MICROSCOPY
Microorganisms: First to evolve -- last to be seen!
Resolving power of the eye
Relevant metric unitsmm = 10-3 meter
mm
nm
Resolution vs
Magnification
Earliest Microscopes• 1590 - Hans & Zacharias Janssen of
Middleburg, Holland manufactured the first compound microscopes
• 1660 - Marcello Malpighi (1628-1694), was one of the first great microscopists, considered the father embryology and early histology - observed capillaries in 1660 . Italian professor of medicine. Anatomist. First to observe bordered pits in wood sections. Gave first account of the development of the seed.
Early Microscopes (Hooke)
© J.Paul Robinson
The Royal Society of London founded in 1616 during the reign of King James I
•1665 - Robert Hooke (1635-1703)- book Micrographia, published in 1665, devised the compound microscope most famous microscopical observation was his study of
thin slices of cork. Named the term “Cell” - He wrote:
“. . . I could exceedingly plainly perceive it to be all perforated and porous. . . these pores, or cells, . . . were indeed the first
microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with
any Writer or Person, that had made any mention of them before this.” Robert Hooke
Oxford University
Overview of discovery
Earliest Microscopes•1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor
city official.
•Leeuwenhoek is incorrectly 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 - however compound microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke
claimed they were too difficult to use - his eyesight was poor.•Discovered bacteria, free-living and parasitic microscopic
protists, sperm cells, blood cells, microscopic nematodes •In 1673, Leeuwenhoek began writing letters to the Royal
Society of London - published in Philosophical Transactionsof the Royal Society
•In 1680 he was elected a full member of the Royal Society,joining Robert Hooke, Henry Oldenburg, Robert Boyle,
Christopher Wren
lens.exe
1670-1690
• Back: Italian compound microscopes - 1670
• Italian Compound microscopes• Back: 1670 (probably Campani)• This microscope was formerly at the
University of Bologna - it