From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
The
A Method
By
H
M.
for Visual
H.
EME
F.
e.g.
M.A.,
WILKINS,
have
w’hich
the
Light
have
classical
work
the
Soret
violet
light
technsic
ins the
potentialities
mains
probably
prefer
are
very
to use
a light
ins the
enzymes
are,
qualitative
It
likely
of
of the
ansd
but
not
of heme
spectrum,
suffi-
in a single
is often
of heme
bright.
source
amsd
used
or its
of violet
emit
light;
a comsvenient
a cheap
medium
most
w’orkers
will
more
will
of
ais obvious
been
arcs
lamp
solutions
such
he useful.
is available,
watt),
hut
a brighter
filter
that
may
the
tsecessary,
and
w-ithin
cells of
havinsg
mercury
mercury
Onse
its
method
a very
thens
pressure
visible
ins tissue
however,
estimation
is isot
tso examples
bright,
source).
bands
heme
bands
a monochromator
(e.g. Osira
125
filter,
high
M.D.
recordinig
are not
of the absorption
is to obtains
A light.
If
is adequate
such
as a compact-source
B.T.H.
250 watt
compact
chloride
founsd
not
CARVALHO,
w’avelensgth
hands
referred
to above.
heme
in insdividual
blood
cells
by
For
w-ill suffice.
problem
Cells
absorption
to identify
These
the longer
to estimate
have
in Living
of the small amounts
violet
end of the visible
a description
lamps
linse at 4047
pressure
lamp
high
used
or photoelectric
the microscope
band
as we
technical
filament
sharp
much
absorption.2’4
recognsised,*
tunsgsten
bright
Soret
but,
S. DE
AND
of Keilins.tm
of photographic
observations
with
is new,
The
bansd
PH.D.
estimations
extreme
as much
as 10 times
higher
thans
As a result,
it has beets possible
measurinsg
of Heme
relatively
beers
ciently
strong
for consvensienst
cell. The Soret
band,
at the
complications
simple
visual
Microscope
Estimation
COMPOUNDS
spectrum
sections,
Violet
be
iodine
expemisive
lamp
nsecessary
ins carbons
(e.g.
tetra-
s’ill suffice
although
copper
nsitrate
ins water
is sometimes
added.5’
6 It is
that. t.he illuminsations
of the microscope
be very
carefully
arransged
amid that
iso light
be lost.
A good achromatic
consdensser
should
be used.
With
a monsochromator,
ams image
of the lamp
is focused
on the insput
slit h)y a
most
importanst
lenss; the
nsear the
output
output
slit is focused
slit to focus
consdensser
diaphragm.
4 inches
focal lensgth
the
coisdenser
by the condenser
ams image
of the
With
should
oni the object;
ansd a lens
prism
of the monsochromator
the compact
source
lamp
and filter,
be used to focus a magnsified
image
diaphragm,
a distatsce
of 2 or more
feet
betw’een
scope
beinsg nsecessary.
Ans apochromatic
objective
is desirable
objectives
may
require
comssiderahle
tube
lensgth
adjustmenst
violet
light.
The microscopist’s
eye may require
a few minutes
tomed
For
to violet
light.
photomicrography,
From
the Instituto
Submitted
We
work
stay
January
to thank
wish
annd
for
ins Brazil
the
additions
Rio
de Biophysica,
20, 1953;
Professor
providing
accepted
Carlos
facilities,
and
of a light
de
Janeiro,
Brazilian
a lens of about
of the sosmrce on
lamp
amid micro-
as achromatic
whets
imsed w’ith
to become
accus-
of sodium
nitrite
ins w’ater
Brazil.
for publication
Chagas
for his
the
filter
is placed
on the
March
friendly
Researchs
29, 1953.
encouragensemst
Counscih
for
durimsg
this
nsade
our
hsavimsg
possil)he.
* Since
this
was written,
Huggins
(1865), to which
Dr.
W.
he referred
K. Metcalf
in 1951.
has
944
drawms
our
at.temstiomi
to
a paper
by
W.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
M.
Fm
J.
due
G.
C.
FIG.
l’:ryt
hsensoglohi
nunserical
tioni
F.
I .-Ilnnnssani honse mssarrow
White.
to
ml.
aperttmre
2.-I
lunsian
ins ervt
hrohlast
hsrohlast
ni. Light
Wat
honie
WILKINS
filters
r air-dried
snnea
nuclei
mssarrow
phase
ansd shows
nomsabsorhi
0.90
niunserical
al)erttmre
solid
S.
and
(‘omit ai ml gransinles
usv(l
to
sons apochiromssat
n’yt oplasnss
AND
snssear,
amid
isolate
-
X
nsg chronisosonses,
quartz
reflect
945
CARVALIIO
fixed
inn met
showing
hsaniol
ahsorpt
4150
approxinssatelv
-
ion
Prepa
A light.
