Lab Index > The Size of Things

The Size of Things:

Calibration & Scale Estimates

Jeff Peng • Elena Hartoonian • Rosalyn Sayaman

CALIBRATION

A standard "Resolution Test Target" (RES-1) calibration slide is used to calibrate microscopic images. The target is divided into 8 groups (Group 0-8), and each group is comprised of 6 elements (Element 1-6). Each element has a rating which gives the density of lines in lines per millimeter. Therefore, the periodicity of the lines in each element is 1/density.

See Specifications table below.

Source: www.newport.com.

Specifications

RES-1  Clear lines on an opaque chrome background (2.0 OD), optical glass substrate 50 x 50 x 1.5 mm 
RES-2  Black lines on heavy white paper, 50 x 50 mm 
Line Pairs per millimeter 
Element Number  Group Number 
0  1  2  3  4  5  6  7 
1  1.00  2.00  4.00  8.00  16.00  32.00  64.00  128.00 
2  1.12  2.24  4.49  8.98  17.96  35.92  71.84  143.70 
3  1.26  2.52  5.04  10.08  20.16  40.32  80.63  161.30 
4  1.41  2.83  5.66  11.31  22.63  45.25  90.51  181.00 
5  1.59  3.17  6.35  12.70  25.40  50.80  101.60  203.20 
6  1.78  3.56  7.13  14.25  28.51  57.02  114.00  228.10 
Line pairs/mm = LP  Line width (mm) = 1/(2LP)   
Space width (mm) = 1/(2LP)  Line length = 5(line width)   
 

For Instance: Group 7 Element 2 at 100x

Group 7 Element 2 has a rating of  143.7 density/mm, so that 1 period = 1 mm/density = 0.0070 mm = 7.0 um.

A period is defined as seen in the image to the right.

 

The following are images taken of the "Resolution Test Target" during our experiment, our goal is to derive an appropriate conversion factor of microns per pixel for measuring the scale of microscopic cells. Pixel resolution is measured in Matlab and the resulting Pixels/Period is the averaged value of 3 measurements. All images displayed below have been reduced by 50% for display purposes.

Magnification Image Period Pixels/Period 1 pixel/mm

100x

Group 6 Element 5

 

 1 mm/101.60 149.2324 pixel/period 1.52e+004

100x

Group 6 Element 6

 

 1 mm/114.00 134.5757 pixel/period 1.53e+004

100x

Group 7 Element 1

 

 1 mm/128.00 119.9310 pixel/period 1.54e+004

100x

Group 7 Element 2

 

 1 mm/143.70  105.9526 pixel/period 1.52e+004

100x

Group 7 Element 3

 

 1 mm/161.30 95.9448 pixel/period 1.55e+004
 
Average pixel/mm
1.53e+004 pixel/mm
Average pixel/um
15.3 pixel/um
 
 
SCALE ESTIMATES: YEAST AND E.COLI

20x

Image displayed reduced by 50%.

100 x

Image displayed reduced by 50%.
 
Estimates of Size
E.coli
Cell # Length (pixel) Length (um) Diameter (pixel) Diameter (um) Volume Cylinder: l*pi*r2 (um3)
1 26.1 1.7 9.5 0.6 0.48
2 33.2 2.2 8.1 0.5 0.43
3 16.9 1.1 10.9 0.7 0.42
4 16.6 1.1 8.8 0.6 0.31
5  21.8 1.4 9.4 0.6 0.40
Avg 22.9 1.5 9.3 0.6 0.41
Yeast
Cell # Diameter (pixel) Diameter (um) Volume Sphere: (4/3)*pi*r3 (um3)
1 73.6 4.8 57.9
2 85.8 5.6 92.0
3 77.7 5.1 65.4
Avg 79.0 5.2 71.8