Proteoglycan

Breakdown

of

Meniscal

Explants

Following

Dynamic

Compression

Using

a

Novel

Bioreactor

Motivated

by

our

interest

in

examining

meniscal

mechanotransduction

processes

we

report

on

the

validation

of

a

new

tissue

engineering

bioreactor

This

paper

describes

the

design

and

performance

capabilities

of

a

tissue

engineering

bioreactor

for

cyclic

compression

of

meniscal

explants

We

showed

that

the

system

maintains

a

tissue

culture

environment

equivalent

to

that

provided

by

conventional

incubators

and

that

its

strain

output

was

uniform

and

reproducible

The

system

incorporates

a

linear

actuator

and

load

cell

aligned

together

in

a

frame

that

is

contained

within

an

incubator

and

allows

for

large

loads

and

small

displacements

A

plunger

with

six

Teflon-filled

Delrin

compression

rods

is

attached

to

the

actuator

compressing

up

to

six

tissue

explants

simultaneously

and

with

even

pressure

The

bioreactor

system

was

used

to

study

proteoglycan

-LRB-

PG

-RRB-

breakdown

in

porcine

meniscal

explants

following

various

input

loading

tests

-LRB-

strain

MPa

-RRB-

The

greatest

PG

breakdown

was

measured

following

compressive

strain

These

strain

and

stress

levels

have

been

shown

to

correspond

to

partial

meniscectomy

Thus

these

data

suggest

that

removing

of

meniscal

tissue

will

result

in

the

breakdown

of

meniscal

tissue

proteoglycans

INTRODUCTION

Mechanical

loading

of

the

meniscus

plays

a

crucial

role

in

the

metabolic

activity

of

fibrochondrocytes

Previous

studies

have

demonstrated

that

increased

load

on

meniscal

tissue

leads

to

an

increase

in

proteoglycan

and

collagen

levels

whereas

unloading

of

meniscal

tissue

causes

a

decrease

in

aggrecan

expression

collagen

formation

and

cell

growth

It

is

not

fully

understood

how

biomechanical

and

biochemical

events

interact

to

produce

changes

in

the

extracellular

matrix

This

lack

in

knowledge

is

in

part

due

to

the

difficulties

associated

with

performing

real

time

meniscal

loading

experiments

in

vivo

Recreating

the

physiological

forces

in

vitro

using

tissue

explants

while

measuring

the

biological

response

provides

one

method

for

observing

the

effect

of

mechanical

stress

on

the

meniscus

however

the

majority

of

commercially

available

bioreactors

may

not

be

suitable

for

application

to

meniscal

loading

studies

The

Biopress

system

-LRB-

The

Biopress

system

Flexcell

International

Hillsborough

NC

USA

-RRB-

uses

air

pressure

applied

to

a

flexible

bottom

under

each

well

The

Biopress

has

been

used

to

apply

pressures

of

MPa

in

previous

studies

on

meniscal

explants

noting

strain

levels

of

approximately

Because

pressures

up

to

MPa

and

strains

ranging

from

to

are

seen

in

the

this

system

may

not

generate

high

enough

pressures

to

mimic

the

full

range

of

meniscal

strains

thought

to

occur

during

active

loading

of

healthy

and

damaged

tissue

in

vivo

Stresses

and

strains

are

approximately

and

MPa

respectively

in

the

normal

healthy

meniscus

but

these

strain

levels

increase

with

a

partial

meniscectomy

to

approximately

and

MPa

Another

biaxial

tissue-loading

device

previously

used

to

compress

articular

cartilage

explants

is

able

to

create

a

maximum

N

axial

force

on

as

many

as

explants

at

once

however

is

limited

to

μm

of

motion

Previous

studies

have

shown

that

following

knee

trauma

such

as

ACL

transection

prior

to

any

articular

cartilage

damage

there

are

signs

of

meniscal

tissue

degeneration

We

have

previously

shown

that

following

partial

meniscectomy

the

remaining

meniscal

tissue

is

subjected

to

an

altered

loading

state

This

altered

loading

state

may

cause

a

direct

breakdown

of

matrix

components

such

as

proteoglycans

-LRB-

PG

-RRB-

or

indirectly

affect

matrix

production

by

induction

of

catabolic

biomolecules

The

objectives

of

this

study

were

-LRB-

-RRB-

to

design

and

build

a

practical

cost-effective

device

for

applying

homogeneous

strains

to

tissue

explants

and

-LRB-

-RRB-

to

utilize

the

system

to

overload

and

underload

meniscal

tissue

and

measure

the

biochemical

output

In

this

communication

we

show

that

the

ensuing

device

is

a

simple

biocompatible

design

that

applies

accurate

and

reproducible

strains

and

is

made

of

components

that

can

be

sterilized

We

utilized

the

bioreactor

to

apply

both

load

and

displacement

controlled

dynamic

compression

tests

Dynamic

strain

compression

tests

showed

PG

breakdown

following

overloading

of

meniscal

tissue

No

significant

changes

in

concentration

of

PG

released

to

the

conditioned

media

was

measured

following

various

levels

of

dynamic

compressive

stress

A

comparison

between

stress

controlled

dynamic

compression

versus

strain

controlled

dynamic

compression

was

also

made

MATERIALS

AND

METHODS

Design

of

Bioreactor

The

frame

consists

of

two

cm

thick

parallel

aluminum

plates

separated

by

cm

diameter

aluminum

support

rods

-LRB-

Fig.

-RRB-

Centered

on

the

bottom

plate

is

a

load

cell

that

is

attached

to

a

six

well

dish

The

system

is

driven

by

a

belt-driven

linear

actuator

utilizing

a

control

package

by

Animatics

-LRB-

Smartmotor

Ultramotion

Mattituck

NY

USA

-RRB-

The

actuator

has

a

maximum

stroke

length

of

cm

and

can

thrust

to

N

It

also

has

a

maximum

speed

of

cm/s

with

bi-directional

repeatability

of

mm

and

a

unidirectional

repeatability

of

mm

Motor

control

was

achieved

by

using

the

SmartMotor

Interface

-LRB-

SMI

-RRB-

This

language

allows

the

motion

of

the

actuator

to

be

controlled

by

the

signal

generated

by

the

load

cell

or

by

the

displacement

of

the

actuator

Displacement

resolution

for

the

actuator

is

μm

FIGURE

A

view

of

the

assembly

of

the

plunger

dish

and

cap

The

test

frame

is

composed

of

two

aluminum

plates

supported

by

aluminum

rods

The

actuator

is

positioned

in

a

centered

hole

in

the

top

plate

and

tighten

into

alignment

with

an

adjustable

collar

A

strain

gage

load

cell

-LRB-

Model

Interface

Scottsdale

AZ

-RRB-

with

a

load

capacity

of

N

-LRB-

sensitivity

of

N

-RRB-

was

utilized

For

tests

that

require

loads

near

or

above

N

a

dimensionally

identical

load

cell

with

N

-LRB-

sensitivity

N

-RRB-

-LRB-

Interface

Scottsdale

AZ

USA

-RRB-

capacity

is

interchangeable

with

the

current

load

cell

This

design

feature

of

interchangeable

load

cells

allows

for

a

larger

range

of

loads

to

be

accurately

measured

A

series

signal

conditioner

-LRB-

Vishay

Instruments

Raleigh

NC

USA

-RRB-

amplifies

the

load

cell

signal

to

produce

a

V

signal

at

the

maximum

load

The

load

cell

is

centered

on

a

cm

thick

aluminum

plate

that

is

the

base

of

the

system

frame

A

threaded

stud

leaving

the

load

cell

connects

to

the

aluminum

dish

via

a

quick

disconnect

pin

The

dish

has

six

mm

deep

wells

equally

spaced

in

a

circular

orientation

Teflon-filled

Delrin

compression

rods

-LRB-

diameter

mm

-RRB-

for

each

well

are

press

fit

into

a

plunger

which

attaches

to

the

actuator

via

a

quick

disconnect

pin

To

ensure

only

one

plunger/dish

orientation

and

to

keep

the

compression

rods

centered

in

each

well

the

plunger

also

features

two

press-fit

aluminum

pins

that

slide

into

matching

holes

in

the

dish

An

aluminum

cap

rests

on

the

shoulder

of

the

dish

and

houses

a

linear

bearing

that

is

press

fit

into

the

cap

Along

the

resting

edge

of

the

cap

four

shallow

grooves

were

machined

to

allow

carbon

dioxide

supply

to

the

explants

during

testing

The

linear

bearing

allows

the

plunger

to

move

up

and

down

within

the

cap

and

restricts

the

plunger

to

vertical

motion

-LRB-

Fig.

