Microscopy dental applications /certified fixed orthodontic courses by Indian dental academy
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Microscopy dental applications /certified fixed orthodontic courses by Indian dental academy
- 1. MICROSCOPY Dental applications INDIAN DENTAL ACADEMY Leader in Continuing Dental Education www.indiandentalacademy.com
- 2. WHY DO WE NEED MICROSCOPES
- 3. CONTENTS…. • History of microscopy • The basics concepts of optics • Optical phenomena of interest to microscopy • Microscopes in dental research - principles, specimen preparation and applications • Choice of microscopes for successful research • Microscopes in clinical dentistry • Conclusion
- 4. A WALK DOWN MEMORY LANE….. Leeuwenhoek- Binnig and Rohrer - Simple microscope Scanning tunnelling Hillier- microscope Galileo - TEM Occhiolino 1609 1674 1938 1981 1590 1665 1931 1957 1983 Jannsen – Ruska – Compound Electron microscope Binnig - microscope Atomic force microscope Robert Hooke - Micrographia Petran and Minsky – Confocal microscopy
- 5. THE BASICS • MAGNIFICATION • IMAGE QUALITY • BASIC OPTICS AND OPTICAL PHENOMENA
- 6. MAGNIFICATION • ENLARGING SOMETHING ONLY IN APPEARANCE • OPTICAL MAGNIFICATION (Power) – Apparent size : True size…….at 25 cm distance • LINEAR AND ANGULAR MAGNIFICATIONS • HIGHER MAGNIFICATION LENSES GENERALLY HAVE HIGH RESOLUTION MAGNIFICATION (M) = M0 x Me • Useful magnification… • Depth of field …… • Field number and size….
- 7. RESOLUTION The ability to differentiate between two closely positioned bright objects. Resolving power line * Light microscope includes phase contrast and fluorescence microscopes. Electron microscope includes transmisson electron microscope.
- 8. THE BASIC CONCEPTS OF OPTICS - OPTICAL PHENOMENA OF INTEREST TO MICROSCOPY REFLECTION Vs REFRACTION refraction reflection
- 9. Diffraction…… light bending around an object Airy diffraction….
- 10. PATHWAYS OF LIGHT…… EYEPIECE AND OBJECTIVE LENS – CONVEX LENSES Converging lens….. Focal point focal length POWER (diopters) = 1 / f
- 11. The real The magnified image…. image.. object object f 2f 2f f IMAGE IMAGE
- 12. Image formed by the microscope….. Eyepiece Objective Image of Object (X1) Object (X) Final image (X2) X1 – a real, inverted and bigger image X2 – a magnified and virtual image
- 13. TOTAL INTERNAL REFLECTION REFRACTION OF LIGHT AT A MEDIUM BOUNDARY,ENOUGH TO SEND IT BACKWARDS AND CAUSE EFFECTIVE REFLECTION OF THE LIGHT Refraction at medium boundary
- 14. THE EVANESCENT WAVE • EVANESCENT = “tending to vanish” • DECAYS EXPONENTIALLY WITH DISTANCE • RADIATION PRESSURE TO ILLUMINATE SMALL OBJECTS • Selective excitation of fluorophores
- 15. FLUORESCENCE
- 16. FOCUS , CONFOCAL AND PARFOCAL FOCUS – • point of convergence CONFOCAL - "having the same focus." PARFOCAL – “having the same focus in the entire range of magnifications”
- 17. Categorization of microscopy …. Illumination methods Imaging method Transmitted Reflected • visible light • laser • electrons • X-rays • non-illumination type
