U.S. patent application number 09/194099 was filed with the patent office on 2002-02-21 for masking background fluorescence and luminescence in optical analysis of biomedical assays.
Invention is credited to BECHEM, MARTIN, KRAHN, THOMAS, PAFFHAUSEN, WOLFGANG, SCHADE, ANDREAS, SCHMIDT, DELF.
Application Number | 20020022274 09/194099 |
Document ID | / |
Family ID | 7795451 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022274 |
Kind Code |
A1 |
KRAHN, THOMAS ; et
al. |
February 21, 2002 |
MASKING BACKGROUND FLUORESCENCE AND LUMINESCENCE IN OPTICAL
ANALYSIS OF BIOMEDICAL ASSAYS
Abstract
In a process for the quantitative optical analysis of
fluorescently labelled biological cells 5, a cell layer on a
transparent support at the bottom 2 of a reaction vessel 1 is in
contact with a solution 3 containing the fluorescent dye 4. The
sensitivity of analytical detection can be considerably improved if
to the fluorescent dye 4 already present in addition a masking dye
9, which absorbs the excitation light 6 for the fluorescent dye 4
and/or its emission light 7, is added to the solution 3 and/or if a
separating layer 10 permeable to the solution and absorbing and/or
reflecting the excitation light 6 or the emission light 7 is
applied to the cell layer at the bottom 2. This process can also be
used for improving the sensitivity in the quantitative optical
analysis of a luminescent biological cell layer. The separating
layer 10 must in this case be composed such that it has a high
power of reflection for the luminescent light 11. Analogously,
these process principles can also be used in receptor studies for
the masking of the interfering background radiation in the
quantitative optical analysis of fluorescently or luminescently
labelled reaction components. In this case, a receptor layer 12 at
the bottom 2 of a reaction vessel 1 is in contact with a solution
(supernatant 3) in which a fluorescent or luminescent ligand 13 is
dissolved. The sensitivity and accuracy of the analytical detection
can be considerably improved here if a masking dye 9 which absorbs
the excitation light 6 for the fluorescent dye and/or its emission
light or (in the case of luminescent ligands) the luminescent light
is added to the supernatant 3. Instead of the masking dye in the
solution 3 or optionally as an additional measure, a separating
layer 10 permeable to the solution 3 and absorbing and/or
reflecting the excitation light 6 and/or the emission light or the
luminescent light can be applied to the cell or receptor layer 12
at the bottom 2.
Inventors: |
KRAHN, THOMAS; (HAGEN,
DE) ; PAFFHAUSEN, WOLFGANG; (LEVERKUSEN, DE) ;
SCHADE, ANDREAS; (ESSEN, DE) ; BECHEM, MARTIN;
(WUPPERTAL, DE) ; SCHMIDT, DELF; (WUPPERTAL,
DE) |
Correspondence
Address: |
NORRIS McLAUGHLIN & MARCUS, P.A.
220 EAST 42nd STREET 30TH FLOOR
NEW YORK
NY
10017
US
|
Family ID: |
7795451 |
Appl. No.: |
09/194099 |
Filed: |
November 20, 1998 |
PCT Filed: |
May 23, 1997 |
PCT NO: |
PCT/EP97/02662 |
Current U.S.
Class: |
436/172 |
Current CPC
Class: |
G01N 33/5005 20130101;
Y10S 436/823 20130101; G01N 33/54393 20130101; Y10T 436/13
20150115; Y10S 436/805 20130101; G01N 33/542 20130101; Y10S 436/80
20130101; G01N 33/5306 20130101 |
Class at
Publication: |
436/172 |
International
Class: |
G01N 033/567 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 1996 |
DE |
196 21 312.6 |
Claims
1. Process for the quantitative optical analysis of fluorescently
labelled biological cells (5) which are applied to a transparent
support at the bottom (2) of a reaction vessel (1) in the form of a
coherent cell layer and are in contact with a solution (3)
containing the fluorescent dye (4), or of luminescent, biological
cells in the form of a coherent cell layer situated on the
transparent support, characterized in that the fluorescent dye (4)
already present in addition to a masking dye (9) which absorbs the
excitation light (6) for the fluorescent dye (4) and/or its
emission light (7) is added to the solution (3) and/or in that a
separating layer (10) which is permeable to the solution and which
absorbs and/or reflects the excitation light (6) for the
fluorescent dye (4) and/or its emission light (7) or, in the case
of the luminescent cell layer, reflects the luminescent light, is
applied to the cell layer.
