0022- 1554/79/271

1-1531$02.00/0

JOURNAL

THE

Copyright

OF

©

HISTOCHEMISTRY

by The

1979

AND

Histochemical

Quantitation

Vol. 27, No. 11, pp. 1531-1533, 1979 Printed in U.S.A.

CYTOCHEMISTRY

Society,

and

Inc.

Resolution

in Electron N.

Department

Electron This

can

microscopy

radioautographic difference

in

NADLER

ofAnatomy,

resolve

technique

J.

resolution

(BR

McGill

structures

is able

to locate

is

what

Microscope 79-131)

University,

Montreal,

with

accuracy

radioactive

label

creates

the

Radioautography’

the order much less

of

with

problem

of

Canada

of

0.2-0.3

nm,

certainty,

quantitation

in

but

the

at best 50 nm. microscope

electron

radioautographs.

Normally, silver opment hit

by

a second

crystal

these

grains

in the

exclusive

The

a grain,

grains

and

constitute

the

radioautography. grains

to

sponsible

the

various

for

these

limits

of the

after

produced an latent

one

silver

grain the

The

other

radioactive

be

crystals, crepancy

to one

Only might

the

is not

only

shared

other uted

re-

content

group

silver is created

is sufficiently

inescapable

much

limitation

Council

work was of Canada.

supported

in the

the

resolution

at the

this

method. silver

particle. not

necessarily

contribute

it

arise of

However,

if to

may

be

easy

enough

the

same

crystal.

has

a

number

from

to resolution

that

nm.

than may

to do

with

by a grant

from

the

Medical

one

emis-

to

adjacent and this

grain

dis-

of quantitation.

the

site

of a grain

radioactive

as

that

long

label

which

as

grain

there

could

is essentially

is no

be attrib-

exclusive

and

radioactivity radius

size

of

this

observations advanced dure,

solid

probability

by

isotope

this

about

50%

probability

and

the

can

sources subtended will

emit

Downloaded from jhc.sagepub.com at INDIANA UNIV on May 10, 2015

versa,

a circle

is for

the

of grains

radius

of each

the

larger

probability of

a point

in that

source

direction,

sources by

line on

the

of the source, as the proce-

100-150

circle as

of

experimental

is about

on

of

circles

Estimates from

such

theoretically

analysis circle;

all

enclosed grain.

a

will

but will the

boundary

probability,

miss for

radius

half

derived

radius

are On

will

50%

are

a resolution

radius

which grains.

rendering

are

derived

If the

of radioactivity,

that

grain

responsible of

grains,

circle

greater

are from

1531

circle.

been

center

used,

use

with

size of circle

the

is shared.

grain

scattered about an artificial and coworkers ( 14). Depending

emulsion

one

angle

radioactivity

drawn

boundary

the

of small

have

a set

a resolution the

structure

that

circle

be

exclusive.

proportion of grains of analyzing these

probability

for

must

than grain

structure

could

usage

the

the one

to make

shared

Common

of grains Salpeter

one

a grain

within

all sources

more

with

of

is only

boundary Vice

close rather

structure

size

which

responsible

that

than

is what

relatively

a radius

center

to reduce the the problem

of grains.

complicated. is,

grain

if there

more

a resolution

greater

of how

any

grain;

radioactivity

to

the

that

of course,

with

decision

about

are

Shared?

it a shared

specify

to that

number

radioactive

Research

draws to

hand,

Alternatively, the

call

one can expect thereby simplifying of

more

extend 100

to

if there

other

represents

be a linear

tissue.

the

site of

end,

problem,

certain include

of the

But,

is a Grain is the

structure

be assigned

grain

deposits

in the

is

even

depends

in the radioautograph

vicinity, the

way

example,

membrane.

so that

consideration

circle

sources

not

the

this

of the

Rather

Thus,

label arising

probably

next

is small, shared,

its the

silver

order

the immediate

membrane

boundary

for

I This

overlying

a second

The

grain

beyond

smaller

will

small,

deposits

directly

(3).

microscope

near

For

give rise to a grain

structure to the

grain.

have

in practice,

in the

by

nm

radioactive

electron

of structures

a shared

may

is exclusive;

and

ordinarily

locally

will

a confluence become

2000

solely

However,

the

released

shared.

