MICROSCOPE UG

MICROSCOPEbasics : 

MICROSCOPEbasics Dr.T.V.Rao MD 9/29/2010 1 Dr.T.V.Rao MD

The History : 

The History Many people experimented with making microscopes Was the microscope originally made by accident? (Most people were creating telescopes) The first microscope was 6 feet long!!! The Greeks & Romans used “lenses” to magnify objects over 1000 years ago. 9/29/2010 2 Dr.T.V.Rao MD

The History : 

The History Hans and Zacharias Janssen of Holland in the 1590’s created the “first” compound microscope Anthony van Leeuwenhoek and Robert Hooke made improvements by working on the lenses Anthony van Leeuwenhoek 1632-1723 Robert Hooke 1635-1703 Hooke Microscope 9/29/2010 3 Dr.T.V.Rao MD

Antioni van Leeuwenhoek : 

Antioni van Leeuwenhoek Leeuwenhoek is called "the inventor of the microscope" Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 Could reach magnifications of over 200x with simple ground lenses 9/29/2010 4 Dr.T.V.Rao MD

How a Microscope Works : 

How a Microscope Works Convex Lenses are curved glass used to make microscopes (and glasses etc.) Convex Lenses bend light and focus it in one spot. 9/29/2010 5 Dr.T.V.Rao MD

How a Microscope Works with.. : 

How a Microscope Works with.. Ocular Lens (Magnifies Image) Objective Lens (Gathers Light, Magnifies And Focuses Image Inside Body Tube) Body Tube (Image Focuses) Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again). 9/29/2010 6 Dr.T.V.Rao MD

Introduction : 

Introduction A microscope is an optical instrument that uses a lens or a combination of lenses to magnify and resolve the fine details of an object. The magnified image seen by looking through a lens is known as a virtual image, whereas an image viewed directly is known as a real image. The object to be magnified is placed under the lower lens, called the objective and viewed through the upper lens, called the eyepiece. 9/29/2010 7 Dr.T.V.Rao MD

Definitions : 

Definitions Absorption When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored light. Refraction Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths Diffraction Light rays bend around edges - new wavefronts are generated at sharp edges - the smaller the aperture the lower the definition Dispersion Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow 9/29/2010 8 Dr.T.V.Rao MD

Refraction : 

Refraction Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. dispersion Short wavelengths are “bent” more than long wavelengths 9/29/2010 9 Dr.T.V.Rao MD

Refraction & Dispersion : 

Refraction & Dispersion 9/29/2010 Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. dispersion Short wavelengths are “bent” more than long wavelengths 10 Dr.T.V.Rao MD

Because of Refraction we cannot shoot something in water : 

Because of Refraction we cannot shoot something in water 9/29/2010 But it is really here!! He sees the fish here…. 11 Dr.T.V.Rao MD

Lenses and the Bending of Light : 

Lenses and the Bending of Light light is refracted (bent) when passing from one medium to another refractive index a measure of how greatly a substance slows the velocity of light direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface 12 9/29/2010 Dr.T.V.Rao MD

Lenses : 

Lenses 9/29/2010 13 focus light rays at a specific place called the focal point distance between center of lens and focal point is the focal length strength of lens related to focal length short focal length more magnification Dr.T.V.Rao MD

Principles in magnification : 

Principles in magnification 9/29/2010 14 Dr.T.V.Rao MD

The Light Microscope : 

The Light Microscope many types bright-field microscope dark-field microscope phase-contrast microscope fluorescence microscopes compound microscopes image formed by action of 2 lenses 15 9/29/2010 Dr.T.V.Rao MD

compound microscopes : 

compound microscopes 9/29/2010 16 In compound microscopes image formed by action of 2 lenses Dr.T.V.Rao MD

The Compound Microscope : 

The Compound Microscope The Optical System Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece. Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus. Eyepiece or Ocular Lens: the lens closest to the eye. Monocular: a microscope having only one eyepiece Binocular: a microscope having two eyepieces. 9/29/2010 17 Dr.T.V.Rao MD

The Compound Microscope : 

The Compound Microscope The Optical System Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece. Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus. Eyepiece or Ocular Lens: the lens closest to the eye. Monocular: a microscope having only one eyepiece Binocular: a microscope having two eyepieces. 9/29/2010 18 Dr.T.V.Rao MD

compound microscope : 

compound microscope 9/29/2010 19 Dr.T.V.Rao MD

Dark field microscope : 

Dark field microscope 9/29/2010 20 Dr.T.V.Rao MD

Principles of Phase contrast Microscope : 

Principles of Phase contrast Microscope 9/29/2010 21 Dr.T.V.Rao MD

Fluorescent Microscope : 

Fluorescent Microscope Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination 9/29/2010 22 Dr.T.V.Rao MD

Electron Microscope : 

Electron Microscope 9/29/2010 23 Dr.T.V.Rao MD

The Bright-Field Microscope : 

The Bright-Field Microscope produces a dark image against a brighter background has several objective lenses parfocal microscopes remain in focus when objectives are changed total magnification product of the magnifications of the ocular lens and the objective lens 24 9/29/2010 Dr.T.V.Rao MD

The Conventional Microscope : 

The Conventional Microscope 9/29/2010 Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press 25 Dr.T.V.Rao MD

Slide 26: 