-RRB-

The

entire

frame

is

small

enough

that

it

can

be

placed

in

a

commercially

available

incubator

to

maintain

physiological

conditions

-LRB-

Model

VWR

West

Chester

PA

USA

-RRB-

FIGURE

A

drawing

of

the

test

frame

shows

the

side

and

top

view

of

the

test

system

The

linear

actuator

is

attached

to

the

plunger

using

a

quick-disconnect

pin

The

dish

is

attached

to

the

load

cell

in

the

same

manner

The

cap

improves

alignment

of

the

plunger

by

utilizing

a

linear

bearing

Accuracy

Evaluation

of

the

System

Ultra-low

pressure

film

-LRB-

Sensor

Products

Inc.

East

Hanover

NJ

USA

-RRB-

was

used

to

measure

well

pressure

during

compression

First

the

repeatability

of

the

pressure

film

was

tested

by

loading

the

film

-LRB-

seven

times

-RRB-

between

flat

platens

in

a

tensile

testing

machine

-LRB-

Model

Instron

Corp.

Canton

MA

USA

-RRB-

to

a

N

target

load

corresponding

to

a

pressure

of

MPa

for

the

given

indentor

size

Calibration

of

the

pressure

film

was

also

done

using

the

tensile

testing

machine

and

included

loading

pieces

of

pressure

film

ranging

from

to

MPa

Pressure

film

analysis

was

completed

using

commercial

software

-LRB-

Scion

Image

National

Institute

of

Standards

and

Technology

Gaithersburg

MD

USA

-RRB-

to

measure

the

density

of

the

pressure

film

samples

Film

was

compressed

between

the

platens

and

a

piece

of

rubber

similar

to

the

rubber

used

for

testing

well

pressure

To

determine

well

pressure

in

the

bioreactor

a

machined

plate

was

set

on

top

of

the

dish

with

a

mm

thick

piece

of

uniform

rubber

Pressure

film

was

placed

on

top

of

the

rubber

and

the

plunger

was

lowered

near

the

surface

of

the

film

Four

tests

were

conducted

each

to

the

same

pressure

-LRB-

MPa

-RRB-

to

determine

the

repeatability

of

the

bioreactor

in

load

control

The

film

from

the

bioreactor

was

analyzed

and

density

measured

to

determine

the

difference

between

each

compression

rod

The

difference

in

film

density

and

the

maximum

percentage

error

was

determined

to

demonstrate

the

accuracy

of

the

system

Determination

of

Displacement

Repeatability

To

further

investigate

the

accuracy

of

the

system

the

gap

between

the

bottom

of

the

compression

rods

and

the

bottom

of

the

wells

was

measured

while

the

system

was

assembled

into

the

bioreactor

This

was

done

by

using

the

actuator

to

compress

commercially

available

Fibre-Strand

body

filler

-LRB-

The

Matin

Senour

Comp.

Cleveland

OH

USA

-RRB-

until

the

gap

between

the

bottom

of

the

compression

rod

and

well

bottom

was

filled

The

actuator

remained

at

this

position

until

the

body

filler

hardened

completely

After

plunger

removal

a

micrometer

-LRB-

μm

resolution

-RRB-

was

then

used

to

measure

the

thickness

of

the

body

filler

This

process

was

repeated

three

times

with

the

same

plunger

and

dish

orientation

Compliance

of

the

System

The

compliance

of

the

system

was

determined

by

placing

a

flat

stainless

steel

plate

over

the

wells

and

running

a

load-deformation

test

in

the

absence

of

menisci

The

test

was

repeated

three

times

and

the

load-deformation

data

recorded

Application

of

the

System

Pigs

used

in

the

experiment

were

weeks

old

sacrificed

h

after

death

-LRB-

received

from

Mayo

Clinic

Rochester

MN

USA

-RRB-

The

explants

were

collected

from

both

the

lateral

and

medial

meniscus

using

sterile

technique

The

explants

were

mm

in

diameter

and

cut

using

a

biopsy

punch

perpendicular

to

the

superior

surface

to

maximize

the

amount

of

superior

tissue

preserved

To

ensure

two

parallel

flat

faces

on

the

cylindrical

explants

a

specialized

cutting

device

was

utilized

Explants

were

held

such

that

a

fixture

containing

two

razorblades

mm

apart

cut

the

tissue

perpendicular

to

the

long

axis

of

the

cylindrical

explant

This

ensured

that

each

explant

was

the

same

height

with

parallel

faces

-LRB-

Fig.

-RRB-

FIGURE

a

Meniscus

following

biopsy

b

Maximum

axial

strain

following

different

partial

meniscectomy

simulated

by

FE

analysis

Various

percentages

-LRB-

and

-RRB-

removed

from

different

portion

of

the

medial

meniscus

-LRB-

A

anterior

C

central

P

posterior

-RRB-

Knee

loaded

with

N

axial

force

Explants

were

incubated

at

°C

-LRB-

-RRB-

for

h

in

growth

media

-LRB-

DMEM/F

FBS

Penn/Strep

-RRB-

which

was

changed

after

h.

For

mechanical

testing

the

explants

were

placed

in

the

six

well

bioreactor

filled

with

μl

of

the

test

media

-LRB-

DMEM/F

FBS

Penn/Strep

-RRB-

The

explants

were

loaded

for

h

at

Hz

at

one

of

the

following

levels

or

strain

or

MPa

unconfined

dynamic

compression

Each

loading

group

consisted

of

six

explants

taken

from

six

different

animals

-LRB-

except

test

animals

-RRB-

Control

explants

were

placed

in

wells

but

not

exposed

to

compression

Previous

experiments

-LRB-

FE

analyses

-RRB-

showed

that

under

two

times

body

weight

the

intact

knee

meniscus

experiences

about

maximum

compressive

strain

Removal

of

of

the

meniscus

minimally

affects

the

maximum

strain

level

however

removal

of

of

the

meniscal

body

increases

the

maximum

strain

to

or

greater

Generally

speaking

strain

is

likely

underloading

the

tissue

is

approximately

physiological

and

is

considered

-LRB-

Fig.

-RRB-

Following

compression

the

explants

were

bisected

into

superior

and

deep

zone

by

cutting

them

in

half

-LRB-

Fig.

-RRB-

weighted

and

placed

in

plates

in

ml

of

fresh

test

media

Samples

were

post-incubated

at

°C

-LRB-

-RRB-

for

h.

Post-incubation

media

was

collected

and

stored

at

°C

for

future

analysis.FIGURE

of

the

meniscus

with

the

direction

of

the

cut

and

explants

showing

superior

and

deep

zones

The

content

of

sulfated

glycosaminoglycan

-LRB-

GAG

-RRB-

released

to

the

conditioned

media

was

assayed

using

dimethylmethylene

blue

-LRB-

DMMB

-RRB-

dye

solution

The

standard

curve

was

generated

with

a

known

concentration

of

shark

cartilage

chondroitin

sulfate

C.

All

samples

were

run

in

duplicates

The

concentration

was

normalized

to

the

wet

weight

of

the

explant

GAG

release

was

then

normalized

to

the

no

load

control

samples

for

each

animal

Data

Analysis

The

concentration

of

the

GAG

released

to

the

media

was

calculated

using

a

standard

curve

-LRB-

-RRB-

The

final

value

was

an

average

from

duplicates

All

data

is

presented

as

mean

standard

error

A

one

way

ANOVA

followed

by

Fisher

's

PLSD

post-hoc

testing

was

used

to

measure

statistical

differences

-LRB-

p

was

considered

significant

-RRB-

Paired

t-tests

were

used

at

each

stress

or

strain

level

to

determine

differences

between

superficial

and

deep

zone

PG

release

to

the

media

RESULTS

Accuracy

Evaluation

of

the

System

The

repeatability

test

showed

an

average

of

MPa

The

pressure

film

demonstrated

equal

pressure

in

each

well

for

each

load

-LRB-

Fig.