may

ines-

silver

silver

grain,

of the

of radioactive

To

of

achieved.

extend

crystal,

radioautograph

ma-

be

the

can

of punctate

and power two

of development

by

to

in

are

can

chemical

deposits

a radioactive

of silver

that

which

process

number

in the

there

to

the

gap

resolving

development,

the

be

grain

until

the

to

method

sites

to stand

that

radiation

and

of the

(15). have

When

microscopy

(7, 9). As a result,

original

the

used,

resolution

necessarily

photon

photographic

radioactive

remain

not

in a membrane

label

the

the

is precipitated

compact

by

electron

filamentous

reason,

silver

problem

reduce

resolution

crystal

of the

hit

if there

the

so adjacent

$ particles greater than

energy and

as the

the will

emissions, directly

at distances

long

from

between

does

low

size as presently

directed

a gap

The

reduction

be

the particles

as

of radioautography

shared from

the

energy

crystal

can

radioactive occur

result

Hence,

Radioautographic

to

of

physical

Here,

density

identify

the

to improve

of this

this

a variable

of the

assign

attempt

a filament

size

For

crystal

deposits

function

to be derived

higher range.

on a finite

structure

with

Of

course,

of

necessarily

Even

can certainly

sions

microscope

somehow

of the

chemical of

bromide

a new grain,

from

to

ultimate

in

borders

However,

The

Notwithstanding,

ordinary

the

and

the

single

the

solution

(8).

image,

beyond

the

silver

by the

advance

grains

in electron

containing

a process

150 nm.

of

shared

is assumed

techniques

to

to at least

order

such grains.

to assign

must

specifically

resulting

limited

grain

one

not

site.

of

it is called

than

direction

may

“H-, grains

not

the

by

precipitation,

more

could

random

image

in

a variety

case,

and

greater

contained grain

which

called

technique.

is that

produced

over

if each

where

Resolution Reaction

or more limitations

One

simple

the

quantitation.

label

in quantitation

is

radioautographic

capable

be

structures

between

dioautography,

for

of grains

to

be proportional

basis

of radioactive

are

consideration

resolution

the

number

Therefore,

will

tissue,

analysis

to the

first

radioautograph

is not

grains.

Limitations

The

the

radioactive

low.

be possible

each

hit,

apt

grain

structure-in

problem

Since

radioactive

not

latent

is sufficiently

in cases

these

devel-

particle

would

it may

structure

range

chemical

radioactive

the

However,

a photographic

after

that

in the

task

upon

silver

to a single

grain.

particle

one

proportion

exclusively

neighbors

is

energy

relative

structures.

attributed

the

if the

is to correlate to the

definite

and

content

objective

structures

one

11, 13),

radioactivity

The

radioactive

produces

ofdeveloped

the

an

one

crystal

(1, 5, 6, 10,

number to

a hit

bromide

to

95%.

nm.

enclose

Estimates

basis

that

represents

the

chance

as advanced

by

Granboulan

the that

1532

NADLER

(6).

Practically,

depth

assuming

an

section,

and

of a tissue

crystals

of

from

the

solid

angle

given

diameter,

center

of the

into

account crystal

not

grain,

the

decision

real

complexity

exposed

angle

estimate

for

circle

in analyzing

the

on a desire

radius

source.

that

the center

of the

(12).

Whatever

to include

mean

ent

proba-

small In

as possible the

The

third

what

proportion that

author

Both

program

author’s

Thus,

first

attributed

to

equations

equal

taken

also

tures

(such

that

circles

probability

another

set

dispersed

structures. content

ratio

of the

the

the

cells,

and

‘251-insulin

as

degree

of dispersion.

tamed

by

graphs

(the

measuring The

same

is corrected to circle-hit the

(4)

point-hit

and

array

in the

and

circle) circles

radioautographic

the

which support

fewer

than

tion

boundary

RESULTS studies

relative the

to

merits

that

much

nearest

AND

reported

of the

possibility

very

be

two

either

simpler

direct

ob-

may

photo-

analysis).

Composites

method

were In

one each

for

grain

analysis

of merely

prepared

analysis may

scoring

compare

and

of different

shapes

tographs uniform

display

tration

varying

each of grains

were

analyzed

among

the

in all

structures

for

square.

