THE

JOURNAL

Copyright

OF

HI5TOCHEMISTRY

@ 1976 by The

AND

Society.

LASER

TECHNIQUE

FOR

of Pathology,

CELL

Squamous

cells

in fluid

Fluid

flow

utilization

systems

rapid

transport

recording wavelengths cellular nation phologic lation

of

Most

cell

cells. laminar into

through

sheath.

the

axial

beam

serves

to

air

may

and,

This

paper

nozzle

in some

cases,

The

cells

tation

in flow of

Several

sheath near the

case

for

spherical at spatial thelial present essing

light

images

field

is

scatter

cells,

because

a unique

This

of

challenge utilizing

cells

pass

work

was

cells

the

their

supported

on

pho-

flow.

(index

vented systems.

Conference Calif., 1975.

shuttered

The

beam

contrast

prove

thickness

‘ania-

variations) the

in cells. in

a

discipline used

filtering

in

operation.

The

a phase

shift.

the

optical

configuration.

continuous off’ and

on

spatially

a fluid-filled

265

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on March 18, 2015

from an argon ion fluorescence mealaser

beam

flow

by

by

as this

9% of its high con-

with

a strobed

is then

Fourier

known

technique

is derived used for cell

The

and through

along

illumination normally into

Nemarski.

is attenuated to thereby providing

1 depicts

surements.

the

to plane

images,

transmit-

implemented The

Figure Laser laser.

optical

to Bright

whereas

modulation

processing.

Epi-

from

flow

belongs

utilized

visualizing

thickness is

illumination amplitude,

excitation

a contract

and

is a Fourier

illumination.

laser

and

optical

trast

for

contrast

are specimens.

a cell.

visualizing

study

proc-

Presented at the Engineering Foundation on Automated Cytology, Pacific Grove,

in

biological

in

direct original

in automated

NO1CB33862. Slit-scan

orien-

representative

techniques

of

non-

measurements the cell size.

shape,

the

tech-

cell

TECHNIQUES

and

for

plane

the

flattened

by DB:

mea-

is certainly

slit-scan

through

National Cancer Institute, 2 Wheeless LL, Kay

affect

measurements fluorescence less than

for

in flow.

optical

cells

satisfactory

valuable Phase

cells and resolutions

systems

Should I

of the

This

technique

orientation

presents

microscopic

provide

tions

orientation

performed.

and

EXPERIMENTAL

measurement.

being

system

recording

gynecologic

assure

surement

forces

in a flow

coherent

tographs

ever,

greatly

the

a

cell

describes

solution. of

important,

on

orientation.

complex

orientation

for photographically

utiliza-

slit-measuring orientation

cell

less

to

slit-scan

is the

of

a

niques

contrast

may

is parallel

understanding

equally

phase

orientation

cell

solution

or visualizing

variations

Cell

The

measured

Control

an

tance

desired

the

One

to cell

recording

tube

of the

provide

requires

contributing

to

plane beam,

slit.

of the flow stream, forcing passage of’ the through the desired geometrical position the detector assembly. It does not. howthe

technique.

of

one

however,

8).

the

(2).

maintain

set

center cells within during

14642

intersects

from or,

into

York

three orthogonal to provide acceptable cell a

least

This

by a coaxial

laser

the

of the is af’f’ected.

of’

beams

The

specimen

plane

(5)

(7,

stream.

such

the

at

corre-

technique

flow

as it emerges

emerges

New

stroboscopic

that

slit

tion

cellular

enveloped

chamber2

stream

technique

flow

an

A focused

stream

a fluid-filled

as

specific cytoplasm)

the

are

permits

or

a sheath

assembly,

coaxial

and

with

within

FLOW’

Rochester,

a laser

contour

illumimor-

use

entering

allowing Detectors

(4. 6). Use of’ slit provides low resolution

position

in a nozzle

the

or more specific

nucleus

systems

control

for

optics,

on cells

(i.e.,

IN JR.

Center,

using

increased

at one on

fluorescence

structures

gaining

from cells information

information

L. WHEELESS, Medical

photographed

parameters.

fluorescence provide components techniques

are

STUDIES

dark field or phase contrast multiple images of a cell on a single permitted by the thickness of the cell stream. Cell orientation, are obtained from these photographs for evaluation of flow sys-

past

of cellular

LEON

AND

of Rochester

cytopathology

of’ cells

quantitation

the

are

in automated

B. KAY

flow

1. pp. 265-268, 1976 Printedin (‘.S..4.

