Pathogens and Disease Advance Access published February 5, 2015
1
Title Page
2
Title:
3
Autofluorescence in Samples Obtained from Chronic Biofilm Infections - “All
4
that glitters is not gold”
5 6
Running Title: Autofluorescence in Chronic biofilm infections
7
Authors:
8
*M.Sc. Steffen Eickhardt1, 2, [[email protected]]
9
M.Sc. Kasper Kragh1, 2[[email protected]]
10
MD Stine Schrøder3 [[email protected]]
11
Ph.D. Steen Seier Poulsen4 [[email protected]]
12
MD, DMSc . Henrik Sillesen5 [[email protected]]
13
Professor Dsc. Ph.D. Michael Givskov 6 [[email protected]]
14
Professor DMSc. Niels Høiby1, 2, [[email protected]]
15
Professor Ph.D. Thomas Bjarnsholt1, 2, [[email protected]]
16
Ph.D. Morten Alhede1, 2, [[email protected]]
17 18
1: Costerton Biofilm Center, Department of International Health, Immunology &
19
Microbiology, The faculty of Health and Medical Sciences, The University of
20
Copenhagen
21
2: The Department of Clinical Microbiology, Rigshospitalet, Denmark
22
3: Ear Nose Throat Department, North Zealand Hospital, Denmark
23
4: The Department of Biomedicine, The faculty of Health and Medical Sciences, The
24
University of Copenhagen
25
5: Dept. of Vascular Surgery, Rigshospitalet, Univ. of Copenhagen
26
6: Singapore Centre on Environmental Life Sciences Engineering (SCELSE),
27
Nanyang Technological University, Singapore
28 29 30
Corresponding Author
31
Steffen Eickhardt
32
Costerton Biofilm Center, Department of International Health, Immunology &
33
Microbiology, The faculty of Health and Medical Sciences, The University of
34
Copenhagen
35
Blegdamsvej 3
36
2200 Copenhagen N
37
Denmark
38 39
Telephone: +45 353 26657
40
Fax: +45 353 27853
41
E-mail: [email protected]
42 43
Word Count:
44
Abstract: 102
45
Manuscript: 1334
46
46 47
Autofluorescence in Samples Obtained from Chronic Biofilm Infections - “All
48
that glitters is not gold”
49
*Steffen Eickhardt1, 2, Kasper Kragh1, 2, Stine Schrøder3, Steen Seier Poulsen4,
50
Henrik Hegaard Sillesen5, Michael Givskov 6, Niels Høiby1, 2. Thomas Bjarnsholt1, 2 &
51
Morten Alhede1, 2
52
Abstract
53
When looking at tissue sections of ex vivo samples, autofluorescence can be a major
54
cause of artifacts and misinterpretations. We here reiterate evidence that
55
autofluorescing granules; often hemosiderin but also ceroid or mucinogen granules
56
are severe obstacles when imaging and diagnosing biofilm infections through
57
fluorescent imaging techniques. We used CLSM with spectral analysis for
58
autofluorescence detection as well as standard histological stains in order to identify
59
the culprit and show that these granules might very well be mistaken for bacterial
60
biofilms. Furthermore, we hypothesize that the increased amount of autofluorescing
61
granules may be a consequence of prolonged inflammation as a consequence of
62
chronic biofilm infections.
63 64
Autofluorescing granules can easily be mistaken as bacterial biofilm.
65
Several techniques are available for detecting bacteria within clinical samples.
66
However, in order to characterize and establish whether a biofilm is present,
67
microscopy
68
accompanied by specific histological stains as well as electron microscopy has been
69
used. Since the development of fluorescence based microscopy and the confocal
70
laser-scanning microscope, more researchers are drawn to this method.
71
However, when studying clinical samples autofluorescence is a problem. The
72
autofluorescence is often present as granules of approximately 1 micron in
73
diameter(1). These granules are spherical and grouped in clusters ranging from 10 to
74
1000 granula, which highly resembles the composition and structure of the in vivo
75
biofilm. In clinical samples, the bacterial biofilm is a tightly packed structure rarely
76
comprised of more than 103 bacteria. The size of most in vivo biofilm is between 10
77
and 500 bacteria(2). Because of the similarities between the autofluorescing
78
granules and the in vivo biofilm, the autofluorescing granules can easily be mistaken
79
as bacterial biofilm. An example of this can be seen in figure 1, a biopsy from the
is
often
preferred.