I)r.
rat ions by
\Vhi(’hl
is
prohahly
()hjective
1.37
320.
air-dried
nuclear
DE
gransules.
ansd
unsfixed.
Cell
4047 A highs
i nsg objective.
at
t
Showing
left
censter
hiensoglohi
ns absorp-
is prohah
fromsi nsonsochromsiat
X 27(X).
ins amia-
or. Objective
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
946
THE
is necessary
to remove
is best to use
the solutions
by
the 3650
timsuum
at 4150 A degrees,
tions, thus obtainsinsg
rather
of
yellow
The
observed,
thus
a large
tsumber
betw’eenm
to adjust
to separate
4047
A amid 3650
the conscenstrations
a bansd of the
A.
of
cons-
the maximum
of the hemoglobins
Soret
hansd absorphigher
absorption
thams that
of the 4047 A line. The
solutions
light..
of the
avoidinsg
MICROSCOPE
visions spectroscope
way it is possible
cuprammomsium
ansd greets
advanstages
LIGHT
A linse amid constinuum
a small
direct
trial.
Ins this
It
additions
VIOLET
violet
light
fixations
of specimenss
may
technsic
artefact.
may
be
desirable
are
several.
No staitsimsg
be prepared
to
eliminate
Liviisg
material
is required
ansd examimsed
traces
and,
of
may
be
as a result,
in a short
time.
The
method
is, to a large
extenst,
specific
for heme
compounsds
(especially
if lack of
ahsorptions
with
white
light
is consfirmed).
The
technsic
may
be readily
transformed
imito a quanstitative
method.
Atsother
advantage
is that,
ow’insg to the short
w’avelensgth
w’ith w’hite
used,
light;
a gaits of 30 per cent ins resolvinsg
pow’er is obtained
as compared
i.e. effective
inscrease
of nsumerical
aperture
from
1.3 to 1.7.
Specimenss
not containsimsg
amid the gain in resolving
The
globin
Attempts
cessful,
to observe
but
he detected
cells
by
this
above
light
focus
used,
gramsules
cytochrome
method
show
may
tsegative
decreases
ins living
amid catalase
sufficienstly
large
exist.
It. mumst
give a most
deceptive
nature
of this absorption
ansd
and
be stained
so that
thens be utihised.
they
absorb
violet
light,
very useful
in stundyinsg
the distribution
of hemoamid ins consfirming
the existensce
of hemoglobin
in
These
containsinsg
ins hivinsg cells oftens
focus.
The spurious
the
may
may
method
has been founsd
ins human
erythroblasts
granules
ins the nucleus.
fixed
smears
(see figs.).
tive
heme
power
below-,
whets
amid in dried
absorption
ins cells
comscentrat.ionss
be stressed
appearance
is show-n
is largely
the
material
that
to
granules
w’hen out of
that it is posi-
of the
aperture
unsuc-
enzymes
refractile
of absorptions
by the fact
imsdepeusdent
consdemsser
w’ere
of heme
and
w’avelength
is opened
of
w’ider.
REFERENCES
1
KEILIN,
plants.
2 THORELL,
tion,
JOPE,
the
D.: On cytochrome
a respiratory
pigment
common
Proc. Roy. Soc., London,
s. B. 98: 312, 1925.
B.: Studies
on the Formation
of Cellular
Substances
London,
Henry
Kimpton,
E. M.: The Ultraviolet
Red
Blood
W.
METCALF,
of intracellular
WooD,
R. W.:
6
BOWEN,
E.
J.:
Cell, London,
K.:
A
sinsphified
hemoglobin.
Physical
Optics,
The
Chemical
1947.
Spectral
Absorption
Butterworths
technic
Blood
New
Aspects
of
during
of Haemoglobins
Scientific
(ed.
Publications,
yeast
1934,
2),
Oxford,
p.
and higher
Blood
Cell
Produc-
inside
and
outside
1949, p. 205.
and its application
spectrography
6: 1114,
1951.
York,
Macmillan,
of Light
to amsimals,
to
the
study
16.
Clarendon
Press,
1946.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1953 8: 944-946
The Violet Light Microscope: A Method for Visual Estimation of Heme in
Living Cells
M. H. F. WILKINS and S. DE CARVALHO
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