-RRB-

There

was

less

than

a

error

-LRB-

Table

-RRB-

FIGURE

Pressure

film

impressions

at

MPa

under

the

six

bioreactor

compression

rods.TABLE

Results

of

pressure

film

verification

MPaPressure

-LRB-

MPa

-RRB-

Test

#Rod

Dev

.10.47730.47680.47710.47710.47680.47680.47700.000220.47680.47690.47680.47680.47690.47720.47690.000130.47730.47680.47690.47700.47690.47710.47700.000240.47730.47680.47690.47680.47690.47680.47690.0002

Determination

of

Displacement

Repeatability

The

micrometer

measurements

from

the

body

filler

showed

that

the

bioreactor

was

extremely

repeatable

Well

had

the

largest

standard

deviation

in

height

mm

whereas

well

had

the

lowest

standard

deviation

Compliance

of

the

System

The

results

of

the

compliance

test

indicate

a

linear

load-deformation

response

-LRB-

-RRB-

The

slope

of

the

load-deformation

curve

was

N/mm

Thus

for

the

range

of

strains

and

stresses

seen

in

this

study

the

compliance

is

negligible

Application

of

the

System

To

determine

an

appropriate

post

incubation

time

preliminary

tests

were

run

PG

concentration

in

the

media

was

monitored

following

and

h

and

and

days

following

dynamic

compression

and

for

controls

samples

-LRB-

Fig.

-RRB-

The

shortest

time

showing

a

strong

signal

was

chosen

to

minimize

the

duration

of

the

experiment

-LRB-

day

-RRB-

FIGURE

PG

concentration

following

different

incubation

time

-LRB-

n

for

controls

-LRB-

C

-RRB-

n

for

compression

test

-LRB-

-RRB-

-RRB-

Data

presented

is

mean

standard

error

The

highest

break

down

of

PG

was

measured

following

compressive

strain

for

both

the

superior

and

deep

zones

of

the

meniscal

explants

-LRB-

Fig.

-RRB-

There

were

significant

differences

between

release

of

GAGs

into

the

media

for

the

overloaded

condition

-LRB-

-RRB-

versus

physiological

loading

-LRB-

-RRB-

for

both

superior

and

deep

zones

No

significant

differences

were

found

between

superficial

and

deep

zones

for

any

compression

level

Explants

exposed

to

pressures

of

either

or

MPa

did

not

show

any

significance

differences

in

either

the

superior

or

deep

zone

for

PG

breakdown

-LRB-

Fig.

-RRB-

FIGURE

PG

concentration

following

different

compression

tests

-LRB-

n

except

test

-LRB-

n

-RRB-

-RRB-

Data

presented

is

mean

standard

error

Statistically

different

than

-LRB-

p

-RRB-

FIGURE

PG

concentration

following

different

load

level

compression

tests

-LRB-

n

-RRB-

Data

presented

is

mean

standard

error

Displacement

controlled

tests

showed

a

rapid

drop

in

load

within

the

first

cycles

with

little

change

in

the

following

cycles

-LRB-

Fig.

-RRB-

For

displacement

tests

at

or

below

strain

loads

settled

just

below

MPa

while

the

strain

test

remained

above

MPa

throughout

the

duration

of

the

test

The

change

in

pressure

measured

from

the

beginning

of

the

test

to

the

end

is

shown

in

Table

Load

controlled

tests

-LRB-

Fig.

-RRB-

showed

a

rapid

increase

in

compressive

displacement

within

the

first

cycles

The

MPa

load

level

reached

maximum

displacement

near

cycles

and

remained

at

that

level

to

the

end

of

the

test

The

MPa

tests

reached

strain

near

cycles

but

steadily

increased

to

strain

by

the

final

cycle

The

differences

in

strain

from

the

start

to

the

end

of

the

tests

can

be

found

in

Table

FIGURE

Stress

vs.

time

for

four

representative

displacement

control

tests

Only

peak

values

during

each

cycle

are

plotted

Stress

was

calculated

by

dividing

the

peak

load

by

the

initial

cross-sectional

area.Table

Change

in

pressures

over

the

duration

of

the

stress-relaxation

tests.StrainPressure

-LRB-

MPa

-RRB-

Data

represents

mean

standard

deviation

n

for

all

groups

Significantly

different

than

-LRB-

p

-RRB-

significantly

different

than

-LRB-

p

-RRB-

FIGURE

Strain

vs.

time

for

two

representative

load

control

tests

Only

peak

strain

values

during

each

loading

cycle

are

plotted

Strain

was

calculated

by

dividing

the

peak

displacement

by

the

original

height

of

the

explant.Table

Change

in

strains

over

the

duration

of

the

creep

tests.Pressure

-LRB-

MPa

-RRB-

Strain

-LRB-

-RRB-

Data

represents

mean

standard

deviation

n

for

all

groups

Significantly

different

than

MPa

-LRB-

p

-RRB-

DISCUSSION

The

explant

compression

system

meets

the

criteria

necessary

to

obtain

a

realistic

representation

of

physiological

forces

present

in

the

knee

joint

This

system

is

able

to

apply

known

pressures

to

six

explants

at

once

which

is

important

when

trying

to

gather

data

for

hypothesis

testing

It

is

capable

of

applying

physiological

and

supraphysiological

levels

of

load

and

displacement

and

has

the

ability

to

test

in

load

or

displacement

control

SMI

programming

allows

for

flexibility

in

frequency

duration

amplitude

and

waveform

The

system

is

small

enough

to

fit

in

a

standard

incubator

and

is

made

of

materials

that

can

endure

autoclaving

and

alcohol

An

important

feature

to

this

system

is

the

ability

to

keep

the

explants

and

media

sterile

from

the

culture

hood

to

the

incubator

The

plunger

dish

and

cap

form

an

enclosure

that

allows

easy

transport

between

the

culture

hood

and

incubator

without

allowing

open

air

and

bacteria

to

infect

the

sample

Since

the

cap

incorporates

a

linear

bearing

it

does

not

need

to

be

removed

for

testing

Utilizing

the

system

features

and

designing

the

correct

protocol

will

help

maintain

a

physiological

loading

sterile

environment

This

bioreactor

is

capable

of

creating

higher

loads

and

greater

displacements

than

previous

systems

used

for

compressing

explants

The

Biopress

system

-LRB-

Flexcell

International

Hillsborough

NC

USA

-RRB-

is

capable

of

loads

as

high

as

N

whereas

the

current

systems

actuator

can

thrust

to

N

An

advantage

of

our

system

over

the

biaxial

tissue-loading

device

presented

in

Frank

et

al.

-LRB-

-RRB-

is

that

it

can

create

displacements

over

mm

with

a

resolution

of

μm

Our

actuator

also

has

a

bi-directional

repeatability

of

μm

compared

to

the

μm

used

in

Sah

et

al.

