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
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
second set of lens
40 or 43X
97 or 100X Be able to identify the structures and their functions Ocular
Low power objective
High power objective
Oil immersion objective Be able to identify the structures and their functions contd… : Arm
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
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
Numerical aperture image larger
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
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
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
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
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
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
-excited by blue
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  C. Julian Chen, Introduction to Scanning Tunnelling Microscopy, Oxford (1993)
 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