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Digital image analysis of trypan blue and fluorescein staining of anterior lens capsules and intraocular lenses
Digital image analysis of trypan blue and fluorescein staining of anterior lens capsules and intraocular lenses Wilfram L. Fritz, MD Purpose: To study the intensity of trypan blue and fluorescein staining over time in anterior lens capsules and various intraocular lens (IOL) materials. Setting: Fritz Eye Associates, Geislingen, Germany. Methods: Excised anterior lens capsules and IOLs were exposed to trypan blue 0.1% and fluorescein 2% solution to assess the time correlation of color saturation. A technique of color differentiation using red⫺green⫺blue analysis was used. Results: In the anterior lens capsules, trypan blue saturation was 17% after 60 seconds and 71% after 24 hours; with fluorescein, there was no visible staining after 5 minutes and 17% saturation after 24 hours. Red and green light transmission was reduced with trypan blue and high with fluorescein. Blue light transmission was reduced with fluorescein. In poly(methyl methacrylate) and silicone IOLs, there was no to minimal staining and in acrylic IOLs, there was intense uptake of both dyes. Conclusions: The implications of this surgical technique are to use a short exposure time with trypan blue and avoid acrylic IOLs in cases of questionabale dye loss to the vitreous. Fluorescein should be used as a dye only when injected subcapsularly. J Cataract Refract Surg 2002; 28:1034 –1038 © 2002 ASCRS and ESCRS
S
ince the first report of anterior capsule staining with fluorescein for improved visualization1 and the subsequent modification of the technique with trypan blue,2 intraoperative dyes have gained wide acceptance. The use of dyes has been expanded to stain preretinal membranes3 and visualize corneal excision planes during surgical keratectomy.4 There is no consensus about the exposure time for optimal staining without unwanted delay or tissue damage. Furthermore, the use of intraocular lenses (IOLs) after a staining procedure raises the question of whether IOL materials can take up the dye. No study addressing this issue has been published. I describe a technique to Accepted for publication October 26, 2001. Reprint requests to Wilfram L. Fritz, MD, Ueberkingerstrasse 14, D 73312 Geislingen, Germany E-mail: [email protected]. © 2002 ASCRS and ESCRS Published by Elsevier Science Inc.
study tissue and IOL material behavior after staining with fluorescein and trypan blue.
Staining Technique Anterior lens capsules that had been excised during standard capsulorhexis were bathed in trypan blue 0.1% solution in a closed plastic container for 30 seconds, 60 seconds, 5 minutes, 30 minutes, 6 hours, and 24 hours. The same procedure was used to stain with fluorescein 2% solution. The tissue was then washed thoroughly for 2 minutes under a flow of balanced salt solution and put on a glass slide with a glass cover. Immediately afterward, digital photographs (Canon D 30, 3 million pixels, exposure time 1/30 second, 800 ISO) were taken using a Zeiss microscope adapter with ⫻1.2 zoom. An x-ray display was used to create a homoge0886-3350/02/$–see front matter PII S0886-3350(02)01238-5
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
neous white light source. The digital images were processed for color and brightness analysis. Adobe Photoshop 5.0 LE software, installed on Windows 98, was used to analyze the color saturation and image brightness and perform red⫺green⫺blue (RGB) light analysis in a region of interest in the tissue sample and in the center of the stained tissue. The region of interest was 5 pixels ⫻ 5 pixels. In a second investigation, a poly(methyl methacrylate) (PMMA) IOL, a hydrophilic silicone IOL, and hydrophilic acrylic IOLs with powers of ⫹25.5 diopters (D) and ⫺2.0 (D) were bathed in trypan blue 0.1% and in fluorescein 2% solution for 180 minutes. The same image analysis was performed.
Image Analysis Images were decoded at the region of interest for image brightness with 100% for a pure image of the light source and 0% for a black cover. Color saturation was analyzed using a saturation plot ranging from 0% (white) to 100% (black) in different color angles. These color angles are not given here; they pertain to the color of fluorescein and trypan blue. However, the color change is given using an RGB light figure, with a relation of 230⫺230⫺230 giving white light. With blue stain, the blue figure is high, whereas green and red are reduced; with yellow stain, the red and green figures are high and the blue is reduced. The change in the 3 numbers allows quantitative analysis of the color changes. A change in the relationship of the 3 numbers indicates a color shift and a reduction in all 3 numbers, a decrease in saturation.
