Antonio Virgilio Failla,
Udo Spoeri,
Benno Albrecht,
Alexander Kroll,
and Christoph Cremer
The authors are with Applied Optics and Information Processing, Kirchhoff Institute for Physics, University of Heidelberg, Im Nevenheimer Feld 227, 69120 Heidelberg, Germany.
C. Cremer (christoph.cremer@kip.uni-heidelberg.de) is also with the Interdisciplinary Centre for Scientific Computing (IWR), University of Heidelberg.
Antonio Virgilio Failla, Udo Spoeri, Benno Albrecht, Alexander Kroll, and Christoph Cremer, "Nanosizing of fluorescent objects by spatially modulated illumination microscopy," Appl. Opt. 41, 7275-7283 (2002)
A new approach to measuring the sizes of small fluorescent objects by use of spatially modulated illumination (SMI) far-field light microscopy is presented. This method is based on SMI measurements combined with a new SMI virtual microscopy (VIM) data analysis calibration algorithm. Here, experimental SMI measurements of fluorescent objects with known diameter (size) were made. From the SMI data obtained, the size was determined in an independent way by use of the SMI VIM algorithm. The results showed that with SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy.
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Here the results of size measurements performed on 40-nm ∅ red beads, 50-nm ∅ green beads, 90-nm ∅ red beads, and 100-nm ∅ green beads are shown. In the first column the actual diameter values of the beads given by the manufacturer are listed (true size). In the second column, the results of size measurements when the background noise was automatically subtracted from the 3D data before the AID computation (wbg) are presented. In the third column the results of size measurements without any previous subtraction of background noise before the AID computation (bg) are presented. Ntot, total number of measurements.
Mean of ensemble plus or minus standard deviation (SD) (see Fig.
5).
Table 2
Numerical Presentation of Nanosizing Results from 50-nm Green Beadsa
N
True Size, -50 nm
ntot
wbg (nm)
bg (nm)
1
54.5 ± 2.3
53.9 ± 2.3
20
2
50.4 ± 1.7
52.6 ± 2.3
22
3
53.7 ± 2.8
53.7 ± 2.2
27
4
54.8 ± 2.0
55.3 ± 2.0
24
Here, the results summarized in Fig.
7 are presented in detail. N indicates a single independent data stack acquisition of an entire field of view (for more details see Section 3). The second column denotes mean values plus or minus the SDM of the size evaluations performed on each data stack (N = 1, 2, 3, 4) when the background noise was subtracted from the 3D data before the AID was evaluated (wbg); the third column denotes mean values plus or minus the SDM of the size evaluations performed on each data stack when the background noise was not subtracted from the 3D data before the AID was evaluated (bg). For example, in the data stack N = 1, twenty single objects in the field of view were registered by SMI microscopy. The mean size (wbg) of these objects was determined to be 54.5 ± 2.3 nm (SDM). In data stack N = 2, twenty-two objects in the field of view were measured; in this case, the mean size (wbg) measured was 50.4 ± 1.7 nm (SDM).
Table 3
Numerical Presentation of Nanosizing Measurements Performed on Singular 40-nm ∅ and 90-nm ∅ Red Beads and Singular 100-nm ∅ Green Singular Beadsa
Nb
40r (nm), λex = 647 nm
90r (nm), λex = 647 nm
100g (nm), λex = 488 nm
1
27.6 ± 8.8
31 ± 11
91.4 ± 3.6
91.6 ± 3.8
93.1 ± 5.0
91.7 ± 4.8
2
20.6 ± 7.2
29.8 ± 7.8
96.3 ± 3.0
96.5 ± 3.2
96.9 ± 8.0
93.9 ± 6.5
3
26.2 ± 6.8
35.6 ± 8.0
92.0 ± 5.1
92.6 ± 5.8
90.9 ± 4.2
89.8 ± 5.1
4
42 ± 14
44 ± 13
89.4 ± 7.2
91 ± 10
105.9 ± 5.7
102.8 ± 4.7
5
28.9 ± 9.2
31.3 ± 9.3
79.8 ± 9.5
75 ± 11
96.9 ± 7.6
95.3 ± 5.6
6
20.7 ± 7.8
29.4 ± 7.6
78.2 ± 6.1
73.3 ± 9.9
97.9 ± 4.4
96.0 ± 3.9
7
36.8 ± 8.4
39.8 ± 4.8
83.1 ± 5.0
82.7 ± 5.3
102.6 ± 8.8
100.3 ± 7.1
8
27 ± 13
34.5 ± 6.8
72.6 ± 6.9
70.9 ± 7.8
9
20.0 ± 8.3
26.4 ± 7.2
93.8 ± 5.9
93.5 ± 6.6
10
32 ± 11
35.1 ± 9.9
91.7 ± 5.1
88.9 ± 8.4
11
35 ± 11
34.7 ± 8.9
66.7 ± 7.4
60 ± 10
12
56.9 ± 5.3
63.2 ± 4.3
84.6 ± 3.7
83.6 ± 9.2
13
31 ± 11
28.6 ± 9.2
85 ± 11
81 ± 13
14
91.9 ± 4.9
92.0 ± 4.9
15
86.5 ± 6.9
86.5 ± 6.9
A
B
A
B
A
B
In this table the results of single bead size evaluation (cf. Fig.
