From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
A Simplified
Technic
to
the
of
Intracellular
W.
K.
that
troscope,
it was
matter,
possible
insciudirig
Sorby
tlesigned
either
be fitted
the
microscope
this
apparatus
see
arid
with
placed
and
Zeiss
marsirfactured
hut,
although
has
beets
ins the
small
adapter
to fit a hanid
little
work
was
on
work,
but
he
did
riot
not
obtains
absorption
It was riot urstil
1936
received
serious
series
gave
mathematical
nic.
of papers
Since
that
elsewhere
on
apply
his
him.
In
biologic
on ultraviolet
time,
a great
to the
deal
of w’ork
to
has
the
simplify
in the
which
at
(see
also
in the visible
spectrum
measurements
at isolated
wavelengths
was the first to use a continuous
source
irs the
The aim of the present
work,
initiated
ins
Spectromicrography
to develop
a simple
readily
haisdled
by
techniic,
ill
relations
to
From
the
a biologist
are
C.
clue
7, 1950;
to
Hanks
to
who
to
Mr.
J. M. Yoffey,
Dr.
C.
J. E. Dann
nsao!e
parts
traisiinsg
for
H.
Burch
of the
done
in
an(1 Mr.
of the
his
which
cats
arid
ins physics;
by
Burch6
Jope’7).
using
capable
asitl
corstenst.
series
Bristol,
to
only
of being
apply
this
of cells arsd
of stains.
its
for luis const
tint
England.
21, 1951.
tIue problem,
suggested
Mr. W. Keohtoroe
L. G. Cooper
for teclunical
1114
and
makes
focisssinig
be made.
The
range
assd
M:oy
apparatus.
arid
tech-
associated
rfhompsorsi;
visible
laboratory
University,
and
are
microscope
Cole
of a
tlepartmeuit
difficulties
publication
who
first
ansd possibilities
stirdy
the hemoglohius
with
the Romansovsky
Bristol
osi solutions
the
mater’ial
irs the
biologic
special
accepted
Professor
encouragement,
l)hysical,
A.
rio
of Anatomy,
November
and
nuatters
with
Department.
thanks
My
ins a good
thiat
ahi(1
has beets relatively
rseglected
apart
by Pollister2r
arid Thos’ell.26
Jopcr7
visible
regions for biologa’
material.
the autumn
of 1948, ss’as twofold:
of spectr’omicrography
the first instance,
to
their
staining
reactions
Subnsitted
advice
technic
available
solu-
labor’afor miru-
of biologic
reflecting
Barer,
tisari
published
mains
of
insvestigationss,
than
ultraviolet,
measurements
achromatic
problem
from
apparatus
beets
The
of a completely
iustroduct-iors
promises
limitations
spectromicrography.
with the lack of achromatic
objectives
difficult
and limits
the wavelengths
recent
other
of
of solutions
other
at tise Kodak
which
hse used
Casperssors7
use
desigrsed
spects’a
mater’ial
spectromicrography
consideration
ultraviolet
year,
great
qosanstities
Gretives
to biologic
applications
that
coult!
to feed
to a mici’oscope,
ahsorptions
apparatus
curves.
that
biologic
consideration.
large
Mr.
aften’
urstil Casperssoss’s
time.
In 1936, ,Jelley,rs
workitsg
designed
anid made
a grating
Spectromicrograph,
eralogic
made
spe(’tros(’ope
the
douse
svhsicls
of mics’oso’opic
named
for determinsirig
other’s
of
suggestions,
on’ (‘0111(1 be irsed
arid
si-Iso
pan’ticles
isis
spec’tros(’ope
spectra
still
1865,
slit. of a spec-
small
Followiusg
vision
Sorhy
irs
of very
disc.
microscope
absorption
chambers
an
used,
direct
light.
the
Sorhy’smethod
much
a
monochromatic
Huggitss’4
by thic
spectra
blood
of a binsocirlar
deter’mine
by
of a microscope
ahsorptions
built-
tube
Application
B.S.
suggested
of a single
Brosvnsirsg
the
first
Its
Hemoglobin
M.B.,
eyepiece
the
spectrum
into
to
the
to
the
solutions
tions
tories,
was
1)y replacing
and
METCALF,
PECTROMICROGRAPHY
stated
did
Spectrography
Study
By
S
of
for
their
assistaruce
advice
ins
aruci to
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
w.