-LRB-

-RRB-

In

addition

the

present

system

is

capable

of

Hz

cyclic

compression

in

a

sinusoidal

wave

using

displacement

or

load

control

The

bioreactor

can

be

used

to

compress

any

tissue

that

fits

under

an

mm

compression

rod

and

in

a

mm

deep

well

All

surfaces

are

machined

to

a

smooth

frictionless

finish

to

ensure

the

sample

is

exposed

to

pure

unconfined

compression

Although

we

believe

this

bioreactor

to

be

an

improvement

over

others

we

realize

that

there

are

some

limitations

that

need

to

be

compensated

for

For

example

this

system

can

only

perform

unconfined

compression

currently

However

the

system

could

be

modified

to

run

confined

compression

tests

as

well

by

outfitting

a

new

plunger/well

assembly

Since

the

stress

tests

were

conducted

at

and

MPa

and

resulted

in

strains

between

and

the

compliance

of

the

system

was

negligible

Similarly

for

the

strain

control

tests

the

loads

were

small

enough

that

the

compliance

of

the

system

was

negligible

However

for

the

and

strain

control

tests

stresses

were

between

and

MPa

Thus

at

the

larger

stress

levels

the

compliance

of

the

system

was

greater

For

instance

during

the

strain

control

test

initially

stress

levels

of

MPa

would

lead

to

approximately

μm

of

compliance

and

thus

for

the

first

of

the

s

test

strains

were

closer

to

instead

of

For

the

strain

test

strains

were

closer

to

for

the

first

s

of

the

test

and

as

the

material

relaxed

the

last

h

and

min

of

the

test

were

at

strain

We

hypothesized

that

PG

breakdown

-LRB-

as

determined

by

GAG

in

the

media

-RRB-

would

be

high

for

both

the

underloading

and

overloading

condition

PG

breakdown

was

significantly

increased

at

strain

Meniscal

tissue

is

a

mechanically

sensitive

and

mechanical

loading

has

been

shown

to

regulate

gene

expression

Hence

we

expected

that

loading

the

tissue

below

normal

physiological

levels

of

magnitude

-LRB-

underloading

-RRB-

would

result

in

PG

breakdown

Lack

of

increased

PG

breakdown

for

control

samples

-LRB-

underloading

-RRB-

is

surprising

and

needs

to

be

further

explored

Perhaps

the

degraded

PG

is

not

being

released

into

the

media

for

the

control

samples

whereas

the

overloaded

samples

have

mechanical

compression

to

help

move

the

broken

down

PG

into

the

media

Future

studies

will

measure

PG

breakdown

in

the

tissue

explants

following

compression

to

determine

the

integrity

of

the

PG

within

the

tissue

These

data

suggest

that

removing

of

meniscal

tissue

and

thereby

increasing

tissue

strains

over

results

in

an

increase

in

PG

breakdown

and

tissue

destruction

Thus

not

only

does

meniscectomy

affect

the

underlying

articular

cartilage

but

the

remaining

meniscal

tissue

appears

to

begin

to

breakdown

possibly

leading

to

a

change

in

meniscal

material

properties

This

data

is

supported

by

previous

work

that

showed

following

an

ACL

transection

degenerative

changes

were

seen

in

the

meniscus

prior

to

any

articular

cartilage

changes

DiMicco

et

al.

showed

an

increase

in

GAG

release

from

bovine

cartilage

that

had

been

exposed

to

an

injurious

level

single

uniaxial

unconfined

compression

Inhibitors

of

biosynthesis

or

degradative

enzymes

did

not

affect

PG

breakdown

suggesting

that

the

breakdown

was

a

mechanical

consequence

of

compression

GAG

release

days

post

injury

was

slowed

by

metalloprotease

inhibitors

Shin

et

al.

showed

that

dynamic

compression

-LRB-

MPa

for

h

at

Hz

-RRB-

increased

both

GAG

synthesis

as

well

as

release

to

the

media

compared

to

unloaded

controls

Our

loading

scheme

was

designed

to

simulate

approximate

physiological

walking

conditions

-LRB-

h

Hz

-RRB-

It

is

difficult

to

compare

our

GAG

data

to

a

study

of

a

single

injurious

insult

or

dynamic

compression

for

h

We

created

an

approximate

daily

physiological

loading

environment

that

might

occur

during

walking

and

simulated

loads

from

unloaded

to

overloaded

due

to

meniscectomy

In

the

future

we

will

investigate

the

mechanisms

of

GAG

release

whether

it

is

mechanical

damage

or

activation

of

enzymatic

activity

It

was

surprising

that

explants

tested

at

MPa

showed

greater

PG

breakdown

than

explants

tested

at

MPa

for

the

superficial

zone

One

possible

reason

for

this

result

might

be

related

to

the

cell

viability

A

compression

of

MPa

may

induce

more

cell

death

than

MPa

of

compression

and

hence

fewer

cells

may

be

available

for

production

of

metalloproteases

that

may

contribute

to

the

breakdown

of

PG

Current

studies

are

underway

to

document

the

degree

of

cell

death

in

the

explants

It

is

interesting

to

compare

the

load

control

results

to

the

displacement

control

results

Based

on

Fig.

the

strain

test

applied

an

initially

very

high

load

-LRB-

MPa

-RRB-

but

equilibrated

at

MPa

Similarly

the

MPa

test

reached

a

steady

state

of

strain

-LRB-

Fig.

-RRB-

Hence

we

would

expect

to

see

similar

levels

of

PG

breakdown

in

the

strain

test

as

well

as

the

MPa

test

This

is

also

true

for

the

strain

test

and

the

MPa

stress

tests

For

example

in

the

deep

zone

the

strain

control

test

resulted

in

ug/ml

per

gram

of

tissue

whereas

the

MPa

test

resulted

in

ug/ml

per

gram

of

tissue

This

large

difference

could

be

due

to

either

lift-off

-LRB-

separation

between

loading

platen

and

sample

during

a

Hz

test

-RRB-

or

the

initially

high

stress

that

was

reached

initially

in

the

displacement

control

tests

In

contrast

to

the

differences

seen

above

the

strain

test

and

MPa

stress

test

resulted

in

and

ug/ml

per

gram

of

tissue

respectively

Previous

computational

studies

of

an

entire

human

knee

joint

have

shown

that

mean

contact

pressures

changed

from

MPa

for

an

intact

healthy

meniscus

to

MPa

following

meniscectomy

Maximum

contact

pressures

on

the

superior

surface

of

the

meniscus

changed

from

to

MPa

when

of

the

meniscus

was

removed

The

differences

between

stress

and

strain

control

studies

noted

above

make

the

data

presented

difficult

to

interpret

due

to

the

non-physiological

nature

of

in

vitro

unconfined

compression

studies

Clearly

this

indicates

that

meniscal

tissue

may

respond

differentially

to

stress

versus

strain

the

duration

of

strain

levels

or

most

likely

the

loading

history

Before

definitive

clinical

implications

can

be

made

regarding

the

effects

of

meniscectomy

on

meniscal

tissue

we

must

first

determine

if

the

levels

of

PG

breakdown

noted

in

this

study

correspond

to

changes

in

the

load-bearing

capacity

of

the

tissue

and

its

function

in

the

knee

joint

One

of

the

limitations

of

this

study

is

lack

of

investigation

of

differences

between

the

medial

and

lateral

menisci

as

well

as

specific

location

within

the

meniscus

from

which

explants

were

harvested

This

likely

resulted

in

higher

standard

deviations

Future

studies

with

a

larger

set

of

animals

are

proposed

in

which

lateral

versus

medial

anterior

versus

posterior

and

inner

radial

versus

outer

radial

differences

could

be

studied

Previous

researchers

have

shown

a

difference

between

inner

and

outer

radial

explants

Bisection

of

explants

has

previously

been

shown

to

release

many

growth

factors

Since

all

samples

in

this

study

were

bisected

the

relative

differences

found

are

still

significant

It

should

be

noted

that

the

measured

response

may

be

due

not

only

to

mechanical

stimuli

but

also

growth

factors

released

due

to

cutting

the

samples

In

addition

to

bisecting

the

samples

other

factors

such

as

FBS

concentration

and

post-incubation

time

may

effect

the

absolute

concentrations

of

GAG

analyzed

in

this

study

Therefore

only

relative

comparisons

between

treatment

groups

should

be

considered

In

summary

this

simple

and

practical

experimental

system

allows

for

reproducible

application

and

quantification

of

homogeneous

stresses

or

strains

to

explants

tissues

thereby

providing

a

systemic

and

quantitative

method

for

correlating

external

mechanical

stimuli

to

cellular

and

molecular

mechanisms

of

mechanotransduction

O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O


Remote

Non-invasive

Stereoscopic

Imaging

of

Blood

Vessels

First

In-vivo

Results

of

a

New

Multispectral

Contrast

Enhancement

Technology

We

describe

a

contactless

optical

technique

selectively

enhancing

superficial

blood

vessels

below

variously

pigmented

intact

human

skin

by

combining

images

in

different

spectral

bands

Two

CMOS-cameras

with

apochromatic

lenses

and

dual-band

LED-arrays

simultaneously

streamed

Left

-LRB-

L

-RRB-

and

Right

-LRB-

R

-RRB-

image

data

to

a

dual-processor

PC

Both

cameras

captured

color

images

within

the

visible

range

-LRB-

VIS

nm

-RRB-

and

grey-scale

images

within

the

near

infrared

range

-LRB-

NIR

nm

-RRB-

by

sequentially

switching

between

LED-array

emission

bands

Image-size-settings

of

for

VIS

for

NIR

produced

cycles/s

-LRB-

cycle

VIS

L&R

pair

NIR

L&R

pair

-RRB-

Decreasing

image-size-settings

-LRB-

for

VIS

and

for

NIR

-RRB-

increased

camera-speed

to

cycles/s

Contrasts

from

below

the

tissue

surface

were

algorithmically

distinguished

from

surface

shadows

reflections

etc.

