep capsule processing. T e total reagent consumed in prepar- ing 12 specimens with mPrep/s capsules was 30 ml, compared to 312 ml calculated for vial processing, providing a 10-fold reduc- tion ( Table 1 ). Table 2 shows that simultaneous staining of 16 grids with mPrep/g capsules required as few as 20 operations, compared to over 100 calculated for droplet staining.


Discussion Conventional processing methods are well established. T us, the mPrep capsule methodology introduced here will be dis- cussed in relation to these known methods. Quality . As shown in Figures 3 – 6 , with mPrep capsule pro- cessing all specimens were well preserved and stained cleanly


www.microscopy-today.com • 2015 September


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