02/06/2014 1 OPTICAL IMAGING TECHNIQUES FOR DENTAL BIOMATERIALS INTERFACES Presented 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.
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02/06/2014
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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.
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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.
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�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).
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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.
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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’.
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�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
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� 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.
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� 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
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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.
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�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
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� 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
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� 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.
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�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.
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� 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.
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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.
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�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.
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�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.
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�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.
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�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
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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
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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.
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�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.
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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