02/06/2014

1

OPTICAL IMAGING TECHNIQUES FOR DENTAL

BIOMATERIALS INTERFACESPresented By:

Dr. Hashmat Gul,

Demonstrator , AMC , NUST,

Dental Materials department.

1. CONFOCAL MICROSCOPY

�The main function of A confocal imaging

system is to improve image contrast.

�There is significant improvements in

resolution, lying somewhere between

that of conventional light microscopy and

TEM/SEM.

�Recent developments have allowed both

clinical imaging and improvements in

resolution at significant depths within a

sample.

02/06/2014

2

WORKING PRINCIPLE

�The expression ‘confocal’ derives from the use of a pinhole aperture in the conjugate focal plane of an objective lens, in both the illuminating and imaging pathways of a microscope.

�The area surrounding the aperture rejects stray light returning from areas that are not in the focal plane of the lens.

�In order to see more than one small patch of the sample some form of scanning device is required.

02/06/2014

3

02/06/2014

4

�ADVANTAGES of High-Resolution CFM

1.Images derived from either the surface of a sample or

beneath the surface.

2.Minimum requirements for specimen preparation.

3.These images are thin (>0.35 μm) optical slices, up to 200 μm

below the surface of a transparent tissue.

�With microscopes running under ‘normal’ conditions,

� The optical section thickness will be >1 μm

� The effective penetration into enamel and dentine = 100 μm.

� The best images derived from structures just below the surface

(<20 μm).

02/06/2014

5

SAMPLE PREPARATION AND MOUNTING

�Most confocal microscopes are of the ‘reflected light’ or ‘epi-

illumination’ type so that samples can be imaged from their surfaces

without the need for thin section preparation.

�ADVANTAGES

� Relatively large intact tooth samples can be placed on the

microscope stage.

� Section the sample once and observe directly the subsurface

structures.

�SAMPLE PREPARATION

�CUT : with a fine diamond saw, running very slowly under water, to give

the best surface finish possible in the ‘as cut’ condition.

�POLISH : It is easier to image internal structures if the sample is lightly

polished, to remove the smear layer(a light-diffusing structure).

�SAMPLE MOUNTING

�For Subsurface Analysis, A coupling/immersion medium is

required.

�Water

� Oil

�Where indicated for the lens being used, cover slips will be

necessary, but these need to be as thin as possible.

02/06/2014

6

2. CONVENTIONAL FLUORESCENCE AND

REFLECTION IMAGING

�In Dental Materials

Research, CFM is used to

highlight the distribution of

components within an

adhesive system with

fluorescent labels.

• It is possible to study the rapidly changing events.

�THE PRINCIPLE OF

FLUORESCENCE

� The absorption of a photon by

a dye molecule that triggers

the emission of another photon

with a longer wavelength and

lower energy.

� The difference in wavelength

is called the ‘Stokes Shift’.

02/06/2014

7

�In Fluorescence Labelling Experiments, it is

important to be aware of

� Any potential Artefacts.

� The Back-scattered Signal (an incoherent light source with a TSM)

� Affect of the resin-based adhesive systems on The Refractive And

Reflective Properties of dentine and enamel.

3. IMAGING WATER TRANSIT IN MATERIALS

�METHODS

� The seal of restorative materials can be judged using high-

resolution micro-leakage studies.

� Fluorescent dyes used to test fluid movement/permeability

02/06/2014

8

� NANO-LEAKAGE

�Griffiths et al. (1999), confirmed that fluorescent dye could

penetrate the porosities within the smear layer as well as the bonded

interface. This is ‘Nano-leakage’.

�Occurs in the absence of gaps through nanometer-sized spaces ( 0.02

μm) and starts at the bottom of the hybrid layer, and spreads

throughout this structure.

�Fluid movement is observed at the junction of the adhesive resin and

hybrid layer during flexure of the restoration and the tooth.

� TO AVOID MISINTERPRETATION OF NANO-LEAKAGE,

� Phase-Separation of Fluorophores: Fluorescent dyes placed in the

pulp chamber must be soluble in Distilled Water or in Phosphate-

buffered Saline. Otherwise, the fluorophores will Phase-Separate

and will not reach the interface.

� Image Artefacts: Solvents, such as Alcohol, should be avoided as

they can impair the integrity of the hybrid layer.

� Size of dye molecule: Mostly very small, may permeate throughout

dentine, the hybrid layer and adhesive layer.

02/06/2014

9

� INCORPORATING DYES INTO DENTINE-BONDING AGENTS

�Increase The Scope Of The Imaging Technique, by analysing

� The morphology of the hybrid layer

� The extension of resin tags.

�Gives Better Image Contrast

� The individual structures within the same specimen can be better

recognized and analyzed .

�Using Two Different Marker Systems & CFM,

�It is possible To Evaluate The Effect Of Pulpal Pressure On

� Adhesive Water Sorption And

� On The Sealing Ability of current adhesive systems.

�TECHNIQUE based upon

� The Silver Staining Nano-leakage Technique Of Sano &

� The Micro-permeability Methods.