Traditionally,
conventional
light
microscopy
80
glandula submandibularis in a patient suffering from sialolithiasis, salivary gland
81
stones.
82
The granules are often composed of cellular debris and break-down products as lipid
83
residues, oxidative modified protein, carbohydrates(3) as well as metals, primarily
84
iron (4,5). The autofluorescing granules have an emission maximum within the
85
orange spectrum, resulting in a yellow to golden emission (6) as can been seen on
86
figure 1c. However, this highly depends on the excitation used (7) and on the origin
87
of the tissue (8). The granules can be observed both intracellular and extracellular.
88
When intracellular, the granules are often located within the perinuclear space, the
89
peribilecanicular region and cytosol (9) as well as the secondary lysosomes and
90
lysosomal compartments (10).
91
That autofluorescence is present in tissue sections is hardly a surprise for anyone
92
who has worked with fluorescent microscopy. The major causes, lipofuscin,
93
hemosiderin and ceroid are well studied and described (11–13). Yet we speculate
94
that autofluorescing granulas can be unintentionally misinterpreted as biofilms and
95
presented as such in publications. This speculation is derived by biofilm images
96
that are presented in low-resolution and are often overexposed to a degree where
97
size, uniformity and morphology of the bacteria are not readily visualized.
98 99
Our aim of this article is to share our experiences, for other biofilm researchers to
100
avoid the pitfalls we have encountered, by: 1) showing that autofluorescing granules
101
are present in chronic biofilm infections. 2) Describing what the autofluorescing
102
granules are 3) showing that these granules easily can be mistaken as biofilm and
103
most importantly 4) introduce guidelines to how to distinguish between biofilms and
104
autofluorescent artifacts.
105 106
Methods:
107
We have investigated ex vivo samples from several types of chronic biofilm
108
infections: as chronic wounds(14), the sinus of cystic fibrosis patients (15), the lungs
109
of cystic fibrosis patients(16) and atherosclerotic plagues. A biopsy from cirrhosis of
110
the liver, known for high amounts of lipofuscin granula, was used as a positive
111
control for autofluorescing granules.
112
We applied histology, conventional light microscopy as well as fluorescent bacterial
113
probes (PNA-FISH) and Confocal Laser Scanning Microscopy with spectral analysis.
114
Consecutive sections were stained both for the presence of bacteria as well as the
115
most described autofluorescing granule, lipofuscin. The fluorescent bacterial stain
116
was PNA-FISH probes targeting Staphylococcus aureus (green) and general Uni-
117
bacterial probes (red) (AdvanDx, Woburn, USA). 4', 6-diamidino-2-phenylindol
118
(DAPI) was used as a counter stain to detect DNA. Sudan Black B and KMnO4 were
119
used as quenchers of autofluorescence along with the fluorescent bacterial probes
120
and DAPI. In order to detect lipofuscin, three specific stains were used according to
121
Bancroft and Gamble 2008 (17). Peroidic Acid Shiff was used in order to detect
122
glycoproteins, Schmorl’s reaction to determine the oxidative state of the sample and
123
Long Ziehl-Neelson to detect acid fastness of the granules. Granules which were
124
positive for three stains as well as yellow golden autofluorescence was considered to
125
be lipofuscin.
126 127
Results:
128
As expected, histology revealed that the positive control from the cirrhotic liver was
129
indeed positive for lipofuscin granules. In three of the four chronic infection samples,
130
the cystic fibrosis lung tissue, the cystic fibrosis sinus tissue and the chronic wound
131
tissue we observed both autofluorescence as well as bacterial biofilms. The histology
132
results showed, that the major culprit of autofluorescing granules in these chronic
133
biofilm infected tissue sections was hemosiderin. No lipofuscin was found in any of
134
the samples. We observed that there appears to be a connection between the
135
severity of the immune response and the amount of hemosiderin present in the
136
sample. Granules such as ceroid and mucinogene granules were also present.
137
Quenchers such as KMnO4 and Sudan Black B were found to work well in removing
138
autofluorescence. However, using quenchers on the samples often ruined
139
histological interpretation and the emission of the studied fluorophores was (i.e. the
140
stain of the actual biofilm) reduced to unusable amounts.