Thus

blood

vessels

were

selectively

enhanced

and

back-projected

into

the

stereoscopic

VIS-color-image

using

either

a

or

conventional

shutter

glasses

Introduction

Visually

guided

procedures

provide

instant

feedback

and

meantime

afford

insights

that

would

otherwise

be

difficult

or

even

impossible

to

obtain

This

is

a

primary

reason

for

mankind

's

continuous

desire

to

extend

vision

beyond

the

boundaries

of

the

human

eye

which

has

resulted

in

many

successful

diagnostic

imaging

modalities

like

X-ray

imaging

endoscopy

thermography

ultrasound

scans

and

MRI

that

all

found

their

way

into

the

clinic

While

investigating

the

principal

feasability

of

a

camera

for

imaging

blood

oxygenation

levels

we

noticed

that

our

multispectral

images

also

contained

information

about

subcutaneous

vasculature

with

improved

contrast

in

the

near

infrared

Near

infrared

imaging

of

superficial

blood

vessels

in

itself

is

not

new

soon

after

infrared

early

electronic

cameras

were

used

for

experiments

Since

then

numerous

near

infrared

imaging

methods

have

been

developed

using

transillumination

mode

and/or

reflection

mode

Our

multispectral

camera

however

provides

the

opportunity

to

obtain

normal

color

images

within

the

visible

range

-LRB-

VIS

-RRB-

which

are

pixel-to-pixel

matched

with

images

obtained

in

the

invisible

near

infrared

range

-LRB-

NIR

-RRB-

By

combining

the

image

information

content

of

both

spectral

bands

with

a

processing

algorithm

we

developed

a

new

technique

that

allows

selective

enhancement

of

superficial

blood

vessels

with

selectable

pigment

suppression

within

a

normal

color

image

The

underlying

principle

does

not

necessarily

require

stereoscopic

image

acquisition

to

derive

increased

vessel

contrasts

from

the

tissue

When

however

developing

a

new

medical

imaging

technique

deriving

image

information

from

patient

tissue

is

not

the

only

issue

Especially

for

visually

guided

procedures

it

is

also

crucial

to

put

effort

in

an

ergonomic

human

interface

and

avoid

conflicts

between

the

proprioceptional

and

visual

perceptions

of

the

user

The

added

value

of

stereoscopic

image

recordings

already

was

recognized

and

successfully

used

to

document

medical

cases

more

than

a

century

ago

We

likewise

reasoned

that

offering

enhanced

blood

vessel

contrast

at

the

cost

of

depth

perception

would

restrict

the

usefulness

of

our

technique

and

therefore

decided

to

realize

a

stereoscopic

version

of

our

vessel

contrast

enhancement

device

Visually

guided

procedures

typically

require

a

combination

of

well

trained

eyes

and

specific

fine-motoric

skills

since

the

coordination

between

eyes

and

hands

is

task

dependent

For

eye-hand

coordination

brain

processes

for

perception

and

action

interact

so

closely

that

they

can

not

be

separated

and

the

influence

of

visual

illusions

-LRB-

like

human

depth

perception

-RRB-

to

motoric

tasks

becomes

stronger

when

input

takes

place

via

lower

levels

in

the

brain

The

match

between

a

human

interface

device

and

the

visual

and

motoric

brain

processes

strongly

defines

whether

a

technology

can

be

applied

intuitively

or

not

A

new

technology

can

be

classified

as

intuitive

if

it

speeds

up

the

learning

process

for

novices

in

a

certain

skill

without

impairing

the

performance

of

persons

already

skilled

in

the

existing

art.

Thus

a

useful

increase

in

vascular

contrast

should

neither

imply

a

task-impairing

decrease

in

depth

perception

-LRB-

like

in

monoscopic

techniques

-RRB-

nor

distort

spatial

clues

for

human

vision

-LRB-

like

shadows

-RRB-

nor

introduce

false

depth

information

-LRB-

like

projection

parallax

-RRB-

As

a

first

reconnaissance

of

practical

feasibility

we

applied

our

new

technique

to

several

visual

clinical

procedures

for

which

we

expected

that

improved

visualization

of

blood

vessels

would

be

of

interest

being

blood

withdrawal

vein

inspection

in

dark

skin

detection

of

veins

through

iodide

inspection

of

varicose

veins

and

nevi

pigmentosum

Methods

Instrumental

Setup

The

instrumental

hardware

setup

is

schematically

drawn

in

Fig.

Two

synchronized

identical

single-chip

CMOS-cameras

-LRB-

Vector

Technologies

Belgium

custom

built

-RRB-

were

equipped

with

apochromatic

lenses

and

two

identical

custom-built

dual-band

LED-arrays

-LRB-

the

Netherlands

-RRB-

These

LED-arrays

were

current

controlled

and

each

had

two

individually

programmable

channels

for

the

emission

of

in

total

Cd

visible

white

light

with

adjustable

color

temperature

-LRB-

consisting

of

broadband

white

LEDs

with

a

yellow

accent

and

broadband

white

LEDs

with

a

blue

accent

-RRB-

as

well

as

one

programmable

channel

for

the

emission

of

near

infrared

radiation

-LRB-

LEDs

nm

max

mW

per

LED

-RRB-

The

light

sources

were

constructed

so

that

the

geometrical

beam

profiles

of

VIS

and

NIR

matched

very

closely

-LRB-

and

thus

also

any

resulting

shadows

and/or

reflections

-RRB-

Left

-LRB-

L

-RRB-

and

Right

-LRB-

R

-RRB-

image

data

was

simultaneously

acquired

and

streamed

to

a

dual

processor

PC

equipped

with

a

stereoscopic

monitor

-LRB-

Sharp

-RRB-

It

was

also

possible

to

connect

a

conventional

cathode

ray

tube

-LRB-

CRT

-RRB-

monitor

equipped

with

shutter

glasses

for

comparison

of

stereoscopic

representation

Figure

Experimental

setup

Two

CMOS-cameras

with

apochromatic

lenses

and

dual-band

LED-arrays

simultaneously

stream

Left

-LRB-

L

-RRB-

and

Right

-LRB-

R

-RRB-

image

data

to

a

dual

processor

PC

Both

cameras

captured

color

images

within

the

visible

range

-LRB-

VIS

nm

-RRB-

and

grey-scale

images

within

the

near

infrared

range

-LRB-

NIR

nm

-RRB-

by

sequentially

switching

between

LED-array

emission

bands

A

schematical

representation

of

image

aquisition

and

data

structure

is

drawn

in

Fig.

