Microscopy

Slide 1: 

Mrs. RANI ASHOK Assistant Professor in Zoology Lady Doak College, Madurai - 2 MICROSCOPY

MICROSCOPY : 

MICROSCOPY SCIENCE WHICH DEALS WITH THE STUDY OF MICRO/ SMALL THINGS, NOT VISIBLE TO THE NAKED EYE.

Microscopy : 

Microscopy THREE GOALS: produce a magnified image of the specimen, separate the details in the image, render the details visible to the human eye or camera. Multiple-lens designs with objectives and condensers (compound) Simple single lens devices that are often hand-held, such as a magnifying glass.

Microscopy: History : 

Microscopy: History Simple Compound

Microscopy: History Contd… : 

Microscopy: History Contd…

Microscopy: History Contd… : 

Microscopy: History Contd…

Microscopy: Importance : 

Microscopy: Importance BIOMEDICAL SCIENCES: overall morphological features of specimens; quantitative tool advances in fluorochrome stains and monoclonal antibody techniques: explosive growth in the use of fluorescence microscopy in both biomedical analysis and cell biology. optical microscope most important biomedical optic EXPLOSIVE GROWTH IN PHYSICAL AND MATERIALS SCIENCES; SEMICONDUCTOR INDUSTRY observe surface features of high-tech materials and integrated circuits FORENSIC SCIENTISTS: hairs, fibers, clothing, blood stains, bullets, and other items associated with crimes

Image formation on Retina : 

Image formation on Retina do~25cm

Simple Magnification : 

Simple Magnification So >>>2f So > 2f So = 2f f< So < 2f

Combination of Lens and Eye : 

Combination of Lens and Eye Simple microscope: bi-convex lens, image perceived by eye as if it were at a distance of 10 inches or 25 centimeters (near point) Image appears on same side of lens as object, cannot be projected onto a screen: virtual image (upright, not inverted). Light reflected from the rose enters the lens in straight lines, refracted and focused by the lens to produce a virtual image on the retina. Image of the rose magnified: perceive actual size of object to be at infinity; eyes trace light rays back in straight lines to virtual image

Concepts Important for Viewing : 

Concepts Important for Viewing Resolution - ability to see objects clearly Depth of Field - depth that focus is clear Contrast Formation - (e.g. absorption contrast) Illumination Source - diascopic vs. episcopic from below (compound) vs. from above (dissecting)

How The Concepts Interact? : 

How The Concepts Interact? As Resolution and Brightness improve, Depth of Field and Contrast Formation are diminished. Vice versa is also true. These are controlled by the iris diaphragm.

Empty Magnification : 

Empty Magnification The effective magnification on an objective lens is 1000 x its numerical aperture. If a 40 x objective has an numerical aperture of 0.65, it has an effective magnification of 650 x. Magnification beyond 650 x is called “empty magnification.”

Resolution : 

Resolution The distance between two objects that is required for the two objects to be distinguished.

Resolution : 

Resolution Actual What We Might See Even if we magnify an image of two objects, we can not distinguish them unless we have adequate resolution.

Contrast : 

Contrast Bright Field Phase Contrast Dark-Field

Compound Microscope : 

Compound Microscope Lens closest to the object:objective. Light from condenser, forms light cone concentrated onto the object (specimen). Light passes through the specimen and into the objective projects a real, inverted, and magnified image of the specimen to a fixed plane within the microscope: intermediate image plane

Compound Microscope contd… : 

Compound Microscope contd… The objective: gathers light from each of the various parts or points of the specimen. focused close enough to the specimen so that it will project a magnified, real image up into the body tube. Distance between the back focal plane of the objective and the intermediate image is termed the optical tube length. mechanical tube length: distance between the nosepiece (where the objective is mounted) to the top edge of the observation tubes where the eyepieces (oculars) are inserted.

Compound Microscope contd… : 

Compound Microscope contd… Eyepiece or ocular: fits into the body tube at the upper end Further magnifies the real image projected by the objective. Eye of observer sees magnified image as if it were at a distance of 10 inches (25 centimeters) from the eye virtual image appears as if it were near the base of the microscope. Photomicrography: enlarged real image projected by the objective. projected on the photographic film in a camera or upon a screen held above the eyepiece.