Eyepiece Body Tube Revolving Nosepiece Arm Objective Lens Stage Stage Clips Coarse Focus Fine Focus Base Diaphragm Light Microscope Parts 9/29/2010 26 Dr.T.V.Rao MD

Slide 27: 

Body Tube Nose Piece Objective Lenses Stage Clips Diaphragm Light Source Ocular Lens Arm Stage Coarse Adj. Fine Adjustment Base Skip to Magnification Section 9/29/2010 27 Dr.T.V.Rao MD

Some Principles : 

Some Principles 9/29/2010 28 Rule of thumb is is not to exceed 1,000 times the NA of the objective Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens Dr.T.V.Rao MD

Binocular Microscope : 

Binocular Microscope 9/29/2010 29 Schematic diagram of a stereoscopic microscope. This microscope is actually two separate monocular microscopes, each with its own set of lenses except for the lowest objective lens, which is common to both microscopes. Dr.T.V.Rao MD

Diagrammatic representation of Microscope : 

Diagrammatic representation of Microscope 9/29/2010 30 Dr.T.V.Rao MD

Slide 31: 

The principle of the compound microscope. The passage of light through two lenses forms the virtual image of the object seen by the eye. 9/29/2010 31 Dr.T.V.Rao MD

Magnification : 

Magnification An object can be focused generally no closer than 250 mm from the eye (depending upon how old you are!) this is considered to be the normal viewing distance for 1x magnification Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed 9/29/2010 32 Dr.T.V.Rao MD

Magnification : 

Magnification To determine your magnification…you just multiply the ocular lens by the objective lens Ocular 10x Objective 40x:10 x 40 = 400 Objective Lens have their magnification written on them. Ocular lenses usually magnifies by 10x So the object is 400 times “larger” 9/29/2010 33 Dr.T.V.Rao MD

Magnification : 

Magnification 9/29/2010 1000mm 35 mm slide 24x35 mm The projected image is 28 times larger than we would see it at 250 mm from our eyes. If we used a 10x magnifier we would have a magnification of 280x, but we would reduce the field of view by a factor of 10x. There used to be things called “slide Projectors” p 34 Dr.T.V.Rao MD

Microscope Resolution : 

Microscope Resolution 9/29/2010 35 Ability of a lens to separate or distinguish small objects that are close together Wavelength of light used is major factor in resolution shorter wavelength  greater resolution Dr.T.V.Rao MD

Infinity Optics : 

Infinity Optics 9/29/2010 Sample being imaged Primary Image Plane Objective Other optics Ocular Other optics Tube Lens Infinite Image Distance The main advantage of infinity corrected lens systems is the relative insensitivity to additional optics within the tube length. Secondly one can focus by moving the objective and not the specimen (stage) Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press 36 Dr.T.V.Rao MD

Objectives : 

Objectives 9/29/2010 Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): Thus high NUMERICAL APERTURE is critical for high magnification In a medium of refractive index n the wavelength gets shorter:n This defines a “resel” or “resolution element” 37 Dr.T.V.Rao MD

Numerical Aperture : 

Numerical Aperture Resolving power is directly related to numerical aperture. The higher the NA the greater the resolution Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin u (n=the lowest refractive index between the object and first objective element) (hopefully 1) u is 1/2 the angular aperture of the objective 9/29/2010 38 Dr.T.V.Rao MD

Numerical aperture : 

Numerical aperture 9/29/2010 A m NA=n(sin m) Light cone (n=refractive index) 39 Dr.T.V.Rao MD

Microscope Objectives : 

Microscope Objectives 9/29/2010 Specimen Coverslip Oil Microscope Objective Stage 60x 1.4 NA PlanApo Standard Coverglass Thickness #00 = 0.060 - 0.08 #0 = 0.080 - 0.120 #1 = 0.130 - 0.170 #1.5 = 0.160 - 0.190 #2 = 0.170 - 0.250 #3 = 0.280 - 0.320 #4 = 0.380 - 0.420 #5 = 0.500 - 0.60 mm 40 Dr.T.V.Rao MD

Slide 41: 

9/29/2010 Refractive Index Objective n=1.52 n = 1.52 n = 1.52 Specimen Coverslip Oil n=1.33 n = 1.52 n = 1.0 n = 1.5 Water n=1.52 Air 41 Dr.T.V.Rao MD

Oil Immersion increases magnification : 

Oil Immersion increases magnification 42 9/29/2010 Dr.T.V.Rao MD

Caring for a Microscope : 

Caring for a Microscope 9/29/2010 43 Clean only with a soft cloth/tissue Make sure it’s on a flat surface Don’t bang it Carry it with 2 HANDS…one on the arm and the other on the base Dr.T.V.Rao MD

Carry a Microscope Correctly : 

Carry a Microscope Correctly 9/29/2010 44 Dr.T.V.Rao MD

Using a Microscope : 

Using a Microscope Start on the lowest magnification Don’t use the coarse adjustment knob on high magnification…you’ll break the slide!!! Place slide on stage and lock clips Adjust light source (if it’s a mirror…don’t stand in front of it!) Use fine adjustment to focus 9/29/2010 45 Dr.T.V.Rao MD

Teaching microscopy is a art : 

Teaching microscopy is a art 9/29/2010 46 Dr.T.V.Rao MD

Slide 47: 

Created by Dr.T.V.Rao MD for “ e “ Learning for Basic Medical Graduates in Developing countries email doctortvrao@gmail.com 9/29/2010 47 Dr.T.V.Rao MD