The

CMOS-camera

detector

chips

were

equipped

with

a

Bayer

filter

mosaic

to

obtain

an

RGB

color

image

within

the

VIS

The

camera

pixels

however

were

also

sensitive

to

near

infrared

radiation

because

all

three

filter

channels

of

the

Bayer

mosaic

-LRB-

red

green

and

blue

-RRB-

were

designed

as

highly

transparant

within

the

NIR

Thus

four

NIR-pixels

were

acquired

for

each

VIS

RGB

color

pixel

group

By

sequentially

switching

between

emission

bands

of

the

LED-arrays

images

within

the

Visual

range

-LRB-

VIS

nm

-RRB-

and

Near

Infrared

range

-LRB-

NIR

nm

-RRB-

were

acquired

in

an

alternating

fashion

VIS

and

NIR

image-size-settings

could

be

varied

independantly

For

encoding

depth

at

image-size-settings

of

for

VIS

and

for

NIR

cycles/s

were

obtained

-LRB-

cycle

VIS

L&R

pair

NIR

L&R

pair

-RRB-

At

the

cost

of

decreasing

image-size-settings

downto

for

VIS

and

for

NIR

camera

speed

could

be

increased

up

to

cycles/s

Encoding

depths

of

and

were

also

available

but

only

used

for

stills

-LRB-

due

to

the

lower

obtainable

framerate

-RRB-

Figure

Schematic

diagram

of

image

aquisition

The

sequentially

acquired

alternating

VIS

and

NIR

raw

image

frames

form

for

the

Left

and

Right

channel

The

NIR

image

size

is

smaller

than

the

VIS

image

size

to

increase

framerate

while

maintaining

an

overview

of

the

imaged

area

Enlarged

details

illustrate

the

NIR

transparency

of

the

Bayer

pattern

RGB-filters

which

are

applied

to

obtain

a

VIS

color

image

The

time

domain

axis

is

expressed

in

image

cycles

Along

this

image

cycle

axis

the

control

signals

for

NIR

and

VIS

LEDs

-LRB-

synchronized

with

respectively

NIR

and

VIS

camera

exposures

-RRB-

are

visualized

Data

Aquisition

Multispectral

stereoscopic

movies

were

recorded

in

several

typical

clinical

settings

for

which

the

technique

was

considered

as

possibly

useful

LED

currents

and

diaphragm

settings

were

chosen

so

that

for

each

movie

saturated

pixels

were

avoided

A

preview

mode

allowed

aiming

adjustment

of

converging

angle

α

and

focusing

of

the

cameras

After

software

triggering

the

stereo-camera

streamed

a

sequence

of

digitally

encoded

image

cycles

to

PC-memory

-LRB-

using

auto-incremental

numbering

-RRB-

All

images

were

automatically

saved

on

a

fast

SATA

harddisk-array

Camera

and

light

source

settings

were

automatically

stored

in

a

text

file

and

located

in

the

same

directory

All

patients

and

volunteers

gave

their

informed

consent

for

filming

as

well

as

for

publishing

the

resulting

image

material

No

diagnosis

or

therapy

was

based

upon

any

of

our

results

Data

Processing

General

Aspects

Processing

was

performed

with

custom

developed

software

-LRB-

programming

language

C++

-RRB-

using

the

stored

text

file

with

camera

and

light

source

settings

as

additional

input

values

Figure

schematically

represents

this

process

Figure

Schematic

diagram

of

image

processing

The

images

captured

within

the

visible

range

-LRB-

VIS

-RRB-

and

the

images

captured

within

the

near

infrared

range

-LRB-

NIR

-RRB-

are

combined

which

reveals

blood

vessel

patterns

below

the

skin

The

left

and

middle

column

focus

on

edge-enhancement

and

suppression

of

superficial

artifacts

the

right

column

serves

to

fill-in

the

blood

vessel

lumen

For

raw

VIS

NIR

images

as

well

as

processed

results

see

figures

and

The

processing

method

allowed

discrimination

between

image

information

obtained

from

the

tissue

surface

versus

image

information

obtained

from

within

the

tissue

In

order

to

achieve

this

a

distinction

was

made

between

shadows

reflections

and

absorption

contrasts

for

both

VIS

and

NIR

Suppressing

Shadows

on

the

Surface

Since

NIR

and

VIS

beams

were

matched

closely

shadows

produced

by

irregular

shapes

at

the

tissue

surface

-LRB-

e.g.

skin

structure

skin

folds

nevi

hair

etc.

-RRB-

or

by

objects

between

the

light

sources

and

the

tissue

-LRB-

fingers

needles

surgical

tools

etc.

-RRB-

also

matched

well

in

both

wavelength

ranges

Our

algorithm

excluded

such

matching

shadows

from

enhancement

and

left

all

useful

aspects

of

shadows

unaffected

-LRB-

e.g.

depth

clues

-RRB-

Useful

Effects

of

Shadows

and

Lighting

Geometry

Due

to

the

fact

that

the

VIS

and

IR

shadows

matched

very

closely

the

algorithm

was

able

to

selectively

enhance

contrast

from

below

the

surface

while

leaving

shadows

on

the

surface

unaffected

-LRB-

thus

not

distorting

these

important

depth

clues

-RRB-

Due

to

the

shallow

angle

of

the

lightbeams

the

skin

texture

was

pronounced

Due

to

the

lighting

from

two

sides

objects

that

were

brought

towards

the

tissue

surface

-LRB-

e.g.

needles

scalpels

probes

etc.

-RRB-

when

lighted

from

both

sides

could

produce

two

separate

-LRB-

not

too

heavy

-RRB-

shadows

These

shadows

met

and

typically

formed

a

V

pattern

when

an

object

touched

the

surface

in

the

middle

of

the

field

of

view

thus

providing

extra

information

for

depth

perception

Enhancing

Absorption

Contrast

of

Blood

Vessel

Walls

Edge

detection

by

a

Prewitt

image

filter

was

performed

on

each

VIS

and

NIR

image

Pixel

positions

containing

edge

information

above

a

certain

adjustable

threshold

in

both

spectral

regions

were

classified

as

surface

artefacts

and

excluded

from

enhancement

The

boundary

regions

of

absorption

contrasts

caused

by

structures

below

the

tissue

surface

produced

edges

that

were

mainly

present

in

the

NIR

image

Enhancement

was

selectively

performed

only

for

pixel

positions

where

the

NIR

image

contained

more

edge

information

than

the

corresponding

VIS

image

These

valid

pixels

identified

the

vessel

boundaries

The

positions

of

these

valid

pixels

were

stored

in

a

NIR

mask

Discarding

Reflections

on

the

Surface

Shiny

areas

that

produced

reflections

and/or

saturated

pixels

also

matched

in

both

wavelength

ranges

This

characteristic

allowed

to

calculate

a

NIR

mask

in

which

superficial

reflections

were

also

excluded

from

enhancement

Neighboring

pixels

of

identified

saturated

pixels

were

excluded

from

enhancement

The

radius

of

this

exclusion

region

was

programmable

but

due

to

the

favorable

anti-blooming

behavior

of

the

CMOS-camera

chips

suppressing

direct

neighbor

pixels

appeared

sufficient

Enhancing

Absorption

Contrast

of

Blood

Vessel

Lumen

The

content

of

blood

vessels

was

separately

enhanced

by

raising

pixel

values

of

the

normalized

NIR

image

to

the

power

of

N

-LRB-

with

N

user

adjustable

between

and

-RRB-

while

discriminating

NIR

pixels

below

a

freely

adjustable

noise

threshold

and

excluding

information

from

identified

shadows

Multiplication

with

the

NIR

mask

then

produced

a

final

enhancement

mask

for

subsequential

backprojection

into

the

VIS

image

by

pixel-to-pixel

multiplication

Suppressing

Contrasts

Originating

from

Melanin

Pigment

Superficial

contrasts

within

the

VIS

originating

from

melanin

pigment

concentrations

could

either

be

filtered

out

or

left

unchanged

Figure

illustrates

the

normalized

distribution

of

the

intensities

for

the

aquired

separate

spectral

bands

-LRB-

R

G

B

and

NIR

-RRB-

in

relation

to

the

intensity

-LRB-

IVIS

-RRB-

of

the

composed

visible

RGB-image

Intensities

were

calculated

using

the

Intel

ippiRGBtoGray

function

Four

clouds

of

data

points

can

be

discerned

being

IR/IVIS

-LRB-

red

channel

-RRB-

IG/IVIS

-LRB-

green

channel

-RRB-

IB/IVIS

-LRB-

blue

channel

-RRB-

and

INIR/IVIS

-LRB-

NIR

channel

-RRB-

By

calculating

the

ratio

of

-LRB-

IR/IVIS

-RRB-

-LRB-

INIR/IVIS

-RRB-

and

comparing

the

result

for

each

pixel

with

an

adjustable

threshold

it

can

be

decided

whether

or

not

to

apply

backprojection

to

a

pixel

This

concerns

the

pixels

located

within

the

overlapping

region

of

IR/IVIS

and

INIR/IVIS

within

Fig.