- 18. MICROSCOPES IN DENTAL RESEARCH • LIGHT MICROSCOPE • PHASE CONTRAST MICROSCOPE • STEREOMICROSCOPE • TOOLMAKER’S MICROSCOPE • FLUORESCENCE MICROSCOPE • TOTAL INTERNAL REFLECTION MICROSCOPE • ELECTRON MICROSCOPE scanning electron microscope • field emission scanning electron microscope transmission electron microscope • CONFOCAL MICROSCOPE • SCANNING PROBE MICROSCOPES atomic force microscope scanning tunneling microscope • X-RAY MICROSCOPE
- 19. LIGHT MICROSCOPE • The simplest form of microscope eyepiece • Types based on illumination – bright, dark, objective rheinberg, phase contrast, differential interference, Hoffman modulation, sample polarised, fluorescence stage condenser • 0.5 – 1mm thin specimen • Image formation due to source of illumination absorption of light by objects in the path
- 20. • Objective lens – 4x, 10x, 40x,100x • Eyepiece – 10x • Resolution : 200 nm (Ernst Abbe’s resolution limitation)
- 21. PHASE CONTRAST MICROSCOPE • Enhances contrast • Altering the Light Waves • The Invisible Can Be Seen • Preparation of Specimen
- 22. STEREOMICROSCOPY • Cherubin d’Orleans – 1671 • Wenham and Greenough -1890 • Optical light microscope • Stereoscopic vision – spatial 3- dimensional images • Designs – Cycloptic (common main objective), Greenough.
- 23. Illumination and other aspects Sources of illumination: – Episcopic and diascopic illumination – Fluorescence illumination – LED (Chambers and Nothnagle – Microscopy and microanalysis – 1997) Total magnification : 2x to 540x Resolution : 1.6 µm
- 24. TOOLMAKER’S MICROSCOPE • Precision measuring instrument • Linear scales • Magnification….. • Illumination : – Brightfield and darkfield – Rheinberg lighting – Polarisation – Phase contrast
- 25. Fluorescence microscope • Concept of fluorescence and epi-fluorescence. • The sample itself is the light source. • Fading - photobleaching •
- 26. Specimen preparation • Fluorescent Dyes • Immunofluorescence
- 27. Total internal reflection microscope • Main disadvantage of fluorescence microscope – background fluorescence • Evanescent wave • 200 nm depth • Cell adhesion
- 28. ELECTRON MICROSCOPE • Development of the electron microscope • Energy of the electrons – 1 KeV to 1 MeV. • Types • Gold standard in dental research….?
- 29. • TRANSMISSION ELECTRON MICROSCOPE – Max Knoll and Ernst Ruska (1931)- (Nobel prize 1986) •SCANNING ELECTRON MICROSCOPE – Zworikyn -1942 • USES – Topographical imaging – Morphology imaging – Substructure analysis – Crystallography – Chemical composition
- 30. BASIC PRINCIPLES Electron source (or) the gun – 5 – 50 KV energy electrons ELECTRON DENSE ELECTRON TRANSLUCENT ELECTRON TRANSPARENT
- 31. Electron optics • Electron beam trajectory – electrostatic lenses (in the electron gun) and magnetic lenses • Double deflection scanning (A.W. Crew – Science – 1961)
- 32. ELECTRON – SPECIMEN INTERACTIONS • BULK SPECIMEN INTERACTIONS • THIN (FOIL) SPECIMEN INTERACTIONS
- 33. SCANNNING ELECTRON MICROSCOPY MAGNIFICATION 20 X TO 65000 X RESOLUTION upto 5 to10 nm
- 34. Specimen preparation for scanning electron microscopy Fixation of specimen Dehydrate and embed in resin Section and Stain Gold / Platinum sputtering (Weimer and Martin – 13th International Congress on Electron Microscopy – 1974)
- 35. SPUTTER COATER • Why sputter coat ? • Thickness…. • Materials used and method…. • In all forms of SEM ??