TECHNIQUE

02/06/2014

10

Deep Dentine Crown Segments with dentine thickness of 0.7– 0.8 mm prepared by removing the occlusal enamel with a slow-speed, water-

cooled diamond saw.

The Roots removed, 1mm below

CEJ

Pulpal Tissue removed

A Standard Smear Layer created(180 grit Silicon Carbide Papers)

The sample attached to a Perspextm Support,

perforated by an 18 SS tube

Connected to A Hydraulic Pressure Device

Different adhesives applied according to the manufacturer’s

instructions

Light-curing

Ammoniacal Silver Nitrate Soln.

delivered at 20 cm H2O for 24 h.

Rhodamine Soln. is delivered for 3 h using the same pressure device.

Samples Sliced into 1 mm slabs

Lightly Polished1200 grit silicon carbide paper,

Ultra-sonicatedfor 2 min

Photo-developed under

UV

Washedwith De-ionized

Water for 30s

Further ultra-

sonicatedfor 2 min.

Examined in Reflection & Fluorescence Mode using a ×100 oil immersion lens with a TSM/

CLSM & the appropriate excitation/ emission filters.

�APPLICATIONS

�Use of fluorescent dyes e.g.

� The diffusion of the Rhodamine dye through the adhesive

interface, from the pulp and the dentinal tubules.

�The Silver Staining Technique shows

� Nano-leakage, within the hybrid layer

� Water Sorption, (Water trees) within the adhesive components.

02/06/2014

11

�The confocal fluorescence

evaluation showing Rhodamine

(fluorescent dye) penetration.

Hybrid layer

Adhesive

�Silver-stained reflection confocal

image of silorane adhesive & dentine

---Silver grains (black dots) dispersion

showing Nanoleakage & Water sorbtion Hybrid zone

Primer layer

Adhesive layer

02/06/2014

12

� Silver-stained reflection confocal

image of Scotchbond 1XT adhesive

(Total-etch/all-in-one system)---

Silver grains (black dots) dispersion showing Nanoleakage & Water sorbtion

Primer layer

Hybrid zone

Adhesive

�Fluorescence confocal image of

scotch bond 1XT adhesive

�ADHESIVE--- The bubbles/blisters

due to water transit=tubular opening

Adhesive

Hybrid zone

02/06/2014

13

� Combined reflection &

fluorescence image of S3

Bond and dentine --- Show the relative sealing ability of different components of an adhesive system

� Silver grains= white reflective

dots.

� Grey background=

fluorescence from Rhodamine

+ water permeation.

Hybrid zone - gap

3M ESPE Silorane Bonding System

4. IMAGING MOISTURE-SENSITIVE MATERIALS

�Confocal microscopy can be used to examine below the surface of samples

without dehydration damage due to vacuum.

�Studies of drying out effects on materials can be made using Dry Objectives.

� To counter surface reflections of drying out cements Immersion Mediums are

used.

�Measuring the rate of crack opening and closure in different environments

will give an indication of their maturation rate.

02/06/2014

14

�A series of confocal reflection images

= the uptake of water and

consequent swelling of a fully

reacted glass ionomer–composite

‘Reactmer---Crack closure over time.

Material Tooth

Crack

�Effects Of Immersion Medium On The Sample

� OIL , keep the sample hydrated,

� GLYCERINE , hygroscopic , the material will lose water.

� WATER , The glycerine can be changed subsequently for water and the effects

of water influx on the same sample can then be studied.

02/06/2014

15

� Such experimental procedures can be applied to imaging

the maturation of

�Glass Ionomer cement

�Poly-acid-modified composites

�Glass Ionomer – Composite-type materials such as ‘Reactmer’

�LIMITATIONS OF FLUORESCENCE & CFM

1. Photo-bleaching of fluorescent probe.

2. Photo-toxicity of fluorescent probes.

3. Non-ideal characteristics of an optical

system of a microscope: chromatic and

spherical aberration.

02/06/2014

16

5. MULTI-PHOTON IMAGING: DEEPER PENETRATION

� LIMITATIONS OF CONFOCAL IMAGING OF DENTAL MATERIALS

�Light-Scattering Properties of the hard tissues & the tooth-colored

restorations.

�Reflective/Opaque Features will interfere with light passing deeply into

the sample, and returning from, the focused-on plane.

�Fluorescence confocal imaging will work well when examining discrete,

isolated, structures within an interface.

�TWO-PHOTON EXCITATION

MICROSCOPY

�Allows imaging of living tissue up to a

very high depth (1mm).

�It uses Red-Shifted excitation light which

can also excite fluorescent dye.

�Titanium: Sapphire Laser used as incident

beam.

02/06/2014

17

�WORKING PRINCIPLE

� The energy of two photons (IR

light) absorbed by the fluorescent

molecule and the energy is

irradiated as a single photon of

shorter wave-length (green).

� The reverse of normal, thus, the

need for pinholes is reduced (no

fluorescence outside the focal

plane).