2. Process for the quantitative optical analysis of fluorescently
or luminescently labelled reaction components in a reaction vessel
(1) filled with a solution (3) in which a fluorescent or
luminescent ligand (13) is dissolved and the solution (3) is in
contact with a receptor layer (12), which is specific for this
ligand (13) and is applied to a transparent support at the bottom
(2) of the reaction vessel (1) or deposited thereon, whose
fluorescent or luminescent radiation (7, 15), which is
characteristic of the receptor-ligand binding, is detected and
analysed through the transparent bottom (2), characterized in that
a masking dye (9) is added to the solution (3) and/or a separating
layer (10) permeable to the solution (3) is applied to the receptor
layer (12), the optical properties of the masking dye (9) and/or of
the separating layer (10) being selected such that the excitation
light (6) for the fluorescent dye (4) of the ligand (13) present in
the solution (3) and/or its fluorescent light (8) or its
luminescent light is absorbed by the solution (3) or the separating
layer (10) or reflected at the separating layer (10).
3. Process according to claim 1 or 2, characterized in that the
separating layer (10) used is a layer of polymeric latex beads.
4. Process according to claim 3, characterized in that the
polymeric latex beads are dyed with a masking dye.
5. Process according to claim 1-2, characterized in that a masking
dye is used which possesses good water solubility and has no
cytotoxic side effects.
6. Process according to claim 1-2, characterized in that in the
case of a replacement of the supernatant (3) containing a
fluorescent dye (4) by a fluorescent dye-free solution (3a) a
masking dye is added which suppresses the non-specific fluorescence
emitted from the stained reaction vessel wall.
Description
[0001] The invention originates from a process for the quantitative
optical analysis of fluorescently labelled biological cells which
are in contact with a fluorescent dye solution or of luminescent
cells which are applied to a transparent support at the bottom of a
reaction vessel in the form of a coherent cell layer, or
alternatively of fluorescently or luminescently labelled reaction
components in a solution in which a fluorescent or luminescent
ligand is dissolved, the solution being in contact with a receptor
layer, which is specific for this ligand and situated on the
transparent support at the bottom of the reaction vessel, whose
fluorescent or luminescent radiation, which is characteristic of
the receptor-ligand binding, is detected and analysed through the
transparent bottom.
[0002] A problem in fluorescence measurement in biomedical assays
is often that the fluorescence changes correlated with the
biological cell action are small compared with the non-specific
background fluorescence. As a result, the resolving power is
greatly restricted. Conventional commercial measuring systems
(fluorescence readers, Dynatech or SLT), cannot solve the problem,
because owing to their optical measuring arrangement (excitation
from `above` through the fluorescent liquid column of the
supernatant) the signal can barely be detected in comparison with
the background. Apparatuses of newer construction (Labsystems),
which illuminate the cells from the back through the transparent
support of the reaction vessel, do have the advantage that on entry
of the excitation light the cells are excited to fluorescence.
Since the excitation light, however, enters further into the
supernatant, which is also fluorescent, the fact that the
non-specific background signal adulterates the cell signal cannot
be avoided. Even very complicated measuring systems (NovelTech,
FLIPR: Fluorescence Imaging Plate Reader) are only able to decrease
this background fluorescence using a special laser illumination
geometry (excitation below about 45.degree.). The reason for the
failure of all problem-solving experiments on the measuring
geometry is the fact that the actual cause of the background
fluorescence cannot be decisively influenced hereby.
[0003] In the receptor binding studies carried out until now using
fluorescently or luminescently labelled ligands, the labelled and
unbound fraction in each case must be removed by processes like
washing. Many coatings, however, are sensitive to these washing
steps. Moreover, the removal of the unbound ligand is associated
with a considerable outlay. The direct measurement of the
receptor-ligand association or dissociation is not possible in this
process.
[0004] The invention is based on the object of improving the
sensitivity of the optical analysis of fluorescently labelled or
luminescent cells in a cellular assay in order to be able to
measure, for example, membrane potential changes which are as low
as possible on the basis of fluorescence changes of
potential-sensitive dyes. In this case, the sensitivity of the
measuring system should be so high that potential changes of below
5 mV can be detected at least qualitatively. In the case of
luminescent cells, an increase in the detection of the luminescence
signal should be achieved. Moreover, the method should be suitable
for screening with a high sample throughput.