- 36. FIELD EMISSION SCANNING ELECTRON MICROSCOPY - Erwin Müller (1936) • Materials science and bio-sciences • metal tip in a low vacuum chamber • Emitter gun – fine wire cathode and concave anode with a hole in the centre • Variation of atomic orientation in emitter tip • Sputtering…. • Resolution 20 Å • Magnification 50,000 X
- 38. Specimen preparation for Transmission electron microscopy ( Auvert – Microscopy and Microanalysis – 2003) Method 1 • Thickness – 500 nm (or) lesser • Fix in plastic (or) isolate and study as solution. • Spread on a support grid coated with plastic. • Heavy metal salt solution -stains -"shadow" around the specimen . •Negative picture
- 39. Method 2 Pre – thinning of samples to 100 – 150 µm Polishing Platinum deposition (standard replica technique) Replication for bulk samples
- 40. CONFOCAL MICROSCOPY • “Confocal imaging” – Nipkow • First stage scanning confocal microscope – Minsky (1957) • Petran, Minsky, Amos and White – Confocal Raman microscope – 1986 • Types -
- 41. CONFOCAL PRINCIPLE • “Confocal” – “having the same focus” • Confocal pinhole • Disadvantage of the pinhole • Compensation…
- 42. Fluorescence in confocal microscopy At focal point Screen with pinhole The pinhole is the CONjugate to the FOCAL point of the lens
- 43. Confocal microscope – working Photomultiplier detector Pinhole aperture Barrier filter Dichroic mirror Laser Pinhole aperture objective Specimen focal planes
- 44. A modification….. The real time tandem scanning microscope (Nipkow disc scanning confocal microscopy) (Watson and Wilson – Journal of microscopy – February 2002) • Nipkow disc – rapid scanning • Advantage – laser / fluorescence / non coherent light (xenon arc) , direct and safe visualisation (Mc cabe and Hewlett-Applied optics – January 1998) • No unwanted back reflections
- 45. Specimen preparation for confocal microscopy Fixation (30 minutes , paraformaldehyde) Fluorophore labelling Elevation of coverslip (Immunocytochemistry – Polak and Noordan) specimen specimen
- 46. PROS AND CONS • Thin optical sections (0.5 – 1.5 • Harmful nature of high µm) intensity lasers • Image formation restricted to a •Laser lines – excitation well defined plane wavelength • Improved contrast and definition • Zoom factor – low • Multi dimensional view scanning rate • 1.2 nm resolution • High cost
- 47. ATOMIC FORCE MICROSCOPE (Scanning force microscope) • Binning, Quate and Gerber – 1986 • Imaging at nanoscale • Abrasion, adhesion, etching, corrosion, friction and polishing
- 48. Working principle • Probe in the end of a cantilever • Flexion of cantilever • Measurement of changes in bending of cantilever by measuring difference A-B ( by Hooke’s law)
- 49. Modes of operation and other aspects • Working modes may be in air (or) vacuum • Tip – sample interaction – Contact mode – Non contact mode – Tapping mode – Lift mode • Non destructive imaging is possible (E.Meyer – Microscopy and science – 1992) • Resolutions : – Vertical : 0.1 Å – Lateral : 30 Å
- 50. Pros and Cons • Three dimensional surface • Image size – area of profile only 100 µm X 100 • No special treatment for the µm sample - 8” diameter and 0.5” • Slow scanning rate thick . (Zhong and Innis – Surface Science – 1993) • Ambient and liquid environment • Very good resolution
- 51. SCANNING TUNNELING ELECTRON MICROSCOPY ( STEM) Binning and Roehrer – 1981….(Nobel Prize -1986) Scanning technique within a transmission arrangement Substrates – extremely flat - gold / platinum coating Operated in vacuum Vertical resolution : 0.1Å, lateral resolution : 1Å Chemical analysis, topography
- 52. X - RAY MICROSCOPY • Kirkpatrick and Baez • Electromagnetic radiation in the X-ray band • Do not reflect (or) refract easily • Charged coupled device (or) a film to detect the X rays that pass through the specimen • Solid – liquid interface analysis (Kaukler et al – NASA materials division research journal – 1999)
- 53. LOADS OF OPTIONS IS ALWAYS CONFUSING !!!!
