Upsala J Med Sci 84: 1-2, 1979 

Electron Probe Micro-X-ray Analyses of Electrolyte 
Composition of Fluid Microsamples 

by Use of a Sephadex Bead 
B. Ove Nilsson and Leif Ljung 

A o m  the Rrprodrrctron R e \ r u t t h  Unit, Biomrdrc t i /  Crntw Uppbuln, SIt e d r n  

Obtaining samples of certain body fluids for determination of the ionic 

content can involve great difficulties if the available amount of fluid is 

small and difficult to reach by a micropipette. For instance, samples of 

uterine secretions of rodents can not be obtained without a serious risk of 

contamination by interstitial fluid or cell sap. 

In order to circumvent these problems, we have developed a new technique 

where we use Sephadex beads as carriers of the fluid to be analyzed. In short, 

a hydrated Sephadex bead is inserted at the place for study and after an 

appropriate time for equlibration between fluid and bead, we have recovered 

the bead in a volatile, hydrophobe silicon oil and then prepared it for micro- 

X-ray analyses in Philips 400 electron microscope. This paper aims to give 

the details of the preparation technique used to obtain a qualitative 

information, the procedures for quantitative analyses will be reported later. 

We have taken the secretion of the mouse uterus as a model, but the technique 

is applicable also for analyses of other body fluids. 

I 

A few Sephadex G 150 beads (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 

are soaked in bidestilled water for about 15 minutes. Six to 8 hydrated beads 

of sizes around 100 um are selected and aspirated in a glass capillary, 

connected by a plastic tubing to a micrometer-controled syringe (Shardlow 

Micrometers Ltd, Sheffield, England). The capillary is laboratory made with an 

inner diameter slightly larger than that of the beads. By the syringe, the 

beads are positioned close to the capillary mouth to minimize the amount of 

water transferred together with the beads. The transfer of the beads is 

performed in a way appropriate for the purpose of the analyses. 

Equilibration between the body fluid and the water of the bead probably 

occurs within some hours, but this has to be tested for each application. 

When analysing the secretion of the mouse uterus, the X-ray spectra 

consequtively obtained displayed a similar configuration after a two-hour 

stay of the beads in the secretion. 

1-792854 I 



The recovery of the beads are made by flushing with a hydrophobe volatile 
silicone oil (DC 200, Dow Corning International Ltd., Brussels, Belgium) 
collecting the beads in a watch-glass. With a preparation microscope, the 

beads are observed in the oil, and single beads are transferred onto specimen 
grids which have been submerged into the oil. The grid with the beads is Slowly 

lifted out of the oil bath and left in room temperature for evaporation of the 
oil. The specimen grids used are nylon grids (Agar Aids, Stansted, England) 

mounted with a Formvar membrane. Grids prepared with Sephadex beads are carbon 

coated before the analyses. 

' 

Energy dispersive micro-X-ray analyses are performed with an Edax-Edith 

equipment attached to a Philips 400 electron microscope for 100 s at 40 kV 
with a fixed intensity and with a take off angle of 30'. The analysing area is 

slightly smaller than the circular projection of the spherical bead. Before the 

analyses, the preparations are checked and mapped with a scanning electron 

microscope (Figs. 1, 2). 

0 1 0 0 S E C  8 7 4 5 5 I N T  
V S : 2 5 0 0  H S :  1 0 E V I C H  

E D B X  

Fig. 1. Sephadex beads prepared 
on a nylon grid with a Formvar 
membrane support. X 900. 

Fig. 2. A representative X-ray 
spectrum from a Sephadex bead 
which was recovered from the 
uterus of a mouse after a 
3-hour stay in the secretion. 
The animal was in an experimen- 
tal delay of implantation and 
had been injected with oestro- 
gen 8 h before the preparation. 

Accepted November 28, 1978 

Address for reprints: 
Ove Nilsson 
Biomedical Centre 
Box 571 
S-751 23 Uppsala 
Sweden