[0005] The invention is furthermore based on the object of
simplifying receptor binding studies based on fluorescently or
luminescently labelled ligands or receptors and making possible
continuous measurement of the receptor binding interaction
(kinetics). Owing to the reduction in the process steps necessary,
this method should be particularly suitable for screening with a
high throughput and for diagnostic applications.
[0006] It was only possible to achieve the required high resolution
with low membrane potential changes after it was possible to
eliminate the cause of the interfering overlapping of the
non-specific background fluorescence and the specific fluoresence
of the cells. The process according to the invention developed for
this purpose is based on the fundamentally new idea of masking the
excitation energy and the fluorescence not originating from the
biological object. To do this, in addition to the fluorescent dye,
a further dye is added which completely absorbs the excitation
light of the fluorescent dye and/or its emission light without
affecting the fluorescence of the cells. By means of this
absorption, the non-specific background signal is masked and the
useful cell signal can be detected with a resolution which was
previously not possible.
[0007] An alternative solution which is within the scope of the
invention is that a separating layer, which is permeable to the
solution and which absorbs and/or reflects the excitation light for
the fluorescent dye and/or its emission light without adversely
affecting the cell properties, is applied to the cell layer. At the
same time, the thickness of the separating layer is selected such
that fluorescence is no longer detectable in the dissolving mixture
with the fluorescent dye but without the cells.
[0008] A further variant of the invention is that the method of the
separating layer according to the invention is also used for
increasing the sensitivity in the quantitative optical analysis of
luminescent (luminous) biological cells which are applied to a
transparent support in the form of a coherent cell layer. For this
purpose, the optical properties of the separating layer permeable
to the solution are selected such that it reflects the luminescent
light as strongly as possible without adversely affecting the cell
properties. In this manner, it is possible to increase the
luminescence intensity and thus the measured effect
considerably.
[0009] The process according to the invention can be used in a
completely analogous manner for the quantitative optical analysis
of fluorescently or luminescently labelled reaction components in a
reaction vessel filled with a solution, the fluorescent or
luminescent ligand being present in dissolved form and the solution
being in contact with a receptor layer which is specific for this
ligand, applied to a transparent support at the bottom of the
reaction vessel or deposited thereon, whose fluorescent or
luminescent radiation, which is characteristic for receptor-ligand
binding, is detected and analysed through the transparent bottom.
In this case, the solution according to the invention of the object
described above is based on the fact that the free ligand which is
in the supernatant, i.e. in solution, and its non-specific
fluorescence or luminescence is masked by an additional dye and/or
by a diffusely absorbing or reflecting separating layer and thus
the cause of the interfering overlapping of the non-specific
background fluorescence and the specific fluorescence of the ligand
in the solution is eliminated. Since the nonbound ligand is masked
in this manner, the measured fluorescence or luminescence is a
direct measure of the ligand-receptor interaction. It can be
measured directly in this process with time resolution.
[0010] In receptor studies, in analogy to the process described
above, the invention thus relates to a process variant in which a
masking dye is added to the solution and/or a separating layer
permeable to the solution is applied to the receptor layer, the
optical properties of the masking dye and/or of the separating
layer being selected such that the excitation light for the
fluorescent dye of the ligand present in the solution and/or its
emission light or its luminescent light is absorbed by the solution
or the separating layer or reflected at the separating layer. In
this case, the thickness of the separating layer is selected such
that fluorescence is no longer detectable in the dissolving mixture
with the fluorescent dye, but without the receptor layer.
[0011] The separating layer preferably consists of polymeric latex
beads (e.g. polystyrene, polyurethane, butadiene, acrylonitrile).
The latex beads can also be dyed with a masking dye, which in this
case must have an adequately high polymer dyeing capacity.
[0012] In the first-mentioned process, the masking dye should be as
well distributed as possible in the solution which also contains
the fluorescent dye in dissolved form. Since, as a rule, the
solvent is water, a masking dye is expediently employed which
possesses good water solubility (>2 g/ml) and has no cytotoxic
side effects.