- 54. SUCCESSFUL RESEARCH.… CHOICE OF MICROSCOPES……. • WHAT DO YOU WANT TO SEE ? • WHAT IS YOUR SPECIMEN SIZE ? • WHAT RESOLUTION DO YOU NEED ? • HOW MUCH ARE YOU READY TO SPEND ??!!
- 55. FEATURES LIGHT SEM TEM CONFOCAL MICROSCOPE MICROSCOPE SPECIMEN 0.1 – 0.5 mm Not specific….. To be less than No specification THICKNESS Ideally upto 3 cm 500 nm thick specimen RESOLUTION 200 nm 5 – 10 nm 0.1 – 0.2 nm 1.2 nm MAXIMUM Upto 2000 x, fixed Can be increased Can be increased 60 x MAGNIFICATION magnification upto 60,000 x upto 70,000 x MAIN USES IN Topography Topography, Same as SEM Topography, DENTAL ARENA morphology, morphology, chemical analysis, substructure substructure analysis MAJOR PROS Easy availability, Very good The best Easiest specimen easy procedure, resolution resolution preparation, economic good resolution MAIN CONS Resolution Gold sputtering, Very thin sample Laser beam, Slow limitation cost factor, only needed, only dead scanning rate, dead cells cells Cost factor Cost factor
- 56. MAGNIFICATION AND MICROSCOPY IN CLINICAL PRACTICE A Quantum leap……!!!!
- 57. FROM “LOUPES” TO “SCOPES”….. • A fascinating moment in dentistry !!!!........ A pinnacle of technology….. • First ever use of magnification and illumination in endodontics – MAGNIFYING LOUPES (surgical telescopes) WITH FIBREOPTIC HEADLAMP • 1953 – Carl Zeiss company – binocular operating microscope • 1981 – Dentiscope – first dental operating microscope • 1993 – surgical operating microscope • Microscope Assisted Precision Dentistry
- 58. SURGICAL OPERATING MICROSCOPE …. ANATOMY • body tube • Eyepiece and binoculars • Magnification changers and power zoom changers • Objective lenses – focal lengths 100 – 400 mm
- 59. THE TRIFECTA ….. Magnify, illuminate and instrument • SURGICAL TELESCOPES : 2 – 3.5 x magnification • AVAILABLE MAGNIFICATIONS….. • HOW MUCH MAGNIFICATION DO WE REALLY NEED….usable power..?
- 60. BASIC INFORMATION RELATING MAGNIFICATION TO OTHER FACTORS……. • f objective lens o magnification factor - magnification – ↓magnification -↓field of view – field of view – ↓illumination o power eyepiece - magnification -↓field of view • f binoculars – magnification o magnification - ↓depth of field – ↓ field of view
- 61. ILLUMINATION IN OPERATING MICROSCOPE Light source xenon halogen bulb quartz halogen bulb Path of light and the “stereoscopic effect” The Beam splitter “co axial” illumination – “GALILEAN OPTICS”
- 62. MICRO INSTRUMENTS • Micro mirrors, explorers, pluggers, burnishers, scalpels • Ultrasonic instruments – CPR, BUC, CT tips
- 63. DOCUMENTATION AND ACCESSORIES….. • Production of quality images / videos / slides • Photo and cine adapters • Handles and LCD screen
- 68. APPLICATIONS…….. IN RESTORATIVE DENTISTRY IN ENDODONTICS • Microfractures • Diagnosis – untreated canals • Locating hidden canals • Caries diagnosis – • Calcified canals microdentistry • Perforation repair • Broken instrument retrieval • Final examination • Surgical endodontics • Patient education
- 69. To conclude….. • Knowledge of physics of microscopes - essential to choose microscopes for research • Clinical practice with operating microscopes ….not a fancy, but a necessity!
- 70. THANK YOU !!