141
Autofluorescent granules and a true biofilm can be seen in figure 1, a tissue sample
142
from the glandula submandibularis in a patient suffering from sialolithiasis (Figure
143
1a). The sample has numerous red fluorescing rod shaped bacteria encased in a
144
matrix containing large amounts of DNA. While the rod shaped red fluorescing
145
bacteria have a clear size and morphology, the orange granules differ slightly in both
146
size and morphology. However, note that when using specific filters, i.e. only looking
147
for green or red fluorescence, the red shaped bacteria only fluoresce in the red filter
148
setting whereas the autofluorescent granules fluoresce with equal intensity in both
149
filters and can thus not be distinguished from the bacteria. As the granules fluoresce
150
strongly in both the green and red spectrum, one could unintentionally misinterpret
151
the granules as being bacteria. In this case, the green and red fluorescence will lead
152
to the false conclusion that both of the PNA-FISH probes have annealed. This visual
153
artifact is seen when using equally intense red emitting and green emitting
154
fluorophores.
155
The granules shown in figure 1 are exited and emit strongly within the spectrum of
156
interest for many of the commonly used fluorophores, like TexasRed, Propidium
157
iodide, Syto 59 and FITC, GFP or Syto 9 etc. (18) . When only imaging the biofilm in
158
the spectrum of interest (for example only looking at emission maxima: 615nm for
159
Texas red or 521nm for FITC), the fluorescence in the sample will not be perceived
160
correctly as autofluorescence and hence lead to a false positive.
161 162
There are different ways to circumvent autofluorescence using quenchers (1,19,20).
163
When using quenching agents to reduce autofluorescence, the autofluorescence
164
needs to be reduced to a point where the emission of the fluorophore is intense
165
enough to penetrate the reduction of the specific bleaching method while retaining
166
the histological information (6). However, one should note that general tissue
167
autofluorescence is often used for navigation in the tissue section for histop-
168
pathological comparison purposes, which is an important source of information in
169
regards to showing the infiltration of the immune systems etc.
170 171
Conclusion:
172
Quenchers as Sudan Black B and KMnO4 can be used to alleviate the
173
autofluorescence, but in our minds the benefits do not outweigh the potential costs in
174
terms of lost information. Therefore, in our opinion autofluorescence should not be
175
quenched, but its presence should be acknowledged and the right questions should
176
be asked when looking at it.
177
We herein reiterate and discuss possible common knowledge. Due to biofilm-like
178
autofluorescent granules in most clinical samples, it is of utmost importance to
179
carefully consider the size and uniformity of potential bacteria when looking for
180
biofilms
181
Individual bacteria should be distinguishable and should not exceed more than 1-2
182
microns in diameter. The size and uniformity of the bacteria must be consistent and
183
coincide with appropriate emission of the fluorophore of choice (i.e. the bacteria
184
should not emit both green and red when they were only stained with green). Special
185
care should be taken when choosing fluorophores, as excitation and emission
186
spectra of different fluorophores overlap and hence complicating the matter.
187 188
188 189 190 191 192
1.
Schnell SA, Staines WA, Wessendorf MW. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem [Internet]. 1999/05/20 ed. 1999;47(6):719–30. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10330448
193 194 195 196
2.
Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sørensen SR, Moser C, Kühl M, et al. The in vivo biofilm. Trends Microbiol [Internet]. 2013 Sep [cited 2014 Mar 19];21(9):466–74. Available from: http://www.sciencedirect.com/science/article/pii/S0966842X1300111X
197 198 199 200
3.
Jolly RD, Palmer DN, Dalefield RR. The analytical approach to the nature of lipofuscin (age pigment). Arch Gerontol Geriatr [Internet]. 2002 May [cited 2014 May 14];34(3):205–17. Available from: http://www.sciencedirect.com/science/article/pii/S0167494301002199
201 202 203 204
4.
Jolly RD, Douglas B V, Davey PM, Roiri JE. Lipofuscin in bovine muscle and brain: a model for studying age pigment. Gerontology [Internet]. 1995/01/01 ed. 1995;41 Suppl 2:283–95. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8821339
205 206
5.
Terman A, Brunk UT. Lipofuscin. Int J Biochem Cell Biol. 2004/05/19 ed. 2004 Aug;36(8):1400–4.
207 208 209
6.
Schnell SA, Staines WA, Wessendorf MW. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem. 1999/05/20 ed. 1999;47(6):719–30.
210 211 212 213
7.
