Fluorescence imaging systems are regularly characterized by their ability to distinguish varying concentrations of fluorophores in a solution or tissue phantom. However, there is inadequate standardization in the field for fluorescence characterization. In this study, we characterize a fluorescence imaging system developed for pathogen detection, regarding its ability to detect a near-infrared dye. During this process, we vary a number of key factors involved in fluorescence imaging, such as the excitation intensity, background level, working distance, volume of fluorescent solution, and type of container used to hold the fluorescent solution. We then analyze the results, with statistical rigor, to determine which factors result in significant changes in fluorescence detection. Notably, we found that using different types of containers to hold the dye solution can have a significant impact on fluorescence detection, while the effects of working distance and excitation intensity can vary. Based on our findings, greater standardization, or at least more thorough reporting of the experimental setup, is recommended to researchers when publishing characterization results of new imaging systems.
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Determineda ICG Concentrations to Achieve a Signal-to-Background Ratio of 1.2, 1.5 or 2.
Intensity (μW/cm2)
WD (cm)
24 Well
48 Well
Tube
SBR
1.2
1.5
2
1.2
1.5
2
1.2
1.5
2
250
20
21.67
55.12
114.29
35.31
103.86
207.95
11.90
29.51
58.33
40
24.10
60.52
120.59
40.30
109.24
205.93
11.45
27.41
54.08
60
24.15
62.93
122.96
38.94
105.80
205.29
11.73
28.08
55.74
500
20
12.36
25.86
54.78
14.73
52.65
112.61
6.72
16.43
32.92
40
13.69
26.39
55.03
19.60
52.01
109.65
5.95
14.24
28.41
60
14.07
26.69
55.62
20.33
49.93
108.35
6.13
14.97
29.36
1000
20
4.50
16.30
28.51
7.80
29.54
70.66
3.47
8.57
17.37
40
6.57
16.81
30.62
10.43
25.87
58.55
3.52
8.07
15.60
60
6.62
16.94
30.63
13.04
28.72
62.58
3.01
8.58
15.91
2000
20
3.88
11.12
19.48
6.70
16.93
48.35
2.35
5.50
10.79
40
4.02
11.19
20.40
7.07
18.52
36.03
1.83
4.50
8.77
60
4.37
11.29
20.59
5.67
16.29
26.97
1.89
4.78
9.67
4000
20
3.10
9.16
17.10
6.14
13.67
36.19
1.33
3.41
7.59
40
4.03
9.70
17.15
6.32
13.91
30.97
1.36
3.39
6.69
60
4.49
9.98
17.21
5.33
10.93
19.64
1.45
3.68
7.33
Testing was conducted using a range of excitation intensities and imaging working distances. The values listed were interpolated from averaged fluorescent intensity readings () taken on a series of dilutions. Different volumes of each dilution were placed into either 24-well plates at 2 mL, 48-well plates at 1 mL or centrifuge tubes at 2 mL for testing. Significant differences in concentrations were found between container type, SBR and intensity. Differences in concentrations between working distances were varied.
Significance, i.e. varying the value resulted in significantly different SBR results, was determined for dye concentration, excitation intensity and volumetric category. Variations in working distance were not found to be significant.
Table 3.
Statistical Grouping Information Using the Tukey Method (, ), for the Variable Factorsa Used During the Dark Room Study
Variable Factor
Values
Statistical Grouping
Variable Factor
Values
Statistical Grouping
Container
Tube
A
Dye concentration (Tube) (nM)
300
A
24-well
B
200
B
48-well
C
100
C
Working distance (tube) (cm)
60
A
50
D
40
A
25
D
E
20
A
12.5
E
F
Working distance (24-well) (cm)
60
A
6.25
E
F
40
A
3.125
E
F
20
A
0
F
Working distance (48-well) (cm)
60
A
Dye concentration (24-Well) (nM)
300
A
40
A
B
200
B
20
B
100
C
Excitation intensity (tube) ()
4000
A
50
D
2000
S
25
D
E
1000
B
12.5
E
500
B
C
6.26
E
250
C
3.125
E
Excitation intensity (24-well) ()
4000
A
0
E
2000
A
B
Dye concentration (48-Well) (nM)
300
A
1000
B
C
200
B
500
C
100
C
250
C
50
C
D
Excitation intensity (48-well) ()
4000
A
25
D
E
2000
A
B
12.5
D
E
1000
B
6.25
D
E
500
C
3.125
E
250
C
0
E
The working distance, excitation intensity, and dye concentration factors were conducted for each container type. Variable values within each factor category (i.e., working distance, excitation intensity, etc.) correspond to settings used during the study, in order to determine system fluorescence detection limits. During the experiments, each factor was varied over the listed range of values in order to determine whether changing the factor resulted in a significantly different detectable fluorescent intensity. The letters assigned to each value indicate to which statistical group the value belongs. Intrafactoral values, which were assigned the same letter or group, were not determined to cause significantly different fluorescence detection from each other. Values assigned to a different group letter, for the same factor, were determined to cause a significantly different fluorescence detection result.
