Titanium Phosphate Glass Microspheres as Microcarriers for In Vitro Bone Cell Tissue Engineering - Biointerfaces: Where Material Meets Biology - page 119

v. Set the calibration scale: Go to Analyse | Set Scale and input ap-
propriate values for Distance in Pixels and Known Distance.
Maintain Pixel Aspect Ratio ¼ 1.0 and Unit of Length ¼ micron.
Make sure that the box next to Global is ticked. Press OK.

d n 3 r 4 n g | 3

NOTE: If the calibration values are not known, use a micrometer
calibration slide to measure a known distance in pixels. After proper
focusing, draw a line on the screen between two divisions on the
slide and measure the length in pixel units by going to Analyse |
Measure. The output measurement is the Distance in Pixels and the
distance between the divisions is the Known Distance in the Set
Scale window.

vi. Measure the diameters in the first and last images: Measure the
diameter of the sphere in the first image of your sequence by
pressing on the circle icon next to the rectangular icon, repeating
Step (iii) to draw a circle roughly around the sphere circumference,
and going to Analyse | Measure. Make a mental note of the ap-
proximate area (say, 22788.789). Repeat the same step for the last
image to get the approximate area (say, 20742.123). Approxi-
mations of both values are needed for the next step.
vii. Obtain the bead diameter: Go to Analyze | Analyze Particles and use
the following values: Size (micron 2 ) ¼ 20 000-25 000, Circularity:
0.0-1.0, Show: Outlines (from drop-down menu). Tick the fol-
lowing boxes: Display Results, Summarize, Exclude on Edges, In-
clude Holes, Record Starts. Press OK on the window that comes up.
Three new windows will appear on the screen. Go to the Results
window. Then go to Edit | Select All and Edit | Copy in order to
paste the values in a suitable spread sheet software.
6. Measure the diameters of three or more microspheres by the above
method and calculate the mass of the sphere by the equation:

.

m sphere ¼ r glass v sphere ¼ r glass 4p(r sphere ) 3 /3

(5.1)

where m sphere is the mass of the sphere (mg); r glass is the density of the
glass (mg mm 3 ); v sphere is the volume of the sphere (mm 3 ); and r sphere is
the radius of the sphere (mm).
7. Calculate the percentage weight loss per unit surface area of the sphere
at each time point using the following equation:

[(m sphere(0) m sphere(t) )/m sphere(0) A t ] 100 (5.2)

where m sphere(0) is the mass of the microsphere at time t ¼ 0(mg);
m sphere(t) is the mass of the microsphere at time t (mg); and A t is the
surface area of the microsphere at time t (mm 2 ).