Results In the anterior lens capsules, the saturation with trypan blue was 2% after 30 seconds and 17% after 1 minute. Thereafter, saturation increased only modestly, reaching 71% after 24 hours (Figures 1 and 2 and Table 1). Image brightness remained constant during this time. With fluorescein, there was no visible staining after 1 minute, 17% saturation after 5 minutes, and 30% saturation after 30 minutes; thereafter, no increase in stain saturation occurred (Figures 3 and 4 and Table 2). Red⫺green⫺blue analysis showed an intense and constant blue light fraction during trypan blue staining; the red and green decrease depended on the exposure time. With fluorescein, red was high, green was slightly less, and blue was strong. In the IOLs, there was no to minimal dye uptake in the silicone, slight uptake in the PMMA (Figure 5), and strong staining in the acrylic with trypan blue (Figure 6) and with fluorescein (Figure 7). Quantitative analysis showed a saturation of 4% in the silicone IOL, 6% in PMMA, 5% in the ⫺2.0 D acrylic, and 10% in the ⫹25.5 D acrylic. The RGB shifts were comparable to those seen in the tissue.
Discussion Several points can be inferred from the data. First, both dyes stain the lens capsule effectively. Trypan blue is superior as it gives a better contrast against a white lens, although the saturation is the same as with fluorescein. About 17% saturation is perceived by the surgeon as a good stain. This was reached after 1 minute with trypan glue and after 5 minutes with fluorescein. There-
Figure 1. (Fritz) Excised anterior lens capsules exposed to trypan blue 0.1%. A: After 1 minute. B: After 5 minutes. C: After 6 hours. D: After 24 hours.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
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LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Figure 2. (Fritz) Color saturation in anterior lens capsules exposed to trypan blue 0.1%.
Table 1. Color saturation of trypan blue 0.1% in anterior lens capsules over time. Saturation/ Color Saturation (%) Brightness (%)
Exposure Time 30 M 2
1M
0.5 H
6H
12 H
24 H
17
24
41
64
71
89
85
85
83
85
89
Red*
228
181
164
126
78
66
Green*
224
198
186
146
114
109
Blue*
227
218
216
212
218
226
*Maximum saturation is 230.
Figure 3. (Fritz) Excised anterior lens capsules exposed to fluorescein 2%. A: After 5 minutes. B: After 30 minutes. C: After 24 hours.
after, intense staining occurred with trypan blue, reaching 71% saturation after 24 hours. This means that lens capsule tissue purposely or inadvertently exposed to trypan blue for a prolonged time becomes impassable to white light. This can lead to severe problems if peripheral retinal disease develops and requires treatment. A short exposure of about 60 seconds and thorough wash1036
out thereafter is mandatory, although an exposure of less than 30 seconds provides unsatisfactory staining. Loss of dye to the vitreous can cause prologed exposure of tissue to the staining substance. A vitrectomy to remove all lost dye should be considered. Fluorescein behaved differently. Even after prolonged exposure, the capsules retained some translu-
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Table 2. Color saturation of fluorescein 2% in anterior lens capsules over time. Exposure Time Saturation/Color
5M
30 M
24 H
Saturation
17
30
26
Brightness
100
93
85
Red
255
237
216
Green
234
217
166
Blue
211
166
160
cency. Moreover, the small molecule was cleared rapidly even if some dye was left behind. However, the small contrast against white lenses makes its use difficult. A satisfactory stain is achieved only with a subcapsular application technique. The results of IOL exposure to trypan blue and fluorescein were surprising, although not unexpected. Poly(methyl methacrylate) and silicone materials have a dense polymer structure. This allows minimal staining
of the surface. Acrylic IOLs, however, take up the dye and stain intensely after prolonged exposure. In a surgical setting, such long exposure times do not occur and stained IOLs have not been reported as problems. However, in a complicated procedure, the staining behavior of IOL materials should be considered. Silicone or PMMA IOLs should be preferred if lost dye is a consideration. This is the first published description of a digital image analysis technique for quantitative analysis of stained tissue and IOL materials. It allows a detailed comparison of stain, color saturation, and relative and absolute changes in color and saturation using RGB light differentiation.
Conclusions Trypan blue effectively stained the anterior lens capsule after a short exposure time of 60 seconds. Fluorescein required about 5 minutes for visible staining. Prolonged exposure to trypan blue causes an intense stain, precluding passage of white light. Acrylic IOLs
Figure 4. (Fritz) Color saturation in anterior lens capsules exposed to fluorescein 2%.
Figure 5. (Ftitz) Intraocular lenses exposed to trypan blue 0.1% for 180 minutes: From left, ⫹25.5 D acrylic, ⫺2.0 D acrylic, ⫹20.0 D PMMA, and ⫹20.0 D silicone.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
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LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Figure 6. (Fritz) The ⫹20.0 D acrylic IOL after exposure to trypan blue 0.1% for 180 minutes.
Figure 7. (Fritz) The ⫹20.0 D acrylic IOL after exposure to fluores-
quickly took up both dyes and should be avoided in cases of questionable or lost dye.