Sparrow JR, Wu Y, Nagasaki T, Yoon KD, Yamamoto K, Zhou J. Fundus autofluorescence and the bisretinoids of retina. Photochem Photobiol Sci [Internet]. 2010/09/24 ed. 2010;9(11):1480–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20862444
214 215 216 217
8.
Seehafer SS, Pearce DA. You say lipofuscin, we say ceroid: defining autofluorescent storage material. Neurobiol Aging [Internet]. 2006/02/04 ed. 2006;27(4):576–88. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16455164
218 219 220 221
9.
Jung T, Bader N, Grune T. Lipofuscin: formation, distribution, and metabolic consequences. Ann N Y Acad Sci [Internet]. 2007 Nov [cited 2014 Dec 16];1119:97–111. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18056959
222 223 224 225
10.
Gerrits PO, Kortekaas R, de Weerd H, Veening JG, van der Want JJ. Regional differences in age-related lipofuscin accumulation in the female hamster brainstem. Neurobiol Aging [Internet]. 2011/05/10 ed. 2012;33(3):625 e1–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21550695
226 227 228
11.
Terman A, Brunk UT. Lipofuscin: mechanisms of formation and increase with age. APMIS [Internet]. 1998/04/09 ed. 1998;106(2):265–76. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9531959
229 230 231 232 233
12.
Haka AS, Kramer JR, Dasari RR, Fitzmaurice M. Mechanism of ceroid formation in atherosclerotic plaque: in situ studies using a combination of Raman and fluorescence spectroscopy. J Biomed Opt [Internet]. 2011/02/02 ed. 2011;16(1):11011. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21280898
234 235 236
13.
Wixom RL, Prutkin L, Munro HN. Hemosiderin: nature, formation, and significance. Int Rev Exp Pathol [Internet]. 1980 Jan [cited 2014 May 19];22:193–225. Available from: http://www.ncbi.nlm.nih.gov/pubmed/7005144
237 238 239 240 241
14.
Fazli M, Bjarnsholt T, Kirketerp-Moller K, Jorgensen B, Andersen AS, Krogfelt KA, et al. Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds. J Clin Microbiol [Internet]. 2009/10/09 ed. 2009;47(12):4084–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19812273
242 243 244 245
15.
Aanaes K, Eickhardt S, Johansen HK, von Buchwald C, Skov M, Høiby N, et al. Sinus biofilms in patients with cystic fibrosis: is adjusted eradication therapy needed? Eur Arch Otorhinolaryngol [Internet]. 2014 Oct 9 [cited 2014 Oct 11]; Available from: http://www.ncbi.nlm.nih.gov/pubmed/25297534
246 247 248 249 250
16.
Kragh KN, Alhede M, Jensen PØ, Moser C, Scheike T, Jacobsen CS, et al. Polymorphonuclear Leukocytes Restrict the Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients. Infect Immun [Internet]. 2014 Aug 11 [cited 2014 Sep 25];IAI.01969–14 – . Available from: http://iai.asm.org.ep.fjernadgang.kb.dk/content/early/2014/08/05/IAI.01969-14
251 252 253
17.
Bancroft JD. Theory and Practice of Histological Techniques [Internet]. Elsevier Health Sciences; 2008 [cited 2014 May 19]. Available from: http://books.google.com/books?id=Dhn2KispfdQC&pgis=1
254 255 256
18.
Sparrow JR, Wu Y, Nagasaki T, Yoon KD, Yamamoto K, Zhou J. Fundus autofluorescence and the bisretinoids of retina. Photochem Photobiol Sci. 2010/09/24 ed. 2010;9(11):1480–9.
257 258 259 260 261
19.
Viegas MS, Martins TC, Seco F, do Carmo A. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Eur J Histochem [Internet]. 2007/06/06 ed. 2007;51(1):59–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17548270
262 263 264 265 266
20.
Baschong W, Suetterlin R, Laeng RH. Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J Histochem Cytochem [Internet]. 2001/11/29 ed. 2001;49(12):1565–72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11724904
267 268
268 269 270 271
Figure 1a, b and c: A micrograph of a tissue sample from the glandula submandibularis in a patient
272
suffering from sialolithiasis. The micrograph contains several clusters of
273
numerous red fluorescing bacteria, 1b and a one area, 1c containing a cluster of
274
autofluorescing granula. Note the similar size and fluorescence of the granula
275
compared to the bacteria. This cluster of autofluorescing granula is easily
276
misinterpreted as a biofilm. The sample has been stained with PNA-FISH and
277
DAPI and obtained by using a 63x 1.4 NA Zeiss objective on a Zeiss 710 CLSM.
