optical imaging technique for dental biomaterials interfaces copy
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PDF, Confocal Microscopy techniques to study Dental Biomaterials interfaces.TRANSCRIPT
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
limits for the wavelengths employed.
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