[0013] According to a further development of the invention, after
the replacement of the supernatant containing a fluorescent dye by
a fluorescent dye-free solution, a further masking dye is added
which suppresses a non-specific fluorescence on the reaction vessel
wall.
[0014] The following advantages are achieved using the
invention:
[0015] The new process described is not tied to a certain measuring
system, but can be used, because it is not a specifically technical
solution, by many commercially available apparatuses. These include
virtually all fluorescence readers which can illuminate and also
measure transparent reaction vessels, e.g. microtitre plates, from
the bottom. With a very low outlay (minimal additional costs only
for the special absorption dyes), it is possible for the first time
by this means to advance in a resolving area, e.g. in the
measurement of potential changes in cell membranes by measurement
of the change in fluorescence of potential-sensitive fluorescent
dyes, which was unachieved until now. For the first time it is
possible even in the case of very low changes to carry out a direct
comparison of the results from various reaction vessels (e.g.
various wells in a microtitre plate), such that the complicated
procedure of the determination of the relative change in a reaction
vessel can be dispensed with. As a result the number of
measurements to be determined, e.g. for kinetic measurements,
decreases. The outlay in terms of time for a measuring programme is
markedly reduced and the possibility created of obtaining identical
results by a simple individual measurement (e.g. end point
determination) with the use of reference to a separate control
batch. The uniformity of the biological batch required in this case
(e.g. homogeneous cell layer) is generally afforded, for example,
for microtitre plates.
[0016] Surprisingly, the use of various water-soluble dyes and also
their mixtures in the very different cells tested showed no
negative effect on the physiology of the cells (e.g. reaction of
the cells in comparison with electrophysiological measurements such
as whole-cell patch-clamp, or effects of the pharmaceuticals
investigated). The use of undissolved dye pigments or inorganic
finely divided particles was also surprisingly well tolerated by
the biological objects.
[0017] As a result of the simple process described for masking the
background fluorescence in quantitative fluorescence measurement in
biomedical assays, connected with an increase in the sensitivity,
e.g. when using potential-sensitive fluorescent dyes, and the
adaptability of this process, e.g. to microtitre plates as reaction
vessels, the use of such measuring techniques will significantly
simplify high-throughput screening, especially no increased
technical expenditure is necessary for the realization of the
advantages outlined, but existing commercial measuring apparatuses
are sufficient for this purpose. In receptor-ligand studies, the
advantage essential to the invention is that, on account of the
masking of the non-specific fluorescence or luminescence, it is no
longer necessary to remove the unbound fraction of the ligands. As
a result, the test procedures are considerably simplified, damage
to and destruction of the sensitive coatings or of the biological
objects such as, for example, cells are avoided and the sensitivity
and thus also the accuracy of the measurement are improved. As a
result of the use of microparticles, the utilizable surface area
for the coating of fluorescently or luminescently labelled ligands
can be significantly increased. By means of suitable measures, e.g.
relatively high specific density or the use of magnetizable
particles, the settlement and concentration of the microparticles
on the transparent support can be achieved. In this case too, the
fluorescence or luminescence of the unbound ligands in the
supernatant is effectively suppressed by the masking.
[0018] Since the interaction between the ligands and the receptor
must not be interrupted by the removal of the unbound fraction, a
continuous measurement of the interaction between ligand and
receptor (kinetics) can be carried out in this manner even in an
individual reaction batch.
[0019] The invention is explained in greater detail below with the
aid of working examples and drawings, wherein
[0020] FIG. 1 shows a reaction vessel for a fluorescence assay
according to the prior art
[0021] FIG. 2 shows the suppression of the background fluorescence
in a fluorescence assay with a masking dye in the supernatant
[0022] FIG. 3 shows the spectral excitation and emission for a
dispersion dye and the spectral absorption of the masking dye
[0023] FIG. 4 shows the site-dependent cell fluorescence without
masking dye
[0024] FIG. 5 shows the site-dependent cell fluorescence with
masking dye
[0025] FIG. 6 shows the suppresion of the background fluorescence
in a fluorescence assay with the aid of a separating layer
[0026] FIG. 7 shows the amplification of the luminescence by
back-reflection from a separating layer
[0027] FIG. 8 shows the wall fluorescence in a fluorescence assay
according to the prior art
[0028] FIG. 9 shows the suppression of the wall fluorescence in a
fluorescence assay with the aid of a masking dye and
[0029] FIG. 10 shows the suppression of the background fluorescence
or luminescence in a fluorescence or luminescence assay for the
investigation of receptor-ligand binding with the aid of a masking
dye in the supematant
[0030] FIG. 1 shows a reaction vessel 1 for a fluorescence assay
with a transparent bottom 2. A fluorescent dye solution 3, in which
the fluorescent dye molecules 4 are indicated schematically, is
found in the reaction vessel 1. The solution 3 is also designated
as the supernatant. The biological cells to be investigated are
arranged on a transparent support on the transparent bottom 2.