278
The bar represents 15 μm.
279 280 281
1
Title Page
2
Title:
3
Autofluorescence in Samples Obtained from Chronic Biofilm Infections - “All
4
that glitters is not gold”
5 6
Running Title: Autofluorescence in Chronic biofilm infections
7
Authors:
8
*M.Sc. Steffen Eickhardt1, 2, [[email protected]]
9
M.Sc. Kasper Kragh1, 2[[email protected]]
10
MD Stine Schrøder3 [[email protected]]
11
Ph.D. Steen Seier Poulsen4 [[email protected]]
12
MD, DMSc . Henrik Sillesen5 [[email protected]]
13
Professor Dsc. Ph.D. Michael Givskov 6 [[email protected]]
14
Professor DMSc. Niels Høiby1, 2, [[email protected]]
15
Professor Ph.D. Thomas Bjarnsholt1, 2, [[email protected]]
16
Ph.D. Morten Alhede1, 2, [[email protected]]
17 18
1: Costerton Biofilm Center, Department of International Health, Immunology &
19
Microbiology, The faculty of Health and Medical Sciences, The University of
20
Copenhagen
21
2: The Department of Clinical Microbiology, Rigshospitalet, Denmark
22
3: Ear Nose Throat Department, North Zealand Hospital, Denmark
23
4: The Department of Biomedicine, The faculty of Health and Medical Sciences, The
24
University of Copenhagen
25
5: Dept. of Vascular Surgery, Rigshospitalet, Univ. of Copenhagen
26
6: Singapore Centre on Environmental Life Sciences Engineering (SCELSE),
27
Nanyang Technological University, Singapore
28 29 30
Corresponding Author
31
Steffen Eickhardt
32
Costerton Biofilm Center, Department of International Health, Immunology &
33
Microbiology, The faculty of Health and Medical Sciences, The University of
34
Copenhagen
35
Blegdamsvej 3
36
2200 Copenhagen N
37
Denmark
38 39
Telephone: +45 353 26657
40
Fax: +45 353 27853
41
E-mail: [email protected]
42 43
Word Count:
44
Abstract: 102
45
Manuscript: 1334
46
46 47
Autofluorescence in Samples Obtained from Chronic Biofilm Infections - “All
48
that glitters is not gold”
49
*Steffen Eickhardt1, 2, Kasper Kragh1, 2, Stine Schrøder3, Steen Seier Poulsen4,
50
Henrik Hegaard Sillesen5, Michael Givskov 6, Niels Høiby1, 2. Thomas Bjarnsholt1, 2 &
51
Morten Alhede1, 2
52
Abstract
53
When looking at tissue sections of ex vivo samples, autofluorescence can be a major
54
cause of artifacts and misinterpretations. We here reiterate evidence that
55
autofluorescing granules; often hemosiderin but also ceroid or mucinogen granules
56
are severe obstacles when imaging and diagnosing biofilm infections through
57
fluorescent imaging techniques. We used CLSM with spectral analysis for
58
autofluorescence detection as well as standard histological stains in order to identify
59
the culprit and show that these granules might very well be mistaken for bacterial
60
biofilms. Furthermore, we hypothesize that the increased amount of autofluorescing
61
granules may be a consequence of prolonged inflammation as a consequence of
62
chronic biofilm infections.
63 64
Autofluorescing granules can easily be mistaken as bacterial biofilm.
65
Several techniques are available for detecting bacteria within clinical samples.
66
However, in order to characterize and establish whether a biofilm is present,
67
microscopy
68
accompanied by specific histological stains as well as electron microscopy has been
69
used. Since the development of fluorescence based microscopy and the confocal
70
laser-scanning microscope, more researchers are drawn to this method.
71
However, when studying clinical samples autofluorescence is a problem. The
72
autofluorescence is often present as granules of approximately 1 micron in
73
diameter(1). These granules are spherical and grouped in clusters ranging from 10 to
74
1000 granula, which highly resembles the composition and structure of the in vivo
75
biofilm. In clinical samples, the bacterial biofilm is a tightly packed structure rarely
76
comprised of more than 103 bacteria. The size of most in vivo biofilm is between 10
77
and 500 bacteria(2). Because of the similarities between the autofluorescing
78
granules and the in vivo biofilm, the autofluorescing granules can easily be mistaken
79
as bacterial biofilm. An example of this can be seen in figure 1, a biopsy from the
is
often
preferred.