Dissecting Scope : 

Dissecting Scope

Choose the right microscope for the job : 

Choose the right microscope for the job Dissecting Scope larger working distance less magnification Compound Light Microscope smaller working distance more magnification

Slide 22: 

Carry a dissecting scope with two hands to your lab desk turn the light on practice focusing on your fingernails or jewelry Carry a compound light microscope with two hands to your lab desk be sure the base is at least a thumbs length in from the edge of the desk. Find the following structures on the scope

Be able to identify the structures and their functions : 

Eyepiece - 10X light tight tube change objectives second set of lens 4X 10X 40 or 43X 97 or 100X Be able to identify the structures and their functions Ocular Body tube Revolving nosepiece Objectives Scanning objective Low power objective High power objective Oil immersion objective

Be able to identify the structures and their functions contd… : 

Arm Base Stage Stage clips / mechanical stage Aperture connects body to base firm foundation for scope Platform for slides holds slide / moves slide hole in stage for light Be able to identify the structures and their functions contd…

Be able to identify the structures and their functions contd… : 

Be able to identify the structures and their functions contd… Condenser Iris diaphragm Coarse focus Fine focus Light focuses light on slide adjusts amount of light focus on scanning and low focus on high and oil illuminate the slide

Know and be able to use the vocabulary of the microscope : 

Know and be able to use the vocabulary of the microscope Magnification Resolution Numerical aperture image larger image clearer ability of lens to gather light

Know and be able to use the vocabulary of the microscope contd… : 

Know and be able to use the vocabulary of the microscope contd… Working distance Depth of field Parfocality between lens & stage bottom to top of slide all objectives in reasonable focus at the same time

Total magnificationocular power multiplied by objective power : 

Total magnificationocular power multiplied by objective power Ocular = 10x scanning = 4x low = 10x high = 40x or 43x oil = 97x or 100x Total magnification 10 x 4 = 40 10 x 10 = 100 10 x 40 = 400 or 10 x 43 = 430 10 x 97 = 970 or 10 x 100 = 1000

THE MICROSCOPE : 

THE MICROSCOPE EYEPIECE

THE MICROSCOPE : 

THE MICROSCOPE ARM

THE MICROSCOPE : 

THE MICROSCOPE BASE

THE MICROSCOPE : 

THE MICROSCOPE BINOCULAR TUBE

THE MICROSCOPE : 

THE MICROSCOPE REVOLVING NOSEPIECE

THE MICROSCOPE : 

THE MICROSCOPE OBJECTIVE LENS

THE MICROSCOPE : 

THE MICROSCOPE MECHANICAL STAGE

THE MICROSCOPE : 

THE MICROSCOPE STAGE CLIPS

THE MICROSCOPE : 

THE MICROSCOPE IRIS DIAPHRAGM

THE MICROSCOPE : 

THE MICROSCOPE COARSE ADJUSTMENT KNOB

THE MICROSCOPE : 

THE MICROSCOPE FINE ADJUSTMENT KNOB

THE MICROSCOPE : 

THE MICROSCOPE LAMP

THE MICROSCOPE : 

THE MICROSCOPE BULB

THE MICROSCOPE : 

THE MICROSCOPE ON/OFF SWITCH

CARE OFTHE MICROSCOPE : 

CARE OFTHE MICROSCOPE Always carry the microscope by the arm and the base.

CARE OFTHE MICROSCOPE : 

CARE OFTHE MICROSCOPE Wrap the cord around the microscope.

CARE OFTHE MICROSCOPE : 

CARE OFTHE MICROSCOPE Always use a dust cover!

Be able to focus properly on a slide on high power in two minutes. : 

Be able to focus properly on a slide on high power in two minutes. We call this the “two minute drill”

Slide 47: 

Clean the microscope with lens tissue only! only!

Turn on the light and adjust it to the lowest amount of light : 

Turn on the light and adjust it to the lowest amount of light

Start with the lowest powerobjective in place. : 

Start with the lowest powerobjective in place.

Place the precleaned slide on the stagewith the specimenin the light from the condenser. : 

Place the precleaned slide on the stagewith the specimenin the light from the condenser.

Bring the stage and the lowest power objective as close together as you can. You should hit the automatic stop. : 

Bring the stage and the lowest power objective as close together as you can. You should hit the automatic stop.

Looking in the ocular, slowly focus away with the coarse focus until you see something, even dirt on the slide. If you need more light, open the iris diaphragm a bit. : 

Looking in the ocular, slowly focus away with the coarse focus until you see something, even dirt on the slide. If you need more light, open the iris diaphragm a bit.

Move the slide slowly until you have centered the object you are looking at. Focus with the coarse focus until it is in the best possible focus. : 

Move the slide slowly until you have centered the object you are looking at. Focus with the coarse focus until it is in the best possible focus.

Turn the revolving nosepiece to the low power objective. Be sure to click it in place. Center the slide. Focus with the coarse focus until it is in the best possible focus. : 

Turn the revolving nosepiece to the low power objective. Be sure to click it in place. Center the slide. Focus with the coarse focus until it is in the best possible focus.

Turn the revolving nosepiece to the high power objective. Be sure to click it in place. Center the slide. Focus with the fine focus until it is in the best possible focus. : 

Turn the revolving nosepiece to the high power objective. Be sure to click it in place. Center the slide. Focus with the fine focus until it is in the best possible focus.

Oil immersion lens : 

Oil immersion lens When magnification is this high, any loss of light will degrade the resolution, making the image fuzzier and harder to see. A small drop of oil, between the slide and the objective lens, will prevent light from scattering and preserve the image.