4) of 40-nm ∅ and 90-nm ∅ red beads and 100-nm ∅ green beads are presented. In each data stack the single beads were arbitrarily labeled with the number Nb (first column). Columns A show the results of size evaluation when the background noise was automatically subtracted before the AID was measured (Swbg); columns B show the same evaluations when no background noise had been subtracted before the AID was measured (Sbg).
Here the results of size measurements performed on 40-nm ∅ red beads, 50-nm ∅ green beads, 90-nm ∅ red beads, and 100-nm ∅ green beads are shown. In the first column the actual diameter values of the beads given by the manufacturer are listed (true size). In the second column, the results of size measurements when the background noise was automatically subtracted from the 3D data before the AID computation (wbg) are presented. In the third column the results of size measurements without any previous subtraction of background noise before the AID computation (bg) are presented. Ntot, total number of measurements.
Mean of ensemble plus or minus standard deviation (SD) (see Fig.
5).
Table 2
Numerical Presentation of Nanosizing Results from 50-nm Green Beadsa
N
True Size, -50 nm
ntot
wbg (nm)
bg (nm)
1
54.5 ± 2.3
53.9 ± 2.3
20
2
50.4 ± 1.7
52.6 ± 2.3
22
3
53.7 ± 2.8
53.7 ± 2.2
27
4
54.8 ± 2.0
55.3 ± 2.0
24
Here, the results summarized in Fig.
7 are presented in detail. N indicates a single independent data stack acquisition of an entire field of view (for more details see Section 3). The second column denotes mean values plus or minus the SDM of the size evaluations performed on each data stack (N = 1, 2, 3, 4) when the background noise was subtracted from the 3D data before the AID was evaluated (wbg); the third column denotes mean values plus or minus the SDM of the size evaluations performed on each data stack when the background noise was not subtracted from the 3D data before the AID was evaluated (bg). For example, in the data stack N = 1, twenty single objects in the field of view were registered by SMI microscopy. The mean size (wbg) of these objects was determined to be 54.5 ± 2.3 nm (SDM). In data stack N = 2, twenty-two objects in the field of view were measured; in this case, the mean size (wbg) measured was 50.4 ± 1.7 nm (SDM).
Table 3
Numerical Presentation of Nanosizing Measurements Performed on Singular 40-nm ∅ and 90-nm ∅ Red Beads and Singular 100-nm ∅ Green Singular Beadsa
Nb
40r (nm), λex = 647 nm
90r (nm), λex = 647 nm
100g (nm), λex = 488 nm
1
27.6 ± 8.8
31 ± 11
91.4 ± 3.6
91.6 ± 3.8
93.1 ± 5.0
91.7 ± 4.8
2
20.6 ± 7.2
29.8 ± 7.8
96.3 ± 3.0
96.5 ± 3.2
96.9 ± 8.0
93.9 ± 6.5
3
26.2 ± 6.8
35.6 ± 8.0
92.0 ± 5.1
92.6 ± 5.8
90.9 ± 4.2
89.8 ± 5.1
4
42 ± 14
44 ± 13
89.4 ± 7.2
91 ± 10
105.9 ± 5.7
102.8 ± 4.7
5
28.9 ± 9.2
31.3 ± 9.3
79.8 ± 9.5
75 ± 11
96.9 ± 7.6
95.3 ± 5.6
6
20.7 ± 7.8
29.4 ± 7.6
78.2 ± 6.1
73.3 ± 9.9
97.9 ± 4.4
96.0 ± 3.9
7
36.8 ± 8.4
39.8 ± 4.8
83.1 ± 5.0
82.7 ± 5.3
102.6 ± 8.8
100.3 ± 7.1
8
27 ± 13
34.5 ± 6.8
72.6 ± 6.9
70.9 ± 7.8
9
20.0 ± 8.3
26.4 ± 7.2
93.8 ± 5.9
93.5 ± 6.6
10
32 ± 11
35.1 ± 9.9
91.7 ± 5.1
88.9 ± 8.4
11
35 ± 11
34.7 ± 8.9
66.7 ± 7.4
60 ± 10
12
56.9 ± 5.3
63.2 ± 4.3
84.6 ± 3.7
83.6 ± 9.2
13
31 ± 11
28.6 ± 9.2
85 ± 11
81 ± 13
14
91.9 ± 4.9
92.0 ± 4.9
15
86.5 ± 6.9
86.5 ± 6.9
A
B
A
B
A
B
In this table the results of single bead size evaluation (cf. Fig.
4) of 40-nm ∅ and 90-nm ∅ red beads and 100-nm ∅ green beads are presented. In each data stack the single beads were arbitrarily labeled with the number Nb (first column). Columns A show the results of size evaluation when the background noise was automatically subtracted before the AID was measured (Swbg); columns B show the same evaluations when no background noise had been subtracted before the AID was measured (Sbg).