Traditionally,
conventional
light
microscopy
80
glandula submandibularis in a patient suffering from sialolithiasis, salivary gland
81
stones.
82
The granules are often composed of cellular debris and break-down products as lipid
83
residues, oxidative modified protein, carbohydrates(3) as well as metals, primarily
84
iron (4,5). The autofluorescing granules have an emission maximum within the
85
orange spectrum, resulting in a yellow to golden emission (6) as can been seen on
86
figure 1c. However, this highly depends on the excitation used (7) and on the origin
87
of the tissue (8). The granules can be observed both intracellular and extracellular.
88
When intracellular, the granules are often located within the perinuclear space, the
89
peribilecanicular region and cytosol (9) as well as the secondary lysosomes and
90
lysosomal compartments (10).
91
That autofluorescence is present in tissue sections is hardly a surprise for anyone
92
who has worked with fluorescent microscopy. The major causes, lipofuscin,
93
hemosiderin and ceroid are well studied and described (11–13). Yet we speculate
94
that autofluorescing granulas can be unintentionally misinterpreted as biofilms and
95
presented as such in publications. This speculation is derived by biofilm images
96
that are presented in low-resolution and are often overexposed to a degree where
97
size, uniformity and morphology of the bacteria are not readily visualized.
98 99
Our aim of this article is to share our experiences, for other biofilm researchers to
100
avoid the pitfalls we have encountered, by: 1) showing that autofluorescing granules
101
are present in chronic biofilm infections. 2) Describing what the autofluorescing
102
granules are 3) showing that these granules easily can be mistaken as biofilm and
103
most importantly 4) introduce guidelines to how to distinguish between biofilms and
104
autofluorescent artifacts.
105 106
Methods:
107
We have investigated ex vivo samples from several types of chronic biofilm
108
infections: as chronic wounds(14), the sinus of cystic fibrosis patients (15), the lungs
109
of cystic fibrosis patients(16) and atherosclerotic plagues. A biopsy from cirrhosis of
110
the liver, known for high amounts of lipofuscin granula, was used as a positive
111
control for autofluorescing granules.
112
We applied histology, conventional light microscopy as well as fluorescent bacterial
113
probes (PNA-FISH) and Confocal Laser Scanning Microscopy with spectral analysis.
114
Consecutive sections were stained both for the presence of bacteria as well as the
115
most described autofluorescing granule, lipofuscin. The fluorescent bacterial stain
116
was PNA-FISH probes targeting Staphylococcus aureus (green) and general Uni-
117
bacterial probes (red) (AdvanDx, Woburn, USA). 4', 6-diamidino-2-phenylindol
118
(DAPI) was used as a counter stain to detect DNA. Sudan Black B and KMnO4 were
119
used as quenchers of autofluorescence along with the fluorescent bacterial probes
120
and DAPI. In order to detect lipofuscin, three specific stains were used according to
121
Bancroft and Gamble 2008 (17). Peroidic Acid Shiff was used in order to detect
122
glycoproteins, Schmorl’s reaction to determine the oxidative state of the sample and
123
Long Ziehl-Neelson to detect acid fastness of the granules. Granules which were
124
positive for three stains as well as yellow golden autofluorescence was considered to
125
be lipofuscin.
126 127
Results:
128
As expected, histology revealed that the positive control from the cirrhotic liver was
129
indeed positive for lipofuscin granules. In three of the four chronic infection samples,
130
the cystic fibrosis lung tissue, the cystic fibrosis sinus tissue and the chronic wound
131
tissue we observed both autofluorescence as well as bacterial biofilms. The histology
132
results showed, that the major culprit of autofluorescing granules in these chronic
133
biofilm infected tissue sections was hemosiderin. No lipofuscin was found in any of
134
the samples. We observed that there appears to be a connection between the
135
severity of the immune response and the amount of hemosiderin present in the
136
sample. Granules such as ceroid and mucinogene granules were also present.
137
Quenchers such as KMnO4 and Sudan Black B were found to work well in removing
138
autofluorescence. However, using quenchers on the samples often ruined
139
histological interpretation and the emission of the studied fluorophores was (i.e. the
140
stain of the actual biofilm) reduced to unusable amounts.
141
Autofluorescent granules and a true biofilm can be seen in figure 1, a tissue sample
142
from the glandula submandibularis in a patient suffering from sialolithiasis (Figure
143
1a). The sample has numerous red fluorescing rod shaped bacteria encased in a
144
matrix containing large amounts of DNA. While the rod shaped red fluorescing
145
bacteria have a clear size and morphology, the orange granules differ slightly in both
146
size and morphology. However, note that when using specific filters, i.e. only looking
147
for green or red fluorescence, the red shaped bacteria only fluoresce in the red filter
148
setting whereas the autofluorescent granules fluoresce with equal intensity in both
149
filters and can thus not be distinguished from the bacteria. As the granules fluoresce
150
strongly in both the green and red spectrum, one could unintentionally misinterpret
151
the granules as being bacteria. In this case, the green and red fluorescence will lead
152
to the false conclusion that both of the PNA-FISH probes have annealed. This visual
153
artifact is seen when using equally intense red emitting and green emitting
154
fluorophores.
155
The granules shown in figure 1 are exited and emit strongly within the spectrum of
156
interest for many of the commonly used fluorophores, like TexasRed, Propidium
157
iodide, Syto 59 and FITC, GFP or Syto 9 etc. (18) . When only imaging the biofilm in
158
the spectrum of interest (for example only looking at emission maxima: 615nm for
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Texas red or 521nm for FITC), the fluorescence in the sample will not be perceived
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correctly as autofluorescence and hence lead to a false positive.
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There are different ways to circumvent autofluorescence using quenchers (1,19,20).
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When using quenching agents to reduce autofluorescence, the autofluorescence
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needs to be reduced to a point where the emission of the fluorophore is intense
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enough to penetrate the reduction of the specific bleaching method while retaining
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the histological information (6). However, one should note that general tissue
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autofluorescence is often used for navigation in the tissue section for histop-
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pathological comparison purposes, which is an important source of information in
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regards to showing the infiltration of the immune systems etc.
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Conclusion:
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Quenchers as Sudan Black B and KMnO4 can be used to alleviate the
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autofluorescence, but in our minds the benefits do not outweigh the potential costs in
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terms of lost information. Therefore, in our opinion autofluorescence should not be
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quenched, but its presence should be acknowledged and the right questions should
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be asked when looking at it.
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We herein reiterate and discuss possible common knowledge. Due to biofilm-like
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autofluorescent granules in most clinical samples, it is of utmost importance to
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carefully consider the size and uniformity of potential bacteria when looking for
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biofilms
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Individual bacteria should be distinguishable and should not exceed more than 1-2
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microns in diameter. The size and uniformity of the bacteria must be consistent and
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coincide with appropriate emission of the fluorophore of choice (i.e. the bacteria
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should not emit both green and red when they were only stained with green). Special
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care should be taken when choosing fluorophores, as excitation and emission
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spectra of different fluorophores overlap and hence complicating the matter.
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1.
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Wixom RL, Prutkin L, Munro HN. Hemosiderin: nature, formation, and significance. Int Rev Exp Pathol [Internet]. 1980 Jan [cited 2014 May 19];22:193–225. Available from: http://www.ncbi.nlm.nih.gov/pubmed/7005144
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Fazli M, Bjarnsholt T, Kirketerp-Moller K, Jorgensen B, Andersen AS, Krogfelt KA, et al. Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds. J Clin Microbiol [Internet]. 2009/10/09 ed. 2009;47(12):4084–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19812273
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Sparrow JR, Wu Y, Nagasaki T, Yoon KD, Yamamoto K, Zhou J. Fundus autofluorescence and the bisretinoids of retina. Photochem Photobiol Sci. 2010/09/24 ed. 2010;9(11):1480–9.
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Viegas MS, Martins TC, Seco F, do Carmo A. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Eur J Histochem [Internet]. 2007/06/06 ed. 2007;51(1):59–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17548270
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Figure 1a, b and c: A micrograph of a tissue sample from the glandula submandibularis in a patient
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suffering from sialolithiasis. The micrograph contains several clusters of
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numerous red fluorescing bacteria, 1b and a one area, 1c containing a cluster of
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autofluorescing granula. Note the similar size and fluorescence of the granula
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compared to the bacteria. This cluster of autofluorescing granula is easily
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misinterpreted as a biofilm. The sample has been stained with PNA-FISH and
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DAPI and obtained by using a 63x 1.4 NA Zeiss objective on a Zeiss 710 CLSM.
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The bar represents 15 μm.
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