�Only at the focal point, there is enough

energy to excite fluorescent dyes with this

long-wavelength light.

�New fluorescent dyes are developed that

produce an optimal fluorescence output for

low illumination intensities e.g. APSS dye.

�Two-photon excitation fluorescence image of the HEMA in scotchbond 1XT adhesive labelled with APSS dye. �Due to the high efficiency of this dye, this high resolution image was recorded in10 s and has a lateral resolution of 760 nm.

02/06/2014

18

�ADVANTAGES of Two-Photon

Excitation

A superior alternative to CFM

� Deeper Tissue Penetration (almost twice)

� Better Resolution (Efficient light detection)

� Greater Accuracy of images

� Reduced Photo-toxicity

6. FLUORESCENCE LIFETIME IMAGING,FLIM

�In standard fluorescence imaging, a sensitive detector, such as CCD, images

emission from a fluorophore.

�Fluorescence Signal Intensity is dependent on

� The intensity of the excitation light

� The concentration of fluorophore.

�Fluorescence Lifetime is,

� Independent of fluorophore concentration,

� But dependent on local environment.

02/06/2014

19

�FLIM allows researchers to obtain precise Quantitative Data about both

� Fluorophore distribution

� Local environment.

�Two Principal Approaches to FLIM implementation exist:

� In the time domain.

� In the frequency domain.

�For Time-domain Measurements,

�A laser or LED excites the sample with femtosecond to nanosecond pulses.

�A Gated Detector i.e. CCD camera system, captures the exponential decay

of the fluorescence.

�The investigator can compute the lifetime of a fluorophore with single

exponential decay by acquiring only two images at two different points in

time after the excitation.

02/06/2014

20

�FLIM can determine

�Adhesive penetration

�Bonding mechanisms to carious dentine.

1.FLIM allows improved

discrimination of different

dyes & adhesive components.

Low-magnification view of the SE Bond dentine–adhesive

interface imaged with wide field fluorescence microscopy:

a. Blue excitation–green emission for the Lucifer yellow(primer);

b. Green excitation–red emission for the Rhodamine(Primer)

�ADVANTAGES

02/06/2014

21

2. FLIM can discriminate weak

fluorescence derived from the

dental substrate & from the

fluorescent dyes with similar

spectral characteristics.

3. FLIM records the decay rate of

the substrate. a. Two-photon microscopy=Poor contrast b/w

Rodamine & Lucifer yellow (similar spectral

characteristics).

b. The FLIM image=a strong contrast due to the large

difference in fluorescence decay.

A. Only the lucifer yellow-labelled primer remains visible;

B. The average fluorescence lifetime of lucifer yellow is 5.3 ns;

C. Poor image Contrast b/w Rodamine & Lucifer yellow;

D. FLIM shows better image contrast.

E. Selectively shows the Rhodamine-labelled primer;

F. The average rhodamine fluorescence lifetime is 2.8 ns,

Lucifer yellow-labelled Primer

Rhodamine-labelled Primer

2 Photon Excitation FLIM

02/06/2014

22

7. HIGH-SPEED IMAGING OF DYNAMIC EVENTSWITHIN MATERIALS

�Imaging of fracture events within materials can be undertaken using

Video Rate Confocal Microscopy.

�APPLICATIONS

TSM using video confocal microscopy has been employed extensively

for the imaging of

� Bur–tooth cutting interactions

� Air abrasion cutting

� The effects of lasers on tooth tissue

� LIMITATIONS

�A significant risk of damage to the end lens of the microscope objective

using such cutting techniques.

�‘In Vivo’ long focal range objectives is used to separate the cutting

laser beam from the lens system of the microscope.

�These lenses have a working range of upto 8 mm.

�Internally focusable elements select the plain of tissue on which to focus.

02/06/2014

23

�RECORDING RATES

�Currently <60 frames per second (twice video rate),

�There is a need for increased speed of imaging and recording:

a feature becoming more available with

� Better EM-CCD camera sensitivity &

� Ever increasing computing power.

DENTINE ABLASION with erbium

YAG laser at 250 mJ, 7 pps, 10 pulses in

total (25 frames per second):

a. Surface after two pulses;

b. During the next pulse;

c. After four pulses;

d. Final image of dentine showing the effect

of sequential pulses

�The Laser Energy Pulses seen�Ablating the tooth tissue &�Debris fields along the cutting path.

02/06/2014

24

ENAMEL ABLATION with

350 mJ, 10 pps, 5 pulses (25 fps).

Progressive structural damage is

shown

ENAMEL

8. CONCLUSION

�The advent of confocal microscopy has undergone a renaissance, especially

within the biological sciences, for high resolution imaging.

�The materials–biological science interface offers, a unique experimental

envelope for pushing the development of new optical microscopic techniques.

�The local environmental advantages for the specimen, enable experiments to

be undertaken with reduced preparation artefact, while modern

developments can take resolution beyond what was once thought to be the

limits for the wavelengths employed.

02/06/2014

25