In general,

there

of results

derived

sets

amy circles assumed

analysis.

The

for 50%

(100-nm

true

content

turn

results

were

the

to

be

tested

radius) of label

method or 95% being

differences

either by

Chi-

among

resolution radius)

proportional

ideal

to

differences

(230-nm

a

distribution

if subjected

using

was concen-

These label

for

no significant

by either

there

structures.

radioactive

out were

radioau-

that

by a uniform

randomized

what

would

method

hypothetical

be produced

the

to obtain

at

15 different com50 nm), but

assumed

the

boundprobability.

to the

relative

using

results

direct

scoring.

and

were

that

of

no significant

or

230-nm

radii

that

be

sources and according there if the subject

ing

maxima

(as

the

more

circles), by

each

complex

be

shared however,

proportion

be for

counts or

methods

L, Salpeter A quantitative

should

be acceptable

a 100-nm

the

230

achieved

closest

about

or

as

less

to it is as valid

±

25-30%

of

direct these

opposed

shared

to grains

than

shared

100 nm simply

of the

(radius

than Under

these

say

circle.

size

grain accurate grain).

if

resolu-

radius

circle

of grains

These

radius

grains,

small,

proportion

defined

grain grain

some

of

circles.

of striking

none;

degree

proportion

a 230-nm

using

the

boundary

be even more center of the

are

to the

as the

boundary

the and

by direct

that

or of a filamentous

should to the

there

is that

30%

affect

obtained

inversely

using

not

of Blackett

results

a probability

might

where

ignored

radius)

does

anticipate

scoring

than

reflects

bound-

50-95%

method

The

shared

a resolution

50 nm

resolution reflecting

resolution

direct

are

of the radii,

the

might vary

finite

10%. not

radius

resolution

by scoring

structures

for

quantitation

The

exceed-

as

available.

LITERATURE

size (about In

it was

as would

were

reso-

the

cases,

source,

(12).

or fewer

theoretically

1. Bachmann microscope.

to the

distributed

cells,

using

and

100-nm

size

by

One would

notion

(±

scoring

author

of all grains

by the

composites of five in size (50-5000 nm)

there small

in number.

composite,

of radioactivity

grains

different varying

in another series each of the same and

involving

structures

33 but

shared

the

either

scoring

to explore

according

Kupifer

by all

follic-

scoring.

radioactive

different.

proposal

the

the

be substituted grains

of hypothetical

series there were of different shape,

and in number; and, posites of 5 structures,

The

methods complex

to

using

that

the

of the

circle,

crystal

touched

attempted

structure.

random. structures,

four

have

radii,

There

case.

100 to 230-nm

of counts

no

the

50%

circumstances, the

DISCUSSION

here

and analyzed

in

from

in thyroid

obtained

or by direct

as a corollary,

boundary

The

are

the radioactive scoring (strictly

are

on

using

obtained

nm

used

first

indicates

for direct

by the circle-hit

numbers

or

differ-

analysis

Fe

were

those was

obtained

range

also

75 nm)

representing

circle-hit

of points

the

were

grains

structure-

the

(2) or by that

original

radioac-

instance

the

circles

of grain

Moreover,

the

shared

for that

volume

boundary

an

as used

in each number

compact

true

the

in

results

of enclosing

studies

struc-

involving

of relative

with (12)

(2)

cells,

100 nm or 230

author

interpretation

grains

be

in resolution more

glial

the

evidence

in

of validity

to

grain

in hepatocytes of

probabilities

Parry

nonlinear

needs

significantly

of

involving

and

statistically Parry

the

circle,

scoring

grains

of

counts

in computing

Hence, ary

content

of dispersed

grain

and

not

mean scoring

CONCLUSIONS

of the

a structure

be enclosed

factor

boundary

with

of relatively

the

among

resolution

unknown).

system

volume

differences

among

of shared

a

of

the

by direct

radioautographs

experiments

by the

true

Account

likely

shared

resolution

superimposing

is

volume

structure

the

result

used

number

number

same

proportion

compared

were

method

investigations,

circles

method

structures total,

obtained

the

structures,

circles.

of

boundary

evaluate all

5% of the

results

either

to For

eliminating

than

of no radius)

in neuronal

of the

is the

associated

the

a given

higher

structure

relative

the

by

a com-

grains

less

“H-N-acetylmannosamine

Blackett

rationale

(which

unknowns.