Table 4.
Analysis of Variance for the Tissue Phantom Studya
Source
DF
Adj SS
Adj MS
F-Value
P-Value
Depth
4
276.49
69.122
95.03
0.00000000000
Distance
2
62.67
31.333
43.08
0.00000000000
Intensity
4
30.53
7.631
10.49
0.00000003571
Concentration
6
783.37
130.561
179.50
0.00000000000
Error
508
369.49
0.727
Total
524
1522.54
Factors resulting in at least some significantly different SBR results when varied included tissue depth, working distance, excitation intensity and dye concentration.
Table 5.
Statistical Grouping Information Using the Tukey Method (, ), for Variable Factorsa Within the Tissue Phantom Study
Variable Factor
Values
Statical Grouping
Volume (mL)
0.25
A
0.5
B
1
C
2
D
Tissue depth (mm)
0
A
1
B
3
C
5
C
10
D
Distance (cm)
20
A
40
B
60
B
Excitation intensity (uW)
500
A
1000
A
250
B
2000
B
4000
B
Dye concentration (nM)
50
A
62.5
B
125
C
250
D
500
E
1000
F
2000
G
Variable values within each factor category (i.e., volume, tissue depth, etc.) correspond to settings used during the study, in order to determine fluorescence dye detection limits. During the experiments, each factor was varied in order to determine whether changing that factor resulted in a significantly different detectable fluorescent intensity. The letters assigned to each value indicate to which statistical group the value belongs. Intrafactoral values, which were assigned the same letter or group, were not determined to cause significantly different fluorescence detection. Values assigned to a different group letter, for the same factor, were determined to cause a significantly different fluorescence detection result.
Tables (5)
Table 1.
Determineda ICG Concentrations to Achieve a Signal-to-Background Ratio of 1.2, 1.5 or 2.
Intensity (μW/cm2)
WD (cm)
24 Well
48 Well
Tube
SBR
1.2
1.5
2
1.2
1.5
2
1.2
1.5
2
250
20
21.67
55.12
114.29
35.31
103.86
207.95
11.90
29.51
58.33
40
24.10
60.52
120.59
40.30
109.24
205.93
11.45
27.41
54.08
60
24.15
62.93
122.96
38.94
105.80
205.29
11.73
28.08
55.74
500
20
12.36
25.86
54.78
14.73
52.65
112.61
6.72
16.43
32.92
40
13.69
26.39
55.03
19.60
52.01
109.65
5.95
14.24
28.41
60
14.07
26.69
55.62
20.33
49.93
108.35
6.13
14.97
29.36
1000
20
4.50
16.30
28.51
7.80
29.54
70.66
3.47
8.57
17.37
40
6.57
16.81
30.62
10.43
25.87
58.55
3.52
8.07
15.60
60
6.62
16.94
30.63
13.04
28.72
62.58
3.01
8.58
15.91
2000
20
3.88
11.12
19.48
6.70
16.93
48.35
2.35
5.50
10.79
40
4.02
11.19
20.40
7.07
18.52
36.03
1.83
4.50
8.77
60
4.37
11.29
20.59
5.67
16.29
26.97
1.89
4.78
9.67
4000
20
3.10
9.16
17.10
6.14
13.67
36.19
1.33
3.41
7.59
40
4.03
9.70
17.15
6.32
13.91
30.97
1.36
3.39
6.69
60
4.49
9.98
17.21
5.33
10.93
19.64
1.45
3.68
7.33
Testing was conducted using a range of excitation intensities and imaging working distances. The values listed were interpolated from averaged fluorescent intensity readings () taken on a series of dilutions. Different volumes of each dilution were placed into either 24-well plates at 2 mL, 48-well plates at 1 mL or centrifuge tubes at 2 mL for testing. Significant differences in concentrations were found between container type, SBR and intensity. Differences in concentrations between working distances were varied.