A
modificatious
rather’
thans
matio’
light
citleol
n’tothen’
usetl
ats(1
to image
thans
gn’eaten’
the
of
that
system
by
originally
a direo’t
suggestetl
whio
feeds
the resultant
ounto tlse slit
microscope
photoelcctn’it’
method
olesignse(!
for
Us wtos
top)tortot
010
tlse
010
tO
be
inuolepenudenstlv
tot
will.
The
optical
1)enich.
siniiltor
ii’as
B
C
IRON
‘
prisnss
The
slit
two
out
swung
firsttllv
-MICROSCOPE
..
Tlsis
14 inschs
viths
was
the
denison’
, rsunuerictol
topert
fluorite
2 nium.
fluorite
toberrat
objective
iO)hi. No
extrtt
fitottersinig
path,
MICROSCOPE
F
TUBE
(Ic-
ouu Lo(’cotrnt
oil
separately
of its
returned
to
as
the
with
througlo
witlu
mentioned
H-
COLLIMATOR
PRISMS
onslv
field
set
ctire
wtos
UI)
riecesstory
tsic!
slit
without
wros
htovirsg
ionss
Thue
onse
thse
ensergenst
of
on!er
ictol
THE
thue
A,
Bot
of
eyepiece
the
This
10 cm.
was
inn length.
it coIns-
Is tons tschronua
t a rid to
toperture
I .3.
Thse
less
from
chronuutttic
difficult
t o nutot oh t lie
the
wtos
o’(!ge
for
tochronutot
riurnerictsl
heanu
center
ensergenst
of the
lens.
sul)stttge
since
it suffered
, tos
it wtos found
when
too
fielol
wtos
eye)iece
wtos
require(!,
the
ursniecesstory.
APPARATUS
beruch
to
of
the
constructed
to ctorsserto
stage.
etoch
a coniu)enistiting
U
as
of
of inntenssity
3,800-6,2(X)
niicroscope
portions
#{176}#{176}
the opt
used
a centering
t rico!,
of topproxinsut(tely
portions
telescope
goc)cl nuechtonsictol
was
irs
HOLDER
loss
of a siml)Ie
better
results,
in t he ruuicn’oscope
by
LENS
L-PLAT(
thtot
to spectrum,
nsoc!el
a small
givens
K’CAM(RA
c’oulol
posit
lensses.
tot tons aperture
from
objective
objective
vitii
since
the
1 .3 , tonsd to very
a)precits1)ly
wtos useo!
used
light
tine
prisnus,
tos to rusonsocultor
inninsersions
of the
tlnstl
prisuiu
telescope
spectrogrttpls
cttse,
lensgtln
ptossed
ure
ansol slit
original
J.
SLIT.
horizonottolly
prisnss
their
visions
oru a stans(l
for s)ectrornicrogrtt)hy.
the
checked
tube
collimating
LENS.
o)riginstollv
direct
flst)Unsted
that
tried
henschs
irs their
a
CORRECTING
G-ADJUSTABLE
ins foctol
above
gave
eyepiece
apptoratus
CO)hfl!)OnelitS.
were
ictol
to standtorcl
mounteol
OBJECTIVE.
LENGH
SETTING
Gretot
were
tonic! then
lenses
E’
tociuro)rnst(tic
used
of
its such
fillet!
which
uusicroscope
Ins tony
was
iiiisge!
1 .-Apparatus
lerss
nuicroscope
to give,
used.
spectroscope
aclsn’onuutotic
8 insclues
Tue
tollertorre
the
CONDENSER.
leusgths
spectrunni.
purpose.
The
wos’(ls, it was
A photographic
opt
was
SlIDE.
foctol
the
frorns
CONDENSER.
toclopteo!
(If
iisstu’uriuenit
light
Zeiss
spectrogra)h
this
ARC.