The

resulting

difference

is

clear

when

comparing

between

Fig.

and

d

with

regard

to

the

visualization

of

nevi

pigmentosum

and

hair

Figure

Relative

intensity

distribution

of

the

different

spectral

bands

Dimensionless

normalized

distribution

of

the

intensities

for

the

aquired

red

-LRB-

IR

-RRB-

Green

-LRB-

IG

-RRB-

Blue

-LRB-

IB

-RRB-

and

near

infrared

-LRB-

INIR

-RRB-

individual

spectral

bands

expressed

in

ratio

to

the

normalized

intensity

-LRB-

IVIS

-RRB-

of

the

composed

RGB-image

The

four

data

clouds

-LRB-

R

G

and

B

labeled

by

their

natural

colors

and

NIR

labeled

as

pink

-RRB-

show

a

generally

marked

separation

but

especially

for

a

number

of

pixels

within

the

red

and

infrared

the

data

clouds

partly

overlap

This

indicates

regions

where

the

VIS

contrast

potentially

is

superior

to

the

NIR

contrast

By

comparing

an

adjustable

threshold

with

the

calculated

ratio

of

-LRB-

IR/IVIS

-RRB-

-LRB-

INIR/IVIS

-RRB-

it

can

be

decided

which

spectral

band

provides

superior

contrast

for

the

pertaining

pixel

and

thus

whether

or

not

it

is

used

for

enhanced

backprojection

Data

Presentation

The

images

could

either

be

displayed

on

an

auto-stereoscopic

liquid

crystal

display

-LRB-

LCD

-RRB-

monitor

or

on

a

conventional

CRT

monitor

equipped

with

shutter

glasses

The

auto-stereoscopic

LCD-monitor

-LRB-

Sharp

-RRB-

was

equipped

with

software

controllable

switching

between

stereoscopic

-LRB-

or

-RRB-

and

normal

-LRB-

monoscopic

or

-RRB-

mode

Monitor

resolution

was

in

monoscopic

mode

-LRB-

XGA

-RRB-

In

stereoscopic

mode

the

available

pixels

were

split-up

in

two

separate

images

-LRB-

L&R

-RRB-

by

activation

of

a

vertical

LCD

parallax

barrier

The

principle

behind

this

technology

is

illustrated

in

Fig.

Figure

Principle

of

applied

autostereoscopic

LCD-monitor

-LRB-

reprinted

with

permission

from

Sharp

-RRB-

In

mode

only

one

camera-channel

is

displayed

-LRB-

either

from

the

L

or

R

camera

-RRB-

and

the

parallax

barrier

is

not

actuated

Both

eyes

of

an

observer

therefore

receive

the

same

image

and

a

conventional

flat

image

with

full

resolution

is

seen

In

mode

both

camera

channels

are

displayed

-LRB-

L&R

-RRB-

and

the

parallax

barrier

is

actuated

The

left

eye

and

right

eye

of

an

observer

now

receive

different

images

and

a

stereoscopic

-LRB-

in-depth

image

-RRB-

with

halve

resolution

is

seen

The

CRT-monitor

-LRB-

iiyama

vision

master

-RRB-

was

used

at

a

resolution

setting

of

in

combination

with

wireless

shutter

glasses

-LRB-

e-Dimensional

-RRB-

and

thus

provided

stereoscopic

information

without

sacrificing

resolution

The

enhancement

algorithm

occurs

on

a

pixel-to-pixel

basis

and

does

not

affect

resolution

Our

device

offered

several

imaging

modes

for

data

presentation

Monocopic

raw

VIS

preview

-LRB-

normal

full

color

vision

-RRB-

Monoscopic

raw

NIR

preview

-LRB-

greyscale

-RRB-

Off-line

stereoscopic

looped

VIS

view

with

and

without

enhanced

blood

vessel

back-projection

-LRB-

with

freely

adjustable

enhancement

settings

-RRB-

Off-line

stereoscopic

looped

raw

NIR

view

or

enhanced

NIR

view

-LRB-

with

freely

adjustable

enhancement

settings

-RRB-

Stereoscopic

stills

in

all

modesMonoscopic

stills

in

all

modes

-LRB-

freely

switchable

between

Left

and

Right

-RRB-

All

modes

offered

the

possibility

for

pause

and

scrolling

forward

or

reverse

frame-by-frame

By

means

of

virtual

slider

controls

and

virtual

pushbuttons

the

user

interface

allowed

freely

adjustable

settings

for

shadow

suppression

pigment

suppression

noise

threshold

and

vessel

lumen

fill-in

contrast

Results

Results

for

Blood

Withdrawal

During

routine

blood

withdrawal

the

inserted

needle

tip

remained

only

slightly

visible

on

the

VIS

image

due

to

skin

surface

deformation

-LRB-

see

Fig.

-RRB-

On

the

raw

NIR

image

however

the

inserted

needle

remained

visible

within

the

tissue

for

a

few

mm

with

some

metallic

reflection

Subcutaneous

bleeding

during

needle

removal

could

be

detected

in

the

raw

NIR

image

while

the

needle

tip

was

still

in

the

tissue

-LRB-

see

Fig.

-RRB-

This

was

clearly

highlighted

by

backprojection

in

the

VIS

image

-LRB-

see

Fig.

-RRB-

The

processing

settings

used

to

obtain

this

backprojection

mainly

laid

the

accent

upon

enhancing

the

absorption

contrast

of

blood

vessel

lumen

whereas

edge

enhancement

was

set

to

minimum

Reflections

on

the

thumb

nail

did

not

lead

to

image

distortion

and

the

blood

volume

in

the

nail

bed

showed

more

contrast

Figure

Routine

blood

withdrawal

Image

pairs

showing

unprocessed

images

for

VIS

-LRB-

a

-RRB-

and

NIR

-LRB-

b

-RRB-

as

well

as

the

result

after

application

of

the

new

image

processing

method

-LRB-

c

-RRB-

Note

the

forked

shadow

-LRB-

which

is

not

effected

by

the

enhancement

algorithm

-RRB-

the

clearly

visualized

subcutaneous

bleeding

and

the

improved

visibility

of

the

needle

tip

Results

for

Dark

Skin

Hardly

any

vascular

contrast

is

present

within

the

VIS

image

-LRB-

see

Fig.

-RRB-

The

NIR

image

however

is

not

affected

by

skin

pigmentation

and

reveals

a

subcutaneous

vascular

pattern

-LRB-

see

Fig.

-RRB-

The

combined

information

results

in

an

enhanced

image

-LRB-

see

Fig.

-RRB-

Figure

Influence

of

skin

pigmentation

A

dark

skin

color

-LRB-

a

-RRB-

has

no

significance

for

the

applied

NIR

wavelength

of

nm

Blood

vessels

provide

good

contrasts

-LRB-

b

-RRB-

and

the

resulting

enhanced

image

-LRB-

c

-RRB-

offers

an

improved

visualization

of

the

vasculature

Results

for

Vein

Detection

Through

Iodide

Iodizing

skin

portions

before

surgery

is

a

common

clinical

procedure

which

darkens

the

skin

and

lowers

the

visibility

of

blood

vessels

Our

method

offers

imaging

right

through

iodide

To

demonstrate

this

we

filmed

a

glass

Petri

dish

placed

horizontally

upon

a

volunteers

arm

while

filling

it

up

to

a

mm

thick

layer

of

iodide

solution

From

the

VIS

image

no

vessels

could

be

detected

through

the

iodide

solution

-LRB-

see

Fig.

-RRB-

and

hardly

any

through

the

empty

Petri

dish

-LRB-

see

Fig.