Significance, i.e. varying the value resulted in significantly different SBR results, was determined for dye concentration, excitation intensity and volumetric category. Variations in working distance were not found to be significant.
Table 3.
Statistical Grouping Information Using the Tukey Method (, ), for the Variable Factorsa Used During the Dark Room Study
Variable Factor
Values
Statistical Grouping
Variable Factor
Values
Statistical Grouping
Container
Tube
A
Dye concentration (Tube) (nM)
300
A
24-well
B
200
B
48-well
C
100
C
Working distance (tube) (cm)
60
A
50
D
40
A
25
D
E
20
A
12.5
E
F
Working distance (24-well) (cm)
60
A
6.25
E
F
40
A
3.125
E
F
20
A
0
F
Working distance (48-well) (cm)
60
A
Dye concentration (24-Well) (nM)
300
A
40
A
B
200
B
20
B
100
C
Excitation intensity (tube) ()
4000
A
50
D
2000
S
25
D
E
1000
B
12.5
E
500
B
C
6.26
E
250
C
3.125
E
Excitation intensity (24-well) ()
4000
A
0
E
2000
A
B
Dye concentration (48-Well) (nM)
300
A
1000
B
C
200
B
500
C
100
C
250
C
50
C
D
Excitation intensity (48-well) ()
4000
A
25
D
E
2000
A
B
12.5
D
E
1000
B
6.25
D
E
500
C
3.125
E
250
C
0
E
The working distance, excitation intensity, and dye concentration factors were conducted for each container type. Variable values within each factor category (i.e., working distance, excitation intensity, etc.) correspond to settings used during the study, in order to determine system fluorescence detection limits. During the experiments, each factor was varied over the listed range of values in order to determine whether changing the factor resulted in a significantly different detectable fluorescent intensity. The letters assigned to each value indicate to which statistical group the value belongs. Intrafactoral values, which were assigned the same letter or group, were not determined to cause significantly different fluorescence detection from each other. Values assigned to a different group letter, for the same factor, were determined to cause a significantly different fluorescence detection result.
Table 4.
Analysis of Variance for the Tissue Phantom Studya
Source
DF
Adj SS
Adj MS
F-Value
P-Value
Depth
4
276.49
69.122
95.03
0.00000000000
Distance
2
62.67
31.333
43.08
0.00000000000
Intensity
4
30.53
7.631
10.49
0.00000003571
Concentration
6
783.37
130.561
179.50
0.00000000000
Error
508
369.49
0.727
Total
524
1522.54
Factors resulting in at least some significantly different SBR results when varied included tissue depth, working distance, excitation intensity and dye concentration.
Table 5.
Statistical Grouping Information Using the Tukey Method (, ), for Variable Factorsa Within the Tissue Phantom Study
Variable Factor
Values
Statical Grouping
Volume (mL)
0.25
A
0.5
B
1
C
2
D
Tissue depth (mm)
0
A
1
B
3
C
5
C
10
D
Distance (cm)
20
A
40
B
60
B
Excitation intensity (uW)
500
A
1000
A
250
B
2000
B
4000
B
Dye concentration (nM)
50
A
62.5
B
125
C
250
D
500
E
1000
F
2000
G
Variable values within each factor category (i.e., volume, tissue depth, etc.) correspond to settings used during the study, in order to determine fluorescence dye detection limits. During the experiments, each factor was varied in order to determine whether changing that factor resulted in a significantly different detectable fluorescent intensity. The letters assigned to each value indicate to which statistical group the value belongs. Intrafactoral values, which were assigned the same letter or group, were not determined to cause significantly different fluorescence detection. Values assigned to a different group letter, for the same factor, were determined to cause a significantly different fluorescence detection result.