LUMINANT
This
scope.
monso(’hro-
1)
fn’ons
o)f the
Fro.
fount!
atlopted,
was
with
lat’gely
to st tirs(ltirol
of the
btosis
sttonso!s were
c!iffererut
D-OBJ(CT
tue
adoptetl,
(fig.
tor’ounso!
The
These
sttohs(!.
Nunuerous
A
tosseroubled
photonsicrogn’tophy.
httnit! spectroscope.
of
Hosggiuss
microscope
images.
lus other’
of the spectrograph.
APPARATUS
whole
betonus
by
his
simplicity.
‘F1se
f2().
1115
METCALF
Ctosperssons
photometen’ises
the
K.
enssure
rounud
the
perfect
horizontal
toxis
tolignsnuenst
of
of
the
the
niicro-
inchivio!ual
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1116
llTavelength
The
tore
spect
ortier
oiled
slide.
even
tonsd
of
showed
at-
5,500
t he
is c’alihrat
spect
iono
\letosurenssenit
ry.
rogrtoIlo
t
loave
0
o)f the
sheet
s nntoo!e
a
ro-
ons
resolut
I Inc
ions
of
A.
he
A suittthle
prisusis
nsicroscope
re!uceol
in
to
of
to
less
than
slit
were
riuicroscope
ruuetho)cl
microscope.
that
of
Tine
frons
of
thse
tnt
i(’t(l
toperture
ctouonel’s
object
nsetosurensenots.
chosen,
anocl the
out
swung
Tloe
011(1 olllject
pltoo’eo!
was
of
co)nsolensser
tonic!
illunoiinsttt.ions
to)
the
mio’roscope
shsowinng
blood
ive
tenoportorily
positions
it.
essential
for true
tol)sorptiofl
fiel!
(If the
speciruien
tOhs(1 thse
focus.
Iohler’s
the
coniolc’nisoer
tins eyepiece
leukocyte
relations
just
iruto
for
field
tonool t lie
cto.rryinsg
brought
adjusted
the
stage,
body
)olynuorphonuclear
slit
\tos
the
specimen
were
illumination
to
on
the
2.-l;sinsol)hile
cons(!enlser
wtovelensg(
photoriset
us
monocular
consclersser
of rnicrospectrogrtopio
Tlse
SPECTHOGttAPIIY
rendered
subsecuenst
tonso! 2,500
l)lace(!
as
risicroscope,
Fit;.
source
t hoe apptortot
A,
Aro inclined
illunuiniauit
irusure
on
3,500
t(t
slide
tine
tloe
the
OF
Spectrornicrographs
ohjeo’t
to
tos to light
siniul)lifie(!
tIIi(!
rts t tokens
the
A.ri
tore
,
of 6,00()
Taking
on
of tons irons
ururnecesstorv
iroru
TECHNIC
Calibration
use
graph
the
SIMPLtFIED
.-‘
of
the
ive,
a conit!itiono
ins(’Iinse(!
optictol
shown
nuonioc’ultor
ptot iO\vay,
ani(!
lIoo!y
piece
to
b-
Ctos-
removed.
of wioite
card
l)lacec!
ins t lie to!)!Iroxinuttte
l)ltorie
of the slit . The
focus
of t hoe riuio’roscopo’
wtos t liens adjusted
so thitot tons irnotoge vtos fornuued
on thoe white
card.
This
wtos thueno rcnssovoJ,
anso! t ho’ inoiage,
which
\vtos h50’,V
5Irosenst
015 thoe
grourl
gltoss
caruierto
screens
tot tlse P15(1 of t lot’ ect
rographo,
was
shstorplv
focussed.
inniage
\tts
spectrogrtoIhs
The
Ph50t
selected
cell
t hoe linse
wtos
oro t he
I Iii
nsove(!
hly nuetonos
ground
gltoss
rel)ltoCed
now
part
Inch’, loruger,
of
chotoniged,
prinstec!
svtoy (l3roc!e#{176}’ P
301
t he
tolonsg
of t iso’ rsno’o’htsnsio’tol
screeno
rel)ro’so’nst
to plsot
Ogrt9hsic
irsg
II.)
.