To focus on oil, first make sure you have focused as well as you can on high power : 

To focus on oil, first make sure you have focused as well as you can on high power Move the revolving nosepiece until you are halfway between high and oil. Place a small drop of oil on the slide where the lens will be. Remember, the more oil you put on, the more there is to clean off later. Move the oil objective in place and focus with the fine focus only!

Slide 58: 

The oil will seep into the microscope and loosen the cement, as well as dry to a scummy mess on the lens. Be sure to clean the lens and the slide with lens tissue when you are done

Types of microscopes : 

Types of microscopes Light microscopes Bright field microscope Dark field microscope Phase contrast microscope Fluorescent microscope Electron microscopes Transmission electron microscope Scanning electron microscope Scanning tunneling microscope

Slide 60: 

I. Dark field contrast microscopy takes advantage of objects that “scatter” light - this requires a special condenser that can “angle” the incident light II. Phase contrast microscopy takes advantage of objects that alter the phase of incident light - This requires “phase rings” in the condenser and in the objective lens III. Fluorescence microscopy take advantage of inherently fluorescent Material of biological objected that can be fluorescenlty labeled.

Slide 61: 

Single Cell Organism ( Tetrahymena) observed with: “Advanced” Light Microscopic Methods

Slide 62: 

Recall that a specimen (e.g a live cell) with few structures that strongly absorb light provides little contrast with bright field microscopy e.g., the single celled protist Tetrahymena

Slide 63: 

Dark Field Microscopy: Some objects can alter the light path by diffraction & or light scattering

Slide 64: 

If the light pathway is angled without a specimen the field is dark... objective lens …but any object that diffracts light will be detected! Dark Field seen condenser lens objective lens

Slide 65: 

back focal plane specimen plane image plane Phase contrast microscopy exploits differences in the phase of light Light as “rays Light as “waves”

Slide 66: 

image plane objective lens condenser lens specimen plane light source occular / projector lens back focal plane No object in the specimen plane Out of phase A special condenser produces light in phase IN phase

Slide 67: 

Isolated condenser with “phase rings”

Slide 68: 

…another set of waves emanate from the object due its interaction with the incident light object When an object is introduced in the specimen plane...

Slide 69: 

The objective lenses (and condenser) contain “phase rings” to exploit differences in the phase of light generated by the object 10x “phase” objective lens 100x “phase” objective lens Lens View: Inside

Slide 70: 

condenser lens specimen plane light source objective lens Phase “ring” Phase “ring” Incident light is in phase

Slide 71: 

objective lens Light emanating from the object are out of phase with the incident light Some objects in cells can alter the phase of light

Slide 72: 

Difference in phase are converted into differences in intensity Phase “ring” Destructive interference Constructive interference objective lens

Slide 73: 

Epi-Fluorescence Microscopy Biological molecules can be labeled with a flourochrome -- a molecule that absorbs light at one wavelength and emits at another, longer wavelength Fluorochrome E.g.

Slide 75: 

Optical System of a Fluorescence Microscope Fluorescein -excited by blue light (450-490) -emits green light (520-560) FLUORESCENT MICROSCOPE

Transmission Electron Microscope : 

Transmission Electron Microscope

Generation of electron beam for TEM : 

Generation of electron beam for TEM

Electron Backscattered DiffractionOrientation Contrast : 

Electron Backscattered DiffractionOrientation Contrast Incident Beam Domain 4 Domain 3 Domain 2 Domain 1 Channeling-in Channeling-out Standard SEM setup: Forescattered BSE Detector (after Prior et al., 1999) Setup for orientation contrast:

Slide 79: 

Normal SEM Setup: EBSD/OC Setup: (after Prior et al., 1999)

Electron Backscattered DiffractionOrientation Contrast : 

Electron Backscattered DiffractionOrientation Contrast (Newman et al., 1999)

Operation of an STM : 

Operation of an STM [1] C. Julian Chen, Introduction to Scanning Tunnelling Microscopy, Oxford (1993) [2] G.A.D. Briggs and A. J. Fisher, Surf. Sci. Rep. 33, 1 (1999)

Single-molecule vibrations : 

Single-molecule vibrations Study vibrations of individual molecules and individual bonds by looking at phonon emission by tunnelling electrons New possibilities for inducing reactions by selectively exciting individual bonds…. Wilson Ho et al., UC Irvine

Applications of different microscopes : 

Applications of different microscopes Bright field – Stained slides Dark field – Live unstained cells Phase contrast – Detection of bacterial components eg. endospores, inclusion bodies like polybetahydroxybutyrate, polymetaphosphate etc. Fluorescent – Microbiological material Transmission electron – 2D and 3D images Scanning electron – 3D images Scanning tunneling electron – At atom level

Guestimate the size of a cell : 

Guestimate the size of a cell The field of view is the circle of white light you see when you look through the ocular This can be measured with a microscopic ruler or micrometer. It will look something like this under the scanning objective. 2 mm or 2000 um

Slide 85: 

Thank You