-RRB-

The

raw

NIR

image

however

clearly

showed

superficial

blood

vessels

even

through

the

Petri

dish

-LRB-

see

Fig.

-RRB-

Figure

Vein

detection

through

iodide

Within

the

visible

range

the

superficial

vasculature

is

only

vaguely

discernable

-LRB-

a

-RRB-

After

filling

the

Petri-dish

with

a

mm

thick

layer

of

iodide

solution

this

fully

blocks

out

the

tissue

view

within

the

visual

range

-LRB-

b

-RRB-

whereas

a

clear

view

of

the

vasculature

remains

possible

at

the

applied

NIR

wavelength

of

nm

-LRB-

fig

-RRB-

Results

for

Varicose

Vein

and

Nevi

Pigmentosum

Inspection

The

device

was

used

to

film

varicosis

patients

during

dermatological

outpatient

clinic

The

visualization

of

varicose

veins

could

be

drastically

improved

Even

in

cases

where

the

blood

vessels

were

covered

by

subcutaneous

fat

they

could

be

detected

quite

well

-LRB-

see

Fig.

-RRB-

By

either

de-activating

or

activating

the

first

NIR

mask

in

the

processing

algorithm

-LRB-

see

Fig.

-RRB-

it

was

also

possible

to

choose

whether

pigment

contrasts

in

the

visual

range

were

left

intact

or

filtered

out

The

difference

is

clear

when

comparing

between

Fig.

and

d

with

regard

to

the

visualization

of

nevi

pigmentosum

and

hair

By

clicking

a

software

screen

button

this

suppression

of

pigment

contrast

could

be

freely

switched

on

and

off

during

viewing

Figure

The

VIS

image

does

not

contain

much

information

about

the

underlying

vascular

pattern

-LRB-

a

-RRB-

The

NIR

image

however

clearly

shows

what

's

hiding

beneath

the

surface

-LRB-

b

-RRB-

Note

that

when

building

the

enhanced

image

the

nevus

which

is

present

in

the

VIS

image

can

freely

either

be

suppressed

as

a

surface

contrast

-LRB-

c

-RRB-

or

be

kept

visible

-LRB-

d

-RRB-

Discussion

From

the

figures

presented

in

this

article

it

is

clear

that

all

experiments

resulted

in

improved

visualization

of

superficial

blood

vessels

Based

upon

the

absorption

coefficients

of

Hb

versus

arteries

provide

superior

contrasts

at

nm

The

presented

images

however

show

enhanced

venous

contrasts

because

the

larger

arteries

are

buried

deeper

underneath

the

skin

Stereoscopic

movies

provide

a

more

lifely

impression

of

the

underlying

but

presentation

of

stereoscopic

movies

is

not

possible

in

a

printed

journal

We

therefore

offer

the

possibility

to

download

viewer

software

and

movies

via

the

internet

For

both

the

autostereoscopic

LCD

and

the

CRT

display

plus

shutter

glasses

we

found

that

the

perception

of

depth

was

less

for

stills

than

for

movies

This

qualitative

observation

matches

research

findings

on

stereoscopic

display

techniques

in

X-ray

and

is

also

consistent

with

the

differences

between

static

and

dynamic

stereoacuity

described

by

Mathias

and

Rudolf

Sachsenweger

Stereoscopic

movies

also

showed

an

increase

in

apparent

image

sharpness

compared

to

stereoscopic

stills

which

may

be

explained

by

Shipley

's

description

of

stereoscopic

contour

integration

over

blur

Compared

to

the

autostereoscopic

LCD-display

the

increase

in

perceived

sharpness

was

much

stronger

when

using

a

CRT

with

shutter

glasses

For

monoscopic

stills

however

the

LCD-display

-LRB-

switched

to

monoscopic

mode

-RRB-

was

superior

to

the

CRT

These

observations

are

in

agreement

with

the

technology

overview

by

and

can

be

explained

by

the

sacrifice

of

resolution

when

using

the

LCD

display

in

autostereoscopic

mode

which

price

has

not

to

be

paid

when

using

the

CRT

with

shutter

glasses

The

beneficial

effect

of

separately

adjustable

enhancement

paths

for

blood

vessel

contours

and

blood

vessel

lumen

which

we

experienced

during

the

development

of

our

processing

algorithm

is

consistent

with

the

findings

of

Yin

et

al.

that

surface-features

and

edge

processes

make

different

contributions

in

determining

an

object

's

unity

and

shape

The

perceived

usefulness

of

stereoscopic

information

however

also

supports

the

concept

formulated

by

Tse

that

mergeable

volumes

rather

than

relatable

contours

are

the

critical

elements

in

completion

With

our

technique

we

have

aimed

to

avoid

parallax

errors

and

loss

of

vessel

contrast

by

shadows

which

are

inherent

to

other

blood

vessel

contrast

enhancement

techniques

projecting

the

vascular

pattern

via

a

projector

onto

the

skin

We

exclude

parallax

errors

because

the

user

truly

looks

beneath

the

tissue

surface

trough

two

camera

's

Absence

of

vessel

contrast

in

shadows

is

avoided

by

lighting

from

two

sides

and

by

discrimination

of

superficial

artifacts

versus

contrasts

originating

from

below

the

surface

This

ability

to

selectively

enhance

contrasts

from

beneath

the

surface

while

preserving

the

natural

depth

clues

of

shadows

on

the

surface

supports

both

static

and

dynamic

transmittance

anchoring

of

the

visual

system

which

is

crucial

for

depth

perception

Preservation

of

natural

shadows

is

furthermore

important

for

the

correct

interpretation

of

depth

clues

by

occlusion

-LRB-

e.g.

from

a

hand

or

an

instrument

positioned

between

observer

and

tissue

-RRB-

thus

precluding

errors

in

depth

perception

Since

only

carefully

balanced

white

light

is

projected

on

the

skin

there

is

no

impairment

of

color

perception

It

is

up

to

the

user

to

freely

switch

between

normal

full

color

vision

color

vision

plus

superimposed

blood

vessel

backprojection

and

stereoscopic

near

infrared

greyscale

vision

-LRB-

with

or

without

enhancement

features

-RRB-

The

embodiment

described

in

this

article

still

has

some

drawbacks

Due

to

the

fact

that

the

pertaining

configuration

aquires

VIS

and

NIR

images

sequentially

motion

artifacts

can

lead

to

a

backprojection

shift.The

deeper

the

vessels

lie

under

the

surface

the

less

we

can

visualize

them

In

the

pertaining

configuration

the

depth

range

is

limited

to

about

mm

Based

upon

the

absorption

coefficients

of

Hb

and

arteries

provide

superior

contrasts

at

nm

The

presented

images

however

show

enhanced

venous

contrasts

because

the

larger

arteries

are

buried

deeper

underneath

the

skin

The

above

mentioned

drawbacks

require

further

development

Fortunately

many

technological

improvents

still

can

be

added

Allthough

the

device

worked

quite

well

under

normal

ambient

lighting

conditions

-LRB-

Lux

-RRB-

the

use

of

a

switchable

filter

might

further

improve

spectral

separation

Furthermore

known

techniques

like

application

of

crossed

polarizers

in

combination

with

more

powerful

light

sources

can

offer

a

considerably

larger

penetration

depth

Conclusions

Compared

to

inspection

with

the

naked

eye

under

normal

lighting

conditions

the

tested

stereoscopic

blood

vessel

contrast

enhancer

offered

improved

visualization

in

all

investigated

settings

providing

the

best

stereoscopic

image

quality

when

using

the

CRT

monitor

with

shutter

glasses

and

the

best

monoscopic

image

quality

when

using

the

LCD

set

to

monoscopic

mode

Our

technique

supports

perception

of

depth

motion

and

discrimination

between

tissue

surface

and

underlying

structures

It

also

has

potential

as

an

educational

tool

by

offering

the

possibility

to

look

and

record

trough

the

eyes

of

an

experienced

specialist

Further

improvements

on

penetration

depth

frame-rate

and

focus

depth-of-field

form

targets

for

momentary

ongoing

further

research

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

contactless enhanced viewing of superficial vasculature

reconnaissance of feasibility for various clinical applications