TIne result
side
the
nstode
ons t be s:onne
idlhi
swunug
of
l)lat
toIptort(tus,
exposure
prisruis
orue
I)y
the
tlf t hoe eel I iit hi t lue l)osi t
nOW
wtos
wtos
tonoy other
toni(! to furt
Pltote
c!ge
screens
ussovinig
Iltot liwtov
Thse
usual
gltoss
\Vitisout
c)microlgr:o
(fig. 2).
A st-eJ)
liv
si t(ge
SO t iotot
t Iso’ posit-ioro
of
its
tue
slit
grouuod
hist((!e.
O)1)t ictol
it
The
cro)sseo!
inst o posit
ion
n’o)grajlhi
tlue
prisniu
iVt(S
tono(l toro exposure
,
11(1W sivunog
Illat
e
.
‘Fhiis
rosa rko
I lie
arid
ant!
e
was
t hoe sI i t l)r((’ isely
of
t hoe spect
of interposirog
slit
it
sllet’t
was
to gist’
the
into
result.eo!
I i ns reltit
rogra)is
developeo!
a series
its a
ion
to
t-tsken.
in
the
of photo-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
w.
graplus
of that
senntet!
the
Tue
portion
of the
al)l)earanCe
difficulty
microscope
at one
noted
K.
field
particular
1117
METCALF
isolated
by
the
slit,
each
orne
of which
repre-
wavelength.
1w Jellev’6
of overexposure
of the
red
accompanied
by underex-
posure
of the violet
due to the greater
dispersion
of the prisms
for violet,
was not- so marked
with thse irons tore as it wris svith the filament
lamp,
owirsg
to the very
high
innternsity
of marsy
of the
lines
emitted
l)’ the irons arc at the violet
end of the spectrum.
This difficulty
was
completely
overcome
by the use of an ultraviolet
filter for a portion
of the spectrogra)hic
exposure
time.
Tine constinnuous
spectrunus
of the tungsten
bulb
was also
made
more
even
by
the use of an Ilford
Micro.
2 filter
for tons additional
part of the exposure
time as well as
1)V slighst.ly
of
tine
over-running
order
As test
ob)jects
cells.
The
UI) o!uite
well
red
Inn order
mine
the
metons
of
the
the
at
consisted
stage,
spectrograph
and
placed
in
the
on
middle
a sensitive
source,
using
apparatus
the
violet
all
the
filters,
etc.
were
of
unstained
of the
spectrum
,
end
humton
showed
it became
necessary
to deterin the spectromicrogrrtphs
by
esserstially
a one
the
of
and
stage,
it.
of
a half
onto
Behind
a constant
light
objective
used
inch
a screen
with
slit
placed
the
The
microammeter.
a 6 volt
duty
by
Potter#{176}2 in
microscope
to
1933.
whole
of
any
which
the
of
was
this
source,
to
a
project
a vertically
mounted
a photoelectric
apparatus
on
cell
was
de-
mounted
the
l)laced
latter
lamp.
It wtts
a circuit
sinssilror
between
the
and
effect
on
to
fitted
of the
to
absorb
apparatus
the
traversing
spectrogrttph
supplied
to
thtot
light
source
sonic
of
the
on photocells.
spectromicrographic
btor
the image
using
wtts
an adverse
tine
to tommy
a microscope
in
chargei
of
such
used
arid
to entthle
stage,
used
being
overheating
htve
one
lenses
was
were
A svtoter-conottoinsing
prevent
was
chttnge
I)ulh
which
radiations
nssicroscope
mechanical
4 amp.
accumulators
infrared
propriate
tine
time
bench.
suggeste!
The
with
of hensoglobin
This
situated
to
a light
tIne
banud
a mechanical
slit
hsttrmful
taken
spectrographs
microdeonsitometer.
heavy
and
first
absorption
with
As
exposure
oni ordinary
a
optical
fronss
with
control
to be moved
tIne
ap-
of
the
knob
accurately
across
slit.
Tue
first
noected
1w
the
consnsecte(!
tue
total
visual
extominationo
of the negatives.