Accordingly, to any

The

to the

more

same

falsely

point-hit

point-hit

(radius

that would

of each

to be attributed

of

derived

Finally,

biological

ular

was

circle

it possible analysis.

of 37-56%;

order

obtained

assess

methods

requiring

of shared

count

plus

number

than

yielding

tivity

the

probability

structures,

grain

main

other

analysis.

structures

The

as membranes)

boundary

the

count

structure. to

the

to

to each two

application,

according

the

exclusive

of the

(2) and

their

of those

estimate,

the

are

a proportion

involved

in each

to

in

how

those

made of grain

content

17-34%.

different

The

be attributed there

Parry

to complete

structures

in the

is equal

should

is to attribute

of radioactivity

Grains concerns

purpose,

tedious

facility

method of the

this and

are

Shared

quantitation

grains

For

of Blackett

(12).

puter

in

of shared

involved.

available,

of the

consideration

structures

each

Analysis

of the

or large

were

The

was

about

structure method

boundary

lution

grains.

each each

radioactive

was

three

the

to minimize

error

the

than

choice that

by by

(resolution

of

of the

the

as many

an endeavor

obtained

error

center

95%

occupied

error where

a

Taking

the

areas the

distance subtends

it is emphasized

and

shared

fact with

radius,

of radioactivity,

average

a point

the

bromide

surface

the

is 230 nm

circle

through silver

the

about

correspond

based

sources

out

the

to do with

boundary

is arbitrary,

of the

work

having

boundary

resolution

can

total

sources

of spherical

necessarily

a working

resolution

of

of point

where

of the

corrections does

developed biity

one crystal

of 5% or less

this

aggregate a monolayer

MM:

CITED Autoradiography evaluation. Lab

with Invest

the electron 14:1041, 1965

2. Blackett NM, Parry DM: A new method for analyzing electron microscope autoradiographs using hypothetical grain distributions. J Cell Biol 57:9, 1973 3. Caro LG: High resolution autoradiography. II. The problem of resolution. J Cell Biol 15:189, 1962 4. Chalkley HW: Method for the quantitative morphologic analysis of tissues. J Natl Cancer Inst 4:47, 1943 5. Fertuck HC, Salpeter MM: Location of acetylcholine receptor by ‘2I-labelled a-bungarotoxin binding at mouse motor endplates. Proc Natl Acad Sci 71 (4):1376, 1974 6. Granboulan P: Comparison of emulsion and techniques in electron microscope autoradiography. Syrup Int Soc Cell Biol, Vol. 4. Edited by CP Leblond and KB Warren. Academic Press, New York, 1965, p 43-54 7. James TH: Some views on the mechanism of development. J Phys Chem 66:2416, 1962 8. Kopriwa BM A comparison of various procedures for fine grain development in electron microscope radioautography. Histo-

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SIMPLIFIED chemie 9. 10. 11. 12.

44:201,

QUANTITATION

IN

1975

Mees CEK, James TH: The theory of the photographic process. (3rd ed.) The Macmillan Company, New York, London, 1966 Nadler NJ: Some theoretical aspects of radioautography. Can J Med Sci 29:182, 1951 Nadler NJ: The quantitative estimation of radioactive isotopes by radioautography. Am J Roentgenol 70:814, 1953 Nadler NJ: The interpretation of grain counts in electron microscope radioautography. Appendix to Hadded et al. J Cell Biol 49:

13. 14.

877, 1971 Pelc SR: Theory of electron autoradiography. 81:131, 1963 Salpeter MM, Bachmann L, Salpeter EE:

microscope 15.

1533

RADIOAUTOGRAPHY

radioautography.

J R Microsc Resolution

J Cell Biol 41:1,

Soc

in electron

1969

Salpeter MM, Fertuck HC, Salpeter EE: Resolution in electron microscope autoradiography. III. Iodine-’25. The effects of heavy metal staining and a reassessment of critical parameters. J Cell Biol 72:161, 1977

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