PHOTOGRAPHY

ORIENTATION

University

technique provides either frame of film at resolutions velocity and acceleration tern performance.

24, No.

Inc.

STROBOSCOPIC

DAVID Department

Vol.

CYTOcHEMISTRY

Histochemical

source a chopper

computer filtered

chamber

is conwheel

control. and into

ficused a Fou-

266

KAY,

rier

plane

where

lenses

behind

a film

plane.

plane

exposure

a frame

the

the

after

of

laser

a single

cell

passes

shutter.

the

strobed

flow stream of’ the film.

surement

region

laser

flow

and

to

The

computer

the

scatter

for

through

the

the the

closes

as

ar’

of

time

the

an

paid

the

coaxial

an

exit

by

a quiescent

recording

A from

capillary.

This

interface

flow

chamber

gap

volume

strong

optical

thereby and

TO

THE

where

the

and

measurement

delay the

a

region exposure

may

is

short.

a

elimi-

accumulate

d

2, A-E

lO-jisec

gion the

axis

the

ing

the

delay

beam

at 2A

1. Optical

system

for

and

A is the

may be recontime for analysis

obtained

cells

flowing

at

chopped

to

tech-

10 cm/ produce

repetition

a superficial

The

edge cell

of the

Figure

always,

2E

squamous

noise and

photography.

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on March 18, 2015

a cell edgewise

is present shows

in Figure rotated

depicts

region

Coherent

cell

is partially

measurement

stroboscopic

the

a 2000-pulses/sec shows

the

of flow.

as

using

with gynecologic of superficial and

camera and Figure 2B shows an squamous cell. Figure 2C depicts flowing through the measurement reand

camera. ages,

laser

d2/ holo-

z

Fraunhofen

DISCUSSION

was

FLOW CHAMBER

FIG.

the

the

together.

laser

if’ the

a distance

diameter

AND

was

pulses

is curled

.

are

and

collimated light A photographic

described images are

laser

Figure

the

hack-

cell

squamous

The

into

large

Filters

orientation.

rate.

works spec-

cell

A

where

cell facing intermediate two cells

opening of

at

in-line

intermediate sec.

minimizing

technique concentrated

between

entrance

pattern

the

of

the flow chamber is by the cell then inter-

is an

Figure

TECHNIQUE

The previously described very well with a moderately

plane

this

nique previously specimens. The

is surrounded

of water, interfaces

(1).

cell is

to

three-dimen-

needed

preceding scattered

RESULTS

aberration.

shutter

of

modified

Fourier

the

cell

of

nozzle into pass through

region

lens Light

is

environment.

The

the

in

holograms

and

laser wavelength. The hologram structed and viewed at a later

inter-

in a fluid-filled

EXTENSIONS

ground

pass

then

imaging

wherein cells emerge from the flow gap measurement region and then

imen

to

builds

cells.

in-line

is

from

measure-

however, a signal levels

be easily

of focus

information

eliminated

Strong

if’ cell

depth

to shutter

the

of

may

imagscatter

exposure

feres with the unscattened forming an interference pattern.

situation.

stream

in an air is solved

system

sional

sense

and

used

enters

exposure

if large

cell the

is then

selection

system

alternate

for

generate

signal.

to optical

imaging

to

a cell

preferential

multiple

gram, was

when

An

one

No background

optical

cells

and aberrations of’ the laser beam for example. at the air-water hound-

attempted

nating

cell

in

provide

shutter.

with

problem

for

long.

converging removed.

of the

of’ measurement

9O

signal

on

region.

too

lasers,

to the images

a sense

receipt

required

attention

refractions will occur,

as

upon

region

laser

Careful faces

detector

delays,

at

scatter

laser

The

scatters both cell

other

The

result

illumi-

chamber

is

two

the images with this approach; problem may occur if the scatter

and begins a background A cell entering the mea-

of’ the

arrives

the

first

ment

the

light

and

the

to block

through

cell

employs

signal.

re-

chamber. The Upon opening

out of the illuminating beam plane to form the stroboscopic

signal.

a

approach

to

are

is used

the

light film

before

cell

a cell

shutter

region of’ the flow of’ events is as follows.

nates the exposure

the

plane (Figure 1). To prevent of’ more than one cell in

measurement sequence laser

Objective

image

images

film images

of film,

beam

occurs.