1)lOt absorption
curves
from these
negatives,
relative
degrees
of density
of the absorption
bands
adjusttoh)le
vice
The
to
of
inosage
bulb.
minute.
Soret
microscope
ron
the
of one
photoelectric
to a 0-74
photocell
This
a vacuum
microamnieter.
perio(ls.
It.
was
tried
and
a barrier
layer
was
obviously
far
a battery
driven
SVtLS
This
coupled
proved
finally
stable
extrenunely
device
microamnieter.
by
perfectly
repltoced
very
adequate
by
had
photovoltaic
insensitive
cathode
but
cell
con-
so it was replaced
D.C. amplifier
to the
follower
a tendency
driven
a battery
selenium
too
to
instability
photomultiphier
over
tube.
This
long
proved
senusitive.
PROCEDURE
Using
it was
by
tine
the standarc!
possih)le
slit.
st#{149}hoichfell
to
(!ennsitometric
nietosure
the
This
could
onuly
within
the
intennsitv
photographic
ensulsion.
l)loo)tonsneterize(!
to
on
enable
method
optictol
be
(lone
tsnsv
with
density
at
the
range
of
onse
negtttive,
too al)sorptionn
the
of any
usual
wrtvelength
the
be
the
about
with
(Brode#{176}
cell
protoj)lasm
lines
of
lines,
drawn
the
those
portions
sufficient
to
of
of
straight
curve
precautions
portions
emitted
gammto
onue
a fair
by
the
curve
hundred,
degree
HO
crossed
tore
of
tiue
can
be
of certoointy.
REsULTS
Thie absorptious
Oxyhemoglobiun
much
its
strouiges
o’euster
at
ausd (‘custer
sous,23 ,Jope,h7
a pou’phoryn
has
spe(’tra
of hsemoglobius
an alpha
batid at 5,790
ahsorptionn
4,150
at 5,590
i)arid
A, whilst
etc.).
This Soret
riusg of conjugated
the
iii
A. Ous rethr(’tiots
the
anid its derivatives
are well kniown.
A, a beta baund at 5,420 A, ausd a very
violet,
the
to hemoglobin,
Soret
bausd
moves
gamma
the
alpha
to 4,250
or Sor’et2t
aund
beta
A (Gamgee,rO
band
with
bands
fuse
Roi)ert-
batsd is shown
by solutions
of all compounds
with
double
h)onsds arid a ceustral
iron atom
(.Jope’7),
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1118
A
SIMPLIFIED
TECHNiC
OF
SPECTROGRAPHY
arid thus is showni
by oxyhemoglohin,
reduced
hemochromogens
hematiri
parahematius
arsd
with
hydrogens
distslfide,
hydrogess
peroxide,
,
hemoglobins,
methemoglobi
nnitric acid,
,
o’arboxyhscmoglobins,
ui a usd i ts o’ompoirnsds
eto’. (Keilius
arid Har-
treero),
Hemoglobin
in
Ins 1877
ansd
hemoglobin
blood
beets
Cell
1879,
from
The
refuted,
lysis
of the
but
suggested
(‘ells.
evidensce
forward
he
and
the
effect-
the
had
si-as revived
Keilin
Spectrographically
inn solutiorss
w’hich
that they
although
that
put
hemoglobin
hint his theory
ley’ on the groisusds
siosns of erythroo’ytes
A re Identical
Hoppe_Selyeri
differed
corpu(’les.
Solution
and
were
the
put
that
fon’was’d
ins 1941
instn’ao’on’ptsso’ular
by
lysis
for’ this
by Atlams,
purely
an
same
optical
year,
Macalbum
ret!
long
sissce
anid
Brad-
fr’om suspensappareust
055
counfirmeol
oune,
of tine
huts
uruable
to obtains
the Sou’et bansd
Soret
hand
immetliately
i)eo’ame
Hartre&#{176} its the
was
theory
obtainsed
their
although
they
work,
si-er-c iris-
WAVELENGTH
FrG. 3.-Absorption
spectra
of two
unfixed
hiunutons red
able to explains
it. Sissce that
time,
Cole,#{176}
arsd Thos’elP#{176}have
wavelength
of the mercury
litnes 4,047
A assd 4,358
A, instao’t
light.
