In vivomonitoring of carbonic anhydrase expression during the growth of larval zebrafish: a new environment-sensitive fluorophore for responsive turn-on fluorescence

Article abstract of DOI:10.1039/d0cc03090b

This study monitors the dynamic progress of a newly developed background-free, target responsive strategy; 2,3-dihydroquinolin-4-imine (DQI) that can instantly respond to environmental changes with fluorescence enhancement, revealing a comprehensive platf

Full text of DOI:10.1039/d0cc03090b

View Article Online
View Journal
ChemComm
Chemical Communications
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Chou, C. Wu,
C. Chen, J. Zhou, Y. Kao, H. Chen and P. Lin, Chem. Commun., 2020, DOI: 10.1039/D0CC03090B.
Volume 54
This is an Accepted Manuscript, which has been through the
Number
January 2018
Pages 1-112
1
4
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Chemical Communications
rsc.li/chemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
towards bifunctional catalysts of tunable basicity
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 5
ChemComm
View Article Online
DOI: 10.1039/D0CC03090B
COMMUNICATION
In Vivo Monitoring of Carbonic Anhydrases Expression on Growth
of Larval Zebrafish: A New Environment-Sensitive Fluorophore for
Responsive Turn-On Fluorescence
Received 00th January 20xx,
Accepted 00th January 20xx
Chih-Hung Chou,a Chang-Yi Wu,b Chun-Lin Chen,b Jun-Qing Zhou,b Yu-Chen Kao,b Hsing-Yin Chen,c
DOI: 10.1039/x0xx00000x
Po-Chiao Lin*a
This study monitors dynamic progress of newly developed conserve the native structure of target proteins and therefore
background-free, target responsive strategy; 2,3-dihydroquinolin- reduce the loss of biological activity.5 New core fluorophores
4-imine (DQI) that can instantly respond to environmental with specific emission wavelengths are always in high demand
changes with fluorescence enhancement, revealing
a
for the study of molecular mechanisms in biochemical and
comprehensive platform for in vivo fluorescence bioimaging of biological systems.
mebrane-bound carbonic anhydrase II on HeLa cells and its For monitoring the protein expression, fluorophores that
expression over the growth larval zebrafish.
instantaneously respond to physical changes in a local
environment, particularly for viscosity and solvent polarity, are
required to monitor real-time biomolecular interactions
without the removal of unbound fluorophore, thereby
reducing background fluorescence.6 The environment-sensitive
fluorophores that exhibit solvatochromism have an emission
wavelength and quantum yield properties that are sensitive to
the local environment. This property makes solvatochromic
fluorophores ideal tools to dynamically measure biomolecular
interactions in which fluorescence is emitted when embedded
in the biomolecule of interest. Recently, several well-known
environment-sensitive fluorophores, including nitrobenzoxa-
diazole (NBD),7 merocyanine dyes,8 6-propionyl-2-dimethyl-
amino naphthalene (PRODAN),9 and sulfamonyl-7-amino-
benzoxadiazole (SBD)10, have been applied to study protein-
ligand and protein-protein interactions. The fluorescent signal
enhancement and the emission wavelength shift provide a
background-free method for in vitro and in cellulo
biomolecular studies. However, the short excitation
wavelength (< 400 nm) required for several environment-
sensitive fluorophores,6 limit their advanced applications.
Although merocyanine dyes have long wavelengths in both
excitation and emission, the large size and subtle change of
fluorescence in response to solvent polarity make them
difficult for use with in cellulo studies.8 Although the colors of
fluorescence are still limited by rational design and synthetic
methods, the success of the fluorophores in a study of
biomolecular interactions encourages us to overcome
structural diversity limitations with tailored emission
wavelength. Therefore, the development of new environment-
sensitive fluorophores, with tailored emission wavelengths, a
significant change of fluorescence in response to
environmental changes, large Stokes shifts and good
fluorescent quantum yields is crucial.
Carbonic anhydrases (CA) are enzymes existing in all organisms
that catalyze the reversible hydration of CO2 to bicarbonate
-
(HCO3 ) and therefore play important roles in the regulation of
physiological pH and therefore plays a crucial role in the
regulation of respiration, bone development, eye function, and
signal transfer in neural networks.1 Remarkably, CA is an
essential enzyme in otolith calcification for zebrafish by
maintaining an appropriate pH of the endolymph. Growth of
several zebrafish orthologs of mammalian CA isoforms have
been studied and screened for expression levels in the inner
ear in developing embryos. Remarkably, the expression of
CA2a and CA15a were up regulated while otolith calcification
began two days post fertilization.2a,b With specific antibody
staining, CA15a was showed low expression compared to CA2a
which is found in inner ear hair cells. Real-time and spatial
monitoring of native CA2a and expression is certainly of great
importance to the study zebrafish development. Therefore, a
target-responsive fluorescence will show instant and specific
protein expression information, for both time and location,
during cell development and zebrafish larvae growth.
Small fluorescent molecules which are of vital importance for
tracking the location of cells and biomolecules can be an ideal
tool for this studies purpose.3 Compared to green fluorescent
protein fusions,4 methods using small fluorescent molecules,
a.Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424,
Taiwan. Email: pclin@mail.nsysu.edu.tw
b.Department of Biological Science, National Sun Yat-sen University, Kaohsiung
80424, Taiwan
c. Department of Medicinal and Applied Chemistry, Kaohsiung Medical University,
Kaohsiung 80708, Taiwan
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
COMMUNICATION
Journal Name
To this purpose, very recently, we reported a Cu(I)-catalyzed changes. To further elucidate the unique solvatochromic effect,
View Article Online
denitrogenative annulation which can collect a series of the density functional theory (DFT) calcuDlaOtIi:o1n0.w10a3s9/pDe0rCfCo0rm30e9d0B.
reduced quinoline analogs, 2,3-dihydroquinolin-4-imines
(DQIs).11 Unknowingly, the aromaticity deficiency and the
short conjugation length of DQIs can provide strong blue green
to yellow fluorescence emission with blue-light excitation
ranging from 400 - 450 nm with high fluorescence quantum
yields up to 66%. The synthetic pathway also allows
explanation of the structural diversity of DQIs which would be
of great importance for biomolecular detection and bioimaging.
Figure 2. Fluorescence enhancement of SA-DQI-directed CAII recognition.
Emission spectra for (a) SA-DQI (1 μM) and SA-DQI (1 μM) / CAII (10 μM) binding,
EZA competition. (b) Emission spectra of negative control 7-OH-DQI (1 μM) and
7-OH-DQI (1 μM) / CAII (10 μM) binding. (c) The emission spectra of SA-DQI (1
μM) treated with CAII in serially diluted concentrations.
The charge transfer character of the excitation produces a
larger dipole moment in the S1 state (10.5 D) than in the S0
state (5.6 D) (Figure 1b). The considerable difference in dipole
moment between S0 and S1 might be subject to subsequent
solvent relaxation. Therefore, the higher-polarity solvents
would reorganize, leading to a relaxed state. Solvent relaxation
can contribute to an emission red-shift accompanied by a
decline in fluorescence quantum yield. With the understanding
of their environment-sensitive properties, DQI have potential
as fluorescence switchable bioprobes that detect subtle
changes in microenvironments.
Figure 1. (a) Fluorescence emission spectra of DQI in various solvents. (b) The
vector of dipole moment of ground state (GS) and excited state (ES). The vector
points from negative charge to positive charge. (c) Absorption and emission
maxima of DQI in various solvents and quantum yields.
To examine the applicability of this new chemical probe,
human carbonic anhydrase II (hCAII) was used as a target
protein. Arylsulfonamide (SA) is the most important class of CA
inhibitors that have high affinity with nanomolar dissociation
constant (KD).1
To selectively recognize hCAII with turn-on fluorescence, a
chemical sensor, SA-DQI, containing an affinity head (SA) and
the environmentally sensitive DQI reporter, was synthesized.12
The fluorescence intensity for SA-DQI in aqueous buffer was
very low (Figure 2a), but addition of hCAII caused fluorescence
to dramatically increased (15-fold), (triplicate average of the
integrated emission spectra). The fluorescence enhancement
is attributed to the microenvironment change when SA-DQI
becomes embedded in the hydrophobic pocket of hCAII. A
blue emission wavelength shift further supports this
To evaluate if DQI is applicable in biomolecular detection,
the photophysical characterization and biocompatibility in
aqueous condition have been carefully studied. The DQI
fluorophore was found to have solvent-dependent emission
(Figure 1a), showing potential for a new series of environment-
sensitive fluorophores. Shown in Figure 1c, DQI exhibited a
clear red-shift emission trend in polar solvents. In contrast, in
less polar solvents such as toluene and n-hexane, the emission
maxima were shifted to 513 and 478 nm, high-polarity solvents
such as methanol and dimethylformamide (DMF) may stabilize
the excited state of DQI, and subsequent solvent relaxation
leads to an energy reduction respectively.
Hence, a significant red emission shift (95 nm) was observed
in methanol compared to n-hexane. A solvent-dependent
effect was also observed when determining the ΦF of DQI. The
ΦF of DQI was determined as 62% in less polar
dichloromethane (DCM). In contrast, the ΦF of DQI measured
in polar protic methanol and highly polar DMF dramatically
decreased to 6 and 7%, respectively. The basic photophysical
properties have been clarified as an intramolecular charge
transfer between C=N moiety of the N-sulfonylimido group
and nitrogen atom in the ring structure. The excited state of
DQI might redistribute electrons, resulting in dipole moment
interpretation
(Figure
S10).
Because
fluorescence
enhancement arises from noncovalent protein-ligand
interactions, the process should be reversible and subject to
competitive quenching. The addition of a strong CA inhibitor,
ethoxzolamide (EZA),13 diminished the fluorescence shown in
Figure 2a. To further examine the role of SA, a control
molecule 7-OH-DQI, of similar structure to SA-DQI but lacks
the sulfonamide group, was synthesized. The 7-OH-DQI
showed much weaker fluorescence signals in both the
presence and the absence of hCAII (Figure 2b), supporting the
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 5
ChemComm
Journal Name
COMMUNICATION
fluorescence enhancement resulting from specific binding of signal is reversible and only occurred when SA-DQI was
View Article Online
DOI: 10.1039/D0CC03090B
arylsulfonamide and hCAII. To further study the fluorescence embedded in the hCAII pocket.
enhancement with stoichiometric hCAII and SA-DQI binding,
SA-DQI (1 μM) was incubated with increasing concentrations
of hCAII and the binding constant for hCAII with SA-DQI was
1.138 ± 0.04 μM (Figure 2c and Figure S3a).
Figure 4. Cells imaging of HeLa cells expressing the CFP
the cell membrane. (a) Transfected HeLa cells were treated with 1
Transfected HeLa cells were treated with 1 M SA-DQI and then subjected to
competition by the addition of 300 M EZA. Scale bars represent 20 m.
–
hCAII
–
PDGFR protein on
μ
M SA-DQI. (b)
μ
μ
μ
Figure 3. (a) Selectivity of SA-DQI (1 μM) with CAII and five other non-targeted
proteins. (b) Viability of the cells after 24 incubation with various
concentrations of SA-DQI.
To exclude the fluorescence enhancement coming from the
interaction of CFP with SA-DQI, a platelet-derived growth
factor reporter (PDGFR)-fused hCAII was expressed on the cell
membrane. The cell surface-localized recombinant hCAII–
PDGFR proteins were treated with SA-DQI (1 μM) and
reasonably present fluorescent signal on membrane (Figure
S8). The DQI-directed background- and washing-free strategy
could be applied to a continuous bioimaging platform for real-
time biomolecular investigation upon specific stimulation.
Since DQI is first revealed in live cell fluorescent imaging, the
cytotoxicity was carefully assessed and the SA-DQI molecule
shows negligible cytotoxicity under imaging conditions,
confirmed using an MTT assay at concentrations of 0.1 - 5 μM
for 24 h (Figure 3b). In figure 4(ii), few dot-like fluorescent
signals outside the cell can be observed which could be SA-DQI
aggregates. Therefore, a better water-soluble SA-conjugated
DQI, SA-TEG-DQI, was synthesized and successfully staining the
transfected hCAII on the cell membrane with very low
background(Figure S7). For endogenous CAs, transmembrane-
type hCAIX and hCAXII are overexpressed for the hypoxia-
inducible factor (HIF-1) in cancer cell has been reported.15 To
image the CA on live cell surface, the A549 cell line was chosen
because of its high endogenous expression of hCAIX under
hypoxic condition. The A549 cells were cultured under hypoxic
mimic (in the presence of deferoxamine mesylate (DFO) (200
μM)) and normoxic condition individually. The fluorescence
intensity enhanced on A549 cell membrane when treated with
SA-TEG-DQI (10 μM) under hypoxic mimic without washing
process (Figure S9a). In the control experiment, the
fluorescence intensity decreased upon addition of the strong
inhibitor, EZA (300 μM) (Figure S9b). Under normaxic condition,
no distinguishable fluorescence signal could be detected for
A549 cell (Figure S9c). Thus, SA-conjugated DQI could not only
detect CA on transfected cell surface but for endogenous
expressed CA cell.
h
An ascending fluorescence trend was observed as hCAII
concentration increased and became increasingly bound to SA-
DQI. The SA-DQI selectivity for hCAII was examined by
incubating the molecule with five non-targeted proteins (10
μM). Figure 3a shows SA-DQI exhibits high selectivity for hCAII
(15-fold) and slight fluorescence increases for, Transferrin (2.5-
fold), and Con A (2-fold). The high selectivity and quantifiable
analysis make ligand-DQI an ideal switchable fluorescent
bioprobe for advanced biomedical applications.
After successfully enhancing selective fluorescence, the SA-
DQI was applied to live cell imaging. HeLa cells were
transfected to express hCAII on a cell membrane. To validate
that the fluorescence enrichment was from the specific
interaction of SA-DQI and membrane-bound hCAII, a cyan
fluorescent protein (CFP)-fused hCAII-platelet-derived growth
factor reporter (PDGFR) (a membrane-anchoring domain that
guides hCAII localization to the cell membrane) was
transfected and expressed on the plasma membrane of the
HeLa cell, showing the location of CAII on the cell membrane.14
Before staining by SA-DQI, the transfected HeLa cells were first
examined at 405 nm (the excitation wavelength of CFP) and
488 nm (the excitation wavelength for SA-DQI). No significant
fluorescence can be observed at 488 nm excitation which
indicates weak background interference before SA-DQI binding
fluorescence enhancement. (Figure S4.)
The cell surface-localized recombinant CFP-hCAII-PDGFR
proteins were treated with SA-DQI (1 μM). Without removing
the unbound SA-DQI, strong fluorescence was observed on cell
membranes using confocal laser scanning microscopy,
reflecting hCAII and SA-DQI binding, (Figure 4a (ii)). Figure 4a (v)
shows the enriched SA-DQI fluorescence (ii) colocalized well
with the CFP-hCAII-PDGFR protein (i) expressed on the plasma
membrane of HeLa cells, which strongly supports the selective
fluorescence turn-on detection of hCAII by SA-DQI. The
Mander’s overlap coefficient of CFP and SA-DQI in HeLa cell
was acquired and analyzed by ZEN 2012 software which shown
in figure 4(a) with the value as 0.70. Subsequently, the
addition of EZA (300 μM) can significantly compete with bound
SA-DQI so that only very weak fluorescence could be detected
(Figure 4b, (ii)). Therefore, the enhancement of the fluorescent
Subsequently, we are interested in applying the
environment-sensitive SA-TEG-DQI for in vivo CA spatial and
temporal expression. Zebrafish is an ideal model for this
purpose because of the well-defined development and
amenability for optical imaging. In vertebrates, CA plays a vital
role in pH regulation. During zebrafish development, CA is
localized in brain, skin, tail, and heart, especially in inner-ear
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
hair cells, and likely provide HCO3- to the endolymph for otolith isoforms could be detected. With the SA-TEG-DQI directed
View Article Online
calcification and maintaining the pH levels.2b
monitoring of in vivo CA expression DaOnI:d10l.1o0c3a9l/iDza0tCioCn03,09th0Be
Tracking proteins of interest in zebrafish is important but formation of otoliths considered being one of the most
challenging in zebrafish embryogenesis; proteins of interest sensitive organs in embryogenesis, can be correlated with CA.
need to be fixed inside the embryo by immunohistochemistry,
In conclusion, the lack of a long conjugation system and
then the signal from fluorescent-conjugated antibody or aromaticity in the reduced quinolinimine form of DQI present
injected fluorescent fused protein, will be observed. Emerging unusual photophysical properties. A solvent-dependent shift in
studies are showing interest for the use of fluorophores to emission and enhanced fluorescence quantum yields, clearly
track proteins in vivo and their real time responses to location support its solvatochromic properties, which can certainly
and expression. To conduct the experiment, zebrafish embryos contribute to the study of biomolecular interactions.
were cultured in E3 media and dechorionated by pronase. Furthermore, a large Stokes shift (~100 nm) can avoid the shot
After six hours post fertilization (hpf), embryos were added to noise from excitation and eliminate the spectral overlap
1-phenyl-2-thiouria (PTU) (0.003%) to inhibit pigmentation. between emission and absorption. Consequently, SA-TEG-DQI
Ten ng SA-TEG-DQI (1.25 mM in 2% dimethyl sulfoxide (DMSO)) was synthesized and successfully used in the selective
was injected into the sinus venosus at 3 days post fertilization recognition and analysis of transfected or endogenous hCA.
(dpf) zebrafish (Figure 5b & 5f). For 3 dpf zebrafish larvae, CA2 The stochiometric fluorescence enhancement with the
dominated expression than other CA isoforms around the formation of hCA/SA-conjugated DQI allows a background-free
brain including the inner ear region.2b,16 Accordingly, figure 5f strategy for in vivo selective protein detection. Remarkably,
showed that the enhanced fluorescence is localized in the SA-TEG-DQI promises a spatial and dynamic monitoring of CA2
brain, showing strong signals at the inner-ear region. This expression on growth of zebrafish larva.
describes the role of CA2 for otolith calcification in the
epithelial cells of otocysts by maintaining an appropriate pH
with bicarbonate.2b In the control experiments, very weak
autofluorescence was observed (Figure 5a & 5e).
Conflicts of interest
There are no conflicts to declare.
Notes and references
1
V. M. Krishnamurthy, G. K. Kaufman, A. R. Urbach, I. Gitlin, K.
L. Gudiksen, D. B. Weibel and G. M. Whitesides, Chem. Rev.,
2008, 108, 946-1051.
2
(a) M. Beier, R. Hilbig and R. Anken, Advances in Space
Research, 2008, 42, 1986–1994; (b) H. Matsumoto, S.
Fujiwara, H. Miyagi, N. Nakamura, Y. Shiga, T. Ohta and M.
Tsuzuki, Mar. Biotechnol., 2017, 19, 430-440.
L. D. Lavis and R. T. Raines, ACS Chem. Biol., 2008, 3, 142-155.
R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509-544.
E. Basle, N. Joubert and M. Pucheault, Chem. Biol., 2010, 17,
213-227.
Figure 5. Images of 3 dpf zebrafish larvae. a) Bright field of control wild-type
zebrafish embryo. b) Bright field of SA-TEG-DQI injection at 3 dpf (n = 8/10). c)
Bright field of TEG-DQI injection at 3 dpf (n = 10/10). d) Bright field of CA2a
morpholino injection at 1–2 cell stage following by SA-TEG-DQI injection at 3 dpf
(n=10/10); e) Fluorescent image of control wild-type zebrafish embryo. f)
Fluorescent image of SA-TEG-DQI injection at 3 dpf (n = 8/10); g) Fluorescent
image of TEG-DQI injection at 3 dpf (n = 10/10). h) Fluorescent image of CA2a
morpholino injection at the 1–2 cell stage following by SA-TEG-DQI injection at 3
dpf (n=10/10). Scale bars represent 200 μm in a-h.
3
4
5
6
7
8
9
G. S. Loving, M. Sainlos and B. Imperiali, Trends Biotechnol.,
2010, 28, 73-83.
Q. Wang and D. S. Lawrence, J. Am. Chem. Soc., 2005, 127,
7684-7685
Two experiments were performed to support the
importance of SA-TEG-DQI in CA2 recognition with immediate
fluorescence enhancement. The TEG-DQI, which removes the
SA ligand from SA-TEG-DQI, was synthesized and injected into
the zebrafish’s sinus venosus at 3 dpf. Consequently, no
significant fluorescence signal can be observed supporting the
A. Toutchkine, V. Kraynov and K. Hahn, J. Am. Chem. Soc.,
2003, 125, 4132-4145.
G. Weber and F. J. Farris, Biochemistry, 1979, 18, 3075-3078
10 Y. -D. Zhung, P. -Y. Chiang, C. -W. Wang and K. -T. Tan,
Angew. Chem. Int. Ed., 2013, 52, 8124-8128.
fluorescence turn-on coming from SA-CA specific binding 11 C .-H. Chou, B. Rajagopal, C. -F. Liang, K. -L. Chen, D. -Y. Jin, H.
-Y. Chen, H. -C. Tu, Y. -Y. Shen and P. -C. Lin, Chem. Eur. J.,
2018, 24, 1112-1120.
12 The synthetic details are in supporting information.
13 J. Olander, S. F. Bosen and E. T. Kaiser, J. Am. Chem. Soc.,
1973, 95, 1616-1621.
(Figure 5c & 5g). A CA2a gene knockdown morpholino
antisense oligomer (MO-CA2a) was used to silence CA2
expression in zebrafish.17 The MO-CA2a was injected into
embryos at the one to two-cell stage and was also used PTU to
inhibit pigmentation in zebrafish at 6 hpf. After 3 dpf, SA-TEG- 14 T. -C. Hou, Y. -Y. Wu, P. -Y. Chiang and K. -T. Tan, Chem. Sci.,
2015, 6, 4643-4649.
DQI (1.25 mM in 2% DMSO) was injected into the sinus
venosus (Figure 5d & 5h). The fluorescence around the otolith
reduced dramatically which compared to SA-TEG-DQI injection
(Figure 5f) suggested knockdown of CA2 reduced the
interaction with SA-TEG-DQI, however, slightly fluorescence
can be observed in the CA2a-knock down zebrafish larvae
compared to control (Figure 5e) which suggests that others CA
15 D. E. B. Swinson, J. L. Jones, D. Richardson, C. Wykoff, H.
Turley, J. Pastorek, N. Taub, A. L. Harris and K. J. O'Byrne, J.
Clin. Oncol., 2003, 21, 473-482.
16 S. Feng, S. Wang, Y. Wang, Q. Yang, D. Wang and H. Li, Gene
Expression Patterns, 2018, 29, 47-58.
17 Y. Ito, S. Kobayashi, N. Nakamura, H. Miyagi, M. Esaki, K.
Hoshijima and S. Hirose, Front. Physiol., 2013, 4, 59.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/D0CC03090B
A customized DQI fluorophore promises in vivo
monitoring of carbonic anhydrases expression over the
growth of larval zebrafish by responsive
fluorescence enrichment.