Fourier

ing

Multiple corded at overlapping the

filtering

the

WHEELESS

up

in as

2D about

traversto

the

the

im-

assorted

LASER

STROBOSCOPIC

267

PHOTOGRAPHY

E FIG.

fringe

on

traceable elements.

patterns

the

2.

optical

at the top of the images tion from the ends ofthe Resolution matel the

in

4

the

stroboscopic

to dust and The horizontal

images

is

x5

50-ji

low

Only

shape,

outline

fore.

should

difficulty

be

specimen and

gleaned

in obtaining images is

of a result

nuclear from rotated

of

applying

diffrac(approxi-

of gynecologic

sional

depth

aperture of the cells

position,

there-

easily

images.

The

cell

obtained

multiple

acceleration

away

from

the

cell

a

two-dimen-

be obtained

using

through

the

measurement

and

acceleration

a single

spacings

obtained

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on March 18, 2015

holoregion

may

photograph.

carry

the

since

the

Cell from

the

earlier.

velocity

is constant. be

a three-dimensional

discussed

information

images also

to

filtering operations are on cell orientation. High images wit h good

from

image

ion

may

may

rotation with

inf’ormat

in flow.

technique

technique

Cell along

cells

Fourier plane for the face three-dimensional

of field

graphic

depth.

the

imaging

cell. The best suited resolution

stream

morphologic

cells

photographs

scratches fringes

are caused by capillary tubes.

due to the low numerical objective used to image the

camera

laser

ji)

throughout

from

A-E,

velocity

time

rotational cell

rotation

he

The and

between velocity between

268

KAY,

images. for

Cell

stability

slit-scan

noted

and

in

all

of

however,

a

the

of’ the

plane

these

cell

images.

image

is

cell

affect

measurement

cell

Current

efforts

of

cell

flow

capillary flow

are

one

will

be

future

an

Laser

utilized

to

attempt

to

the

assess

flow

fluid-filled

force

alignment

stroboscopic

photography

the

success

of

desirable

potential

exists

cell

orientation

for

using

techniques

tion

two-dimensional

flow

system

this

and

at

occurring

one

at

orthogonally laser

transferred

flow.

primary

slit-scan

measurement

and

flow.

a high

strobe

The in

flow or

A cell speed

A is

illumination

stations

the

wavelengths

to

in

stations

other.

of

resolu-

measurement

positioned

direction

photo-

moderate

two

station

the

achieved,

strobe

information

the

occurring

separate

to obtain

with

envisioned,

be laser

could stream

vidicon,

be

along

8.

would

be

linear

ar-

at

additional from

prescreening

an on

the

second

Processing morphologic image

which

cell

classification

sta-

would inforwould

be (3).

CITED

1. Boettner EA, Thompson BJ: Multiple exposure holography of moving fibrous particulate matter in the respiratory range. Optical Engineering: March/April, 1973. 2. Bonner WA, Hullett HR, Sweet RG, Herzenberg LA: Fluorescence activated cell sorting. Rev Sci Instrum 43:404, 1972 3. Cambier JL, Wheeless LL: The binucleate cell: Implications for automated cytopathology. Acta Cytol 19:281, 1975 4. Crissman H, Mullaney P, Steinkamp J: Methods and applications of flow systems for analysis and sorting of mammalian cells, Methods for Cell Biology. Edited by DM Prescott. Academic Press, New York, 1974 5. Goldsmith HL: Red cell motions and wall interactions in tube flow. Fed Proc 30:1578, 1971 6. Wheeless LL, Hardy JA, Balasubramanian N: Slit-scan flow system for automated cytopathology. Acta Cytol 19:45, 1975 7.

using

separated

image

in

array

processing.

LITERATURE

shaping

elliptical

in

to derive available

should greatly

achieving

proper

detector

image

used

useful

performed.

toward An

flow.

other

for

mation

modifications.

graphic

the

directed

incorporated in

Should the

being

through

plane.

and may

on

be

indicates to the

geometry.

chamber

to

which

unstable as it

orientation

is being

Occasionally,

seen

is usually possible,

chamber

ray

tion

are

is perpendicular

if

the

orientations

measurements

This orientation be prevented,

desired

favorable

fluorescence

WHEELESS

Wheeless

LL, Patten SF: Slit-scan cytofluorometry. Acta Cytol 17:333, 1973 Wheeless LL, Patten SF, Onderdonk MA: Slitscan cytofluorometry: data base for automated cytopathology. Acta Cytol, in press

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on March 18, 2015