Ilyina
cells
showed
ol)tainsed
the
Soret
is clearly
fs’om
hansd.
sohstionis
of actual
The
shown
experimenstal
certaiunly
hemoglohius,
have
a mixture
Soret
of the
arid
Theu’e
readinugs,
methemoglobin,
their
3,
Son’et
appears
to
is therefot’e
harstl
are
due
insac(’uracy
of any
curves
; secoundly,
of
varying
ausd
maxima
ausd
at
probably
slightly
quantities
traces
differeist
to
at the
al)SOrb
ansd fiusally
of irsta(’t ret!
at
the
expected
he
idetutical
tio
evidensce
that
as they
are
differ’ from osse assothes-.
graphs
have beets left deliberately
factors.
Firstly,
the insevitable
the chances
of ohtairsirng
smooth
is almost-
pn’esenno’e
irs figure
of hemoglobiui.
ans(I extracellular
hemoglobits
The sharp
peaks
of the
which
shoivus that
ret! cells tb
Jopeni
usinig a coustirsiroiss
sour’ce
(‘olsfin’med
tlsis ai)sor’ptions,
ct al.hi h)y Liii irigensious
device
showed
that esens suspesssiosis
w’avelensgth
sults
cells.
with
a o’ombiuiations
photographic
the hemoglobin
of
oxyhemoglobin,
of
other
wavelensgths.
that
irstrathe
re-
of several
te(’hniie
reduces
(‘ells
in these
retluced
den’ivatives,
Smooth
all
curves
of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
K.
cats
however
plotting
he
the
readily
obtained
by
METCALF
averagirsg
HEMOGLOBIN
BETWEEN
EOsIN
Manuy
workers,
w’lseus doitig
smears,
series
by
literature
estimate
the depth
as to the
Lillie2#{176}has
showun,
the
readiusgs
ins steps
CONTENT
differential
the
house
amouust
otnt
an
extensive
cosiur
azure
STAINED
OF
marroii
CELLS
coinrits
of hemoglobins
of
both
the
bands
barsds
the
find
of
Romarsovsky
eosiuss
austi
its the greens arstl red
shsoisld thseor’etically
cells
550
banid
are,
with
44
methyl
os’sky staiins,
ani(l the P(’ttk
45
46
of hemoglobini,
appreciable
Fr’om matirn’e
peak
43
spectra
of course,
its the
antI
Inas
compouuntls
regions
insterfere
of tise
with
CELL
4-Absorption
Soret
stains
erythsroid
stains
thse azure
‘-.
42
the
nno evidence
CELL
FIXED
41
AND
ous Romansovsky
ins cells
however,
stuthies
staius,
has-c separately
ideustifiable
al)sorptiots
spectrum
respectively.
Neither
of these
the
arid
STAINING
of eosiss staiusinsg.
I conk!,
validity
of this methiod.
carried
ins sU(’h
Fr;.
A
of 50
averages.
RELATIONSHiP
stained
1119
48
49
of
fixed
on
effect
(fig.
ervthrocvtes
50
Soret
52
red
chemical
in methyl
the
SI
human
unless
dissolvet!
alcohiol
47
53
54
cells
55
56
baustl,
so the
si-as
57
stained
58
ansd
comh)insationi
alcohol,
-.
q
iusvestigatet!.
SI
unstained.
occirrs.
effects
o
Romanovsky
of the
of fixation
It appears
to have
4).
stainsed
with
Wright’s
modifio’ations
of the
Romaus-
absorption
curves
were O1)taitsed
which
showed
both the Soret band
due to eosirs (fig. 4). lus these cells, the ratio of tise height
of thse Soret
to flue height
of the
cosius
peak
was
conistanst
withins
the
limit-s
of the
tech-
tu.
Humans
borue marr’osv
smean’s is-crc
aniti (‘ytoplasm
of cells of the erythroid
globius
cell
cousfeunt
its this
amounits
being
as
iuntlicatetl
series,
of hemoglobiss
preseust
its the
a late
by
the
stainset!
with
sen’ies were
height
of
the
Wrighst’s
stairs ant! the ssucleus
iusvestigated
as to their hemoSoret
peak.