Light (excitation light) 6 is shone through the bottom 2 in order
to excite the cells 5 to fluorescence. A background fluorescence
radiation 8, which originates from the likewise excited fluorescent
dye molecules 4 in the supernatant 3, overlaps the fluorescent
light 7 emitted by the cells 5. Only the fluorescent light 7,
however, is decisive for the bioanalytical investigation and
analysis of the cells 5. Since, however, in all known fluorescence
analysis apparatuses the background fluorescence 8 is additionally
determined, small fluorescence differences of the cells 5 are lost
in the strong background fluorescence 8, which leads to a marked
sensitivity loss.
[0031] This disadvantage can be avoided by the procedure according
to the invention of FIG. 2 by suppressing the background
fluorescence by a masking dye in the supernatant 3. The background
fluorescence 8 present in FIG. 1 is completely absorbed in the
supernatant according to FIG. 2. The masking dye (schematically
designated by 9) added to the supernatant 3 can either be present
in dissolved form or in finely divided disperse phase
(colour-pigmented systems). Preferably, however, soluble dyes are
employed, because in this case the addition can be carried out
particularly simply with the aid of a pipette and because, in
contrast to a pigment system, the physical effects of particle size
distribution and of sedimentation processes and layer thickness
inhomogeneities do not have to be taken into account.
[0032] The following demands are made on the properties of a dye of
this type:
[0033] when using a soluble absorption dye, good water solubility
for use in biological assays
[0034] no membrane permeability of the dye in order to avoid
staining of the cells
[0035] high specific absorption in the excitation and/or emission
wavelength range of the fluorescent dye
[0036] no toxic side effects (avoidance of cell damage)
[0037] A solubility of >2 mg/ml is regarded as good water
solubility. The cell toxicity can be determined with the aid of
known test procedures (e.g. cytotoxicity test). FIG. 3 shows the
optical (spectral) properties of a fluorescent dye and of a masking
dye in a graph. Curve A shows the spectral distribution of the
excitation light, curve B the spectral distribution of the emitted
fluorescent light for the commercially available dispersion
fluorescent dye bis(1,3-dibutylbarbituric acid)trimethaneoxonol
(Dibac.sub.4(3)) and curve C the spectral transmission (absorption
spectrum) of the masking dye used (Brilliant Black BN, C.I. 28440,
Food Black 1, e.g. Sigma B-8384). It is recognized that the masking
dye is almost completely absorbed in the wavelength range of the
excitation and emission of the fluorescent dye.
[0038] Contrast enhancement or increase in sensitivity can be even
better understood with the aid of FIGS. 4 and 5. To demonstrate the
action of the masking dye on the nonspecific background
fluorescence, two video recordings of the same image section were
made before and after adding 100 mg/ml of the soluble masking dye
Brilliant Black in the presence of the potential-sensitive
fluorescent dye Dibac.sub.4(3) (5 mM). Both times, the same video
line was assessed by image analysis and the two fluorescence
intensity profiles shown over the identical sections in the
reaction vessel. The region Z in this case corresponds to the
region in which the cell layer, i.e. the biological sample, is
found, while right of this in the zone , to the greatest part, the
fluorescence signal originating from the supernatant is measured.
The measuring range of the recording system (8 bit) is between 0
(black) and 255 (white). For the unmasked recording, a contrast
ratio of about 1:3.6 results (intensity ratio of the darkest and
lightest image portions) and in the case of the masked recording a
contrast ratio of about 1:14.4. This corresponds to a contrast
increase by a factor of 4.