3. Takeda M. [Technique for peeling of epiretinal membranes]. [Japanese] Jpn J Clin Ophthalmol 2001; 55: 72⫺73 4. Georgiadis N, Kardasopoulos A, Bufidis T. The evaluation of corneal graft tissue by the use of trypan blue. Ophthalmologica 1999; 213:8⫺11
References 1. Fritz WL. Fluorescein blue, light-assisted capsulorhexis for mature or hypermature cataract. J Cataract Refract Surg 1998; 24:19⫺20 2. Melles GRJ, de Waard PWT, Pameyer JH, Beekhuis WH. Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7⫺9
1038
cein 2% for 180 minutes.
From Geislingen, Germany. The author does not have a financial interest in any product mentioned.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
S
ince the first report of anterior capsule staining with fluorescein for improved visualization1 and the subsequent modification of the technique with trypan blue,2 intraoperative dyes have gained wide acceptance. The use of dyes has been expanded to stain preretinal membranes3 and visualize corneal excision planes during surgical keratectomy.4 There is no consensus about the exposure time for optimal staining without unwanted delay or tissue damage. Furthermore, the use of intraocular lenses (IOLs) after a staining procedure raises the question of whether IOL materials can take up the dye. No study addressing this issue has been published. I describe a technique to Accepted for publication October 26, 2001. Reprint requests to Wilfram L. Fritz, MD, Ueberkingerstrasse 14, D 73312 Geislingen, Germany E-mail: [email protected]. © 2002 ASCRS and ESCRS Published by Elsevier Science Inc.
study tissue and IOL material behavior after staining with fluorescein and trypan blue.
Staining Technique Anterior lens capsules that had been excised during standard capsulorhexis were bathed in trypan blue 0.1% solution in a closed plastic container for 30 seconds, 60 seconds, 5 minutes, 30 minutes, 6 hours, and 24 hours. The same procedure was used to stain with fluorescein 2% solution. The tissue was then washed thoroughly for 2 minutes under a flow of balanced salt solution and put on a glass slide with a glass cover. Immediately afterward, digital photographs (Canon D 30, 3 million pixels, exposure time 1/30 second, 800 ISO) were taken using a Zeiss microscope adapter with ⫻1.2 zoom. An x-ray display was used to create a homoge0886-3350/02/$–see front matter PII S0886-3350(02)01238-5
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
neous white light source. The digital images were processed for color and brightness analysis. Adobe Photoshop 5.0 LE software, installed on Windows 98, was used to analyze the color saturation and image brightness and perform red⫺green⫺blue (RGB) light analysis in a region of interest in the tissue sample and in the center of the stained tissue. The region of interest was 5 pixels ⫻ 5 pixels. In a second investigation, a poly(methyl methacrylate) (PMMA) IOL, a hydrophilic silicone IOL, and hydrophilic acrylic IOLs with powers of ⫹25.5 diopters (D) and ⫺2.0 (D) were bathed in trypan blue 0.1% and in fluorescein 2% solution for 180 minutes. The same image analysis was performed.
Image Analysis Images were decoded at the region of interest for image brightness with 100% for a pure image of the light source and 0% for a black cover. Color saturation was analyzed using a saturation plot ranging from 0% (white) to 100% (black) in different color angles. These color angles are not given here; they pertain to the color of fluorescein and trypan blue. However, the color change is given using an RGB light figure, with a relation of 230⫺230⫺230 giving white light. With blue stain, the blue figure is high, whereas green and red are reduced; with yellow stain, the red and green figures are high and the blue is reduced. The change in the 3 numbers allows quantitative analysis of the color changes. A change in the relationship of the 3 numbers indicates a color shift and a reduction in all 3 numbers, a decrease in saturation.
Results In the anterior lens capsules, the saturation with trypan blue was 2% after 30 seconds and 17% after 1 minute. Thereafter, saturation increased only modestly, reaching 71% after 24 hours (Figures 1 and 2 and Table 1). Image brightness remained constant during this time. With fluorescein, there was no visible staining after 1 minute, 17% saturation after 5 minutes, and 30% saturation after 30 minutes; thereafter, no increase in stain saturation occurred (Figures 3 and 4 and Table 2). Red⫺green⫺blue analysis showed an intense and constant blue light fraction during trypan blue staining; the red and green decrease depended on the exposure time. With fluorescein, red was high, green was slightly less, and blue was strong. In the IOLs, there was no to minimal dye uptake in the silicone, slight uptake in the PMMA (Figure 5), and strong staining in the acrylic with trypan blue (Figure 6) and with fluorescein (Figure 7). Quantitative analysis showed a saturation of 4% in the silicone IOL, 6% in PMMA, 5% in the ⫺2.0 D acrylic, and 10% in the ⫹25.5 D acrylic. The RGB shifts were comparable to those seen in the tissue.
Discussion Several points can be inferred from the data. First, both dyes stain the lens capsule effectively. Trypan blue is superior as it gives a better contrast against a white lens, although the saturation is the same as with fluorescein. About 17% saturation is perceived by the surgeon as a good stain. This was reached after 1 minute with trypan glue and after 5 minutes with fluorescein. There-
Figure 1. (Fritz) Excised anterior lens capsules exposed to trypan blue 0.1%. A: After 1 minute. B: After 5 minutes. C: After 6 hours. D: After 24 hours.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
1035
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Figure 2. (Fritz) Color saturation in anterior lens capsules exposed to trypan blue 0.1%.
Table 1. Color saturation of trypan blue 0.1% in anterior lens capsules over time. Saturation/ Color Saturation (%) Brightness (%)
Exposure Time 30 M 2
1M
0.5 H
6H
12 H
24 H
17
24
41
64
71
89
85
85
83
85
89
Red*
228
181
164
126
78
66
Green*
224
198
186
146
114
109
Blue*
227
218
216
212
218
226
*Maximum saturation is 230.
Figure 3. (Fritz) Excised anterior lens capsules exposed to fluorescein 2%. A: After 5 minutes. B: After 30 minutes. C: After 24 hours.
after, intense staining occurred with trypan blue, reaching 71% saturation after 24 hours. This means that lens capsule tissue purposely or inadvertently exposed to trypan blue for a prolonged time becomes impassable to white light. This can lead to severe problems if peripheral retinal disease develops and requires treatment. A short exposure of about 60 seconds and thorough wash1036
out thereafter is mandatory, although an exposure of less than 30 seconds provides unsatisfactory staining. Loss of dye to the vitreous can cause prologed exposure of tissue to the staining substance. A vitrectomy to remove all lost dye should be considered. Fluorescein behaved differently. Even after prolonged exposure, the capsules retained some translu-
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Table 2. Color saturation of fluorescein 2% in anterior lens capsules over time. Exposure Time Saturation/Color
5M
30 M
24 H
Saturation
17
30
26
Brightness
100
93
85
Red
255
237
216
Green
234
217
166
Blue
211
166
160
cency. Moreover, the small molecule was cleared rapidly even if some dye was left behind. However, the small contrast against white lenses makes its use difficult. A satisfactory stain is achieved only with a subcapsular application technique. The results of IOL exposure to trypan blue and fluorescein were surprising, although not unexpected. Poly(methyl methacrylate) and silicone materials have a dense polymer structure. This allows minimal staining
of the surface. Acrylic IOLs, however, take up the dye and stain intensely after prolonged exposure. In a surgical setting, such long exposure times do not occur and stained IOLs have not been reported as problems. However, in a complicated procedure, the staining behavior of IOL materials should be considered. Silicone or PMMA IOLs should be preferred if lost dye is a consideration. This is the first published description of a digital image analysis technique for quantitative analysis of stained tissue and IOL materials. It allows a detailed comparison of stain, color saturation, and relative and absolute changes in color and saturation using RGB light differentiation.
Conclusions Trypan blue effectively stained the anterior lens capsule after a short exposure time of 60 seconds. Fluorescein required about 5 minutes for visible staining. Prolonged exposure to trypan blue causes an intense stain, precluding passage of white light. Acrylic IOLs
Figure 4. (Fritz) Color saturation in anterior lens capsules exposed to fluorescein 2%.
Figure 5. (Ftitz) Intraocular lenses exposed to trypan blue 0.1% for 180 minutes: From left, ⫹25.5 D acrylic, ⫺2.0 D acrylic, ⫹20.0 D PMMA, and ⫹20.0 D silicone.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002
1037
LABORATORY SCIENCE: DIGITAL IMAGE ANALYSIS OF TRYPAN BLUE AND FLUORESCEIN SATURATION
Figure 6. (Fritz) The ⫹20.0 D acrylic IOL after exposure to trypan blue 0.1% for 180 minutes.
Figure 7. (Fritz) The ⫹20.0 D acrylic IOL after exposure to fluores-
quickly took up both dyes and should be avoided in cases of questionable or lost dye.
3. Takeda M. [Technique for peeling of epiretinal membranes]. [Japanese] Jpn J Clin Ophthalmol 2001; 55: 72⫺73 4. Georgiadis N, Kardasopoulos A, Bufidis T. The evaluation of corneal graft tissue by the use of trypan blue. Ophthalmologica 1999; 213:8⫺11
References 1. Fritz WL. Fluorescein blue, light-assisted capsulorhexis for mature or hypermature cataract. J Cataract Refract Surg 1998; 24:19⫺20 2. Melles GRJ, de Waard PWT, Pameyer JH, Beekhuis WH. Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999; 25:7⫺9
1038
cein 2% for 180 minutes.
From Geislingen, Germany. The author does not have a financial interest in any product mentioned.
J CATARACT REFRACT SURG—VOL 28, JUNE 2002