The
orthochromatic
erytht’oblast,
coistairset!
both
mi its (‘ytoplasm
arid its its nucleus;
nitrcletns
(fig.
5).
most
mature
considerable
less, hsowever,
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1120
A
It must
SiMPLIFIED
be usiderstood,
however,
up of the actual
tent
of those
smaller
small
portions
consceritratiots
erythroblast,
still
suggest
that
ther
less
rocytes,
there
was
height
to
which
was
present
hemoglobius
plus the
conutenst
hemoglobin
lie above
ansd
its an
irs a polychromat-ic
is-hens
hemoglobin
ratio,
Soret
cells
of the
However,
apparerut
cositenit
cytoplasm
present
more
The
of all these
the
peak.
is-as
SPECTROGRAPHY
ear’hiei’
arid
Soret
the
irs the
peak
corresponded
peak
state
nucleus.
to eosin
being
44.3
of affairs
below
ratio
±
per’
irs the
tinud
poitut
3.7
nucleus
censt
fur-
corsstasst
its the
si-as
it is-as
n’etiuir’es
s’emainsed
to the
it. A
ins tlse o’ytoplasm
would
)o55ihly
type
This
peak,
of the
cons-
orthsochsu’omatic
er’ythn’oblast,
ins a proerythroblast.
last cell type
there
si-as more
hemoglobin
The absorption
curves
of this last. cell
investigation.
cytoplasm
of the
of hemoglobin
only just detectable
Isi all except
the
than
ins f-he nucleus.
OF
that
the
hemoglobin
is made
nucleus
the
TECHNIC
matirre
that
for
er’yth-
of tine
cosin
conssitleretl,
it was
7
‘06
‘05
0
NUCLEUS
E
CYTOPLASM
-
0
03
02
01
0<
;
WAS/Ft
38
Fr;.
39
4041
42
5.-Absorption
found
that
tso apparent
43
44
45
spectra
of
FN(TH
46
47
48
ruucleus
49
5051
and
there
was relatively
much
correlatioss
between
the
more
tivo
52
53
cytoplasns
54
55
of
56
57
58
eosins thass hemoglobin
sets of figures.
Thus,
siderinig
the cytoplasm
of erythroid
cells is it justifiable
to estimate
hemoglobits
by the depth
of eosins staining.
Outside
these limitationss,
of the method
is very
opens to question.
THE
GRANULES
The eosinsophilic
isles which
stains
between
to
suggest
IN
THE
leukocytes
deeply
with
E0sIN0PHILIc
GRANULOCYTES
59
6061
orthochroniic
OF
62
erythsrobltist.
arsd thser’e was
only si-hers cnithe
THE
amounit
of
tine salidit-y
CAMEL
ins the camel
constaini
chiaractes’istio’ally
oval gsanseosiss. The similarity
ins shape
arsti staiusiuig
n-eactiori
these gransules
anid the circulating
eryfhrocytes
led Durans-.Jorda
that
the former
gave rise to the latter’.
Absor’pt.ions
cur’s-es
ins 1947,
fs’om cells
camel’s
blood
showed
that. the oval erythn’ocytes
had typical
Sos’et bassds,
but
that there
si-as iso trace
of the Soret
band
irs the curve
of the eosirsopisile
gr’antslocytes.
This
does isot, of coun’se,
definsitely
disprove
Dun’ari-,Jortla’s
Isypothiesis,
of
but
leaves
the
theory
supported
only
by
the
oval
shape
of the
gi’anuules
arid
of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
K.
ii-.
the
erythrocvtes.
fur’thscr
It
stage
of
could
(‘ors(’eiiai)lv
developmeust
1121
METCALF
still
dus’itug
h)c postirlated
wisich
that
hemoglobin
there
si-as
was
some
formed
its
the
grtsuiules.
Atu iustcresting
sideliuse
to thse ai)o\e
region
4,350 A in the eosiunophile
is the absence
of any absorptioss
maxima
grausirlocyte.
Agnen3 extracte(1
vertloperox-
ins tine
idase
from
wavelength.
emi)yemtt
pus
He stsggested
particinlarly
appear
the
to h)c
eosiusophils.
such
t50
Fiusahly,
the
From
sirbstansce
IN
hEMOGLOBIN
formations
austl sisoss-ed
that
it had an ahsorptions
that
it came
from
the grassules
of the
RED
ability
(1F:LL5
of tiried
was
absou’ptious
CAN
is-hens
iuise.stigate(I.
Figun’e
curses
ins (‘amel’s
CARBOXYHEMOGLOBIN
up
o’ari)ors
UNSTAINED
Fr;.
42
6.-Absorpt
carbons
43444546
ioni
474649
sl)ect
rto of
5051
sttoinse(l
siould
mounoxitle
withs
the
for a shiort. time its nun atmosphere
that
this (toes ins fao’t occur’.
5253
54
015(1 unsstainie!
rIhe
-
STAINED
394041
thsere
TO
to take
pla(’e(l
6 shows
shownn,
eosiusopisils.
(111.-iNGE
1)100(1 tells
of carboxyhemoglobins
of (‘otol gas
the
pu’esent
peak
at that
graunnlocytcs
--
55
red
56
57
cells
5859606i
tofter
62
treatment
with
nnsonsOxi(le.
Soret
i)austl
and
uuan’rower’
(Iiffets
Tue
other
of
abso)r’ptioun
from
those
tius(I ius isas-irig
cun’se
sisows
of
that
o’an’boni
shossun
oune mains
eveti
l)t’(’\i0115lY
ceuster’
fixeol
ausd
iui hx’iusg
at 4,200
staitned
much
higher,
A iusstead
steeper
of several
er’ytiirocytes
remains
peaks.
ctspable
mounoxide.
SurrArn
I
A simplifieol
.
2.
3,
asud
is unot affected
4,
‘I1he ratio
en’vthsrocytes
.5, There
t’tst
ili
6.
fs’om
microspe(’tn’ographs
for
A uionsre(’o)n’diuig
mio’r’odeussitometeu’
The Soret
i)ats(I of hiemoglobius
the
Both
the
by fixinug
tue
(If
aui(I
is
the
tlried
cytoplasm
(‘ous(’euntrationn
of immature
hsemoglobius
gr’austslorytes
titstl fixed
atmosphen’e.
irs the
visible
r’ausge
is described.
the ab)ove is discussc(l.
iun inidividual
erythrocytes
or’ staisninig.
hsemoglobiuu
detectable
t50
eosirnophsile
use
for’ use siith
is (icmonsstral)lc
aul(I stainned
or
to)
staiuniusg
CO5lti
n’etl cells
i)ut
sertlopert)xi(Itise
usot
ito
is conist.anst
its the
in
nsucleiss.
thie grarsisies
pres-
of thuc o’amel.
eu’\’thit’o(’ytes
Oats take
tsp (‘ttrh)Ons mousoxide
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1122
A
SIMPLIFIED
TECHNIC
OF
SPECTROGRAPHY
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G.
1 ADAMs,
2
3
A. ,
oxyhaemoglobin.
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BARER,
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0 COLE,
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by
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BRACKETT,
ultraviolet
the
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:
Beitrage
13:
zur
85-87,
new
16
Note
W. :
E.
JoI’E,
: The
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ultra-violet
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AND
vol.
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rays
ion
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of the
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solar
spec-
Roy.
Soc.
LtOII.
& Clins.
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Blutfarbstoffe.
of
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microscopic
D. L.
RUBINSTErN,
spectral
: Secretion
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E. F.
London
AND
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mi-
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the
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inside
Butterwort-h,
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A. :
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the
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arid
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der
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19 KErLIN,
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no DURAN-JORDA,
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violet
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A. A., RAvrKovrcH,
R.B.C.,
21
the
Scient.
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Rend. Acad. d. Sc. URSS 48: 325, 1945.
JELLEY,
E. E. : An improved
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1951 6: 1114-1122
A Simplified Technic of Spectrography and Its Application to the Study of
Intracellular Hemoglobin
W. K. METCALF
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