[0039] According to FIG. 6, an alternative possibility of improving
the ratio of useful to background signal is covering of the cell
layer with a finely divided optical separating layer 10. The
separating layer 10 expediently consists of a finely divided
inorganic white pigment, such as, for example TiO.sub.2 or
Al.sub.2O.sub.3. By means of this, not only the background
fluorescence radiation from the supernatant 3 is screened off, but
also the measurable quantity of cell fluorescence is increased by
reflection from the inorganic particles.
[0040] Alternatively, the separating layer can consist of polymeric
latex beads having a diameter preferably in the range from 200 nm
to 5 mm. Suitable polymers are, for example, polystyrene,
polyurethane, butadiene, acrylonitrile. The latex beads can also be
dyed with a suitable masking dye to which the same criteria apply
as for the absorption dye added to the solution (see above). A
suitable class of dye is, for example, .RTM.Resoline.
[0041] In luminescence assays (luminous cells), the requirements
fundamentally consist of detecting the specific very low light
intensity of a biological cell with high sensitivity.
[0042] By applying a reflecting separating layer 10 according to
FIG. 7, it is possible, analogously to the method for suppression
of the background fluorescence (according to FIG. 6), to increase
the luminescence signal of the biological cells. In this
connection, radiated fractions 11 of the undirected luminescent
light are reflected in the direction of the detector and thus
increase the specific measuring signal.
[0043] In a large number of other fluorescent test procedures on
biological cells, it is possible, in contrast to dispersion dyes,
to remove the fluorescent dye from the supernatant after staining
of the cells by solution exchange. The fluorescent dye FURA2-AM is
cleaved, for example, into the free dye after penetrating into the
cell and in this case loses its cell membrane permeability. As a
result, a concentration of the impermeable fluorescent dye in the
cell occurs. In this case, the fluorescent supernatant 3 can be
replaced by a fluorescent dye-free solution 3a without changing the
specific cell fluorescence. The non-specific background
fluorescence of the supernatant is removed in this manner.
FURA2-AM, however, stains reaction vessels persistently (wall
fluorescence) and thus produces another non-specific fluorescence
signal which is comparable with the background fluorescence of
dispersion dyes. This situation is shown in FIG. 8. In this case,
the background fluorescence radiation 8 is attributed to the
fluorescent dye molecules 4 adhering to the vessel walls. By
incorporation of masking dyes in the fluorescent dye-free
supernatant 3a, this non-specific fluorescence signal can also be
completely suppressed.
[0044] In FIG. 10, a working example analogous to FIG. 2 is
additionally shown in which the biological layer applied to a
transparent support at the bottom 2 of the reaction vessel 1
consists of receptors 12 which enter into a specific bond with the
fluorescently or luminescently labelled ligand 13 present in the
supernatant (solution) 3. The bound ligands are designated here by
14. In the case of the unlabelled solution, the primary light 6
shone through the bottom 2 excites the fluorescently labelled
ligands 13 and 14 to fluorescence. In the case of luminescently
labelled ligands, the primary light 6 is inapplicable. Analogously
to the implementation according to FIG. 2, a masking dye which
takes care that the fluorescent or luminescent radiation emitted
from the unbound ligands 13 is completely absorbed in the solution
is in turn added to the solution 3. The fluorescent or luminescent
radiation 15 determined at the bottom 2, i.e. the measured effect,
is therefore very predominantly attributed to the ligands 14 bound
to the receptors 12 and is not adulterated by the background
radiation of the unbound ligands 13 in the solution 3. The
measuring signal is therefore a direct measure of the strength of
the ligand-receptor binding. In this case, the layer thickness of
the receptor layer is in the nm range, while the dimensions of the
supernatant situated above it are in the order of magnitude of
several mm.
[0045] According to FIGS. 6 and 7, the separating layer 10
permeable to the solution can be employed in the investigation of
ligand-receptor binding in an entirely analogous manner to the
masking or suppression of the background fluorescence or
luminescence. In this case, the interfering background fluorescence
or luminescence is screened off by the separating layer 10 and in
the case of luminescent ligands radiated fractions 11 of the
luminescent light originating from bound ligands are reflected in
the direction towards the reflector and thus the useful signal is
increased.
[0046] The non-fluorescent or luminescent reaction component to be
assessed with respect to its binding strength in classical
pharmacological receptor binding studies was not shown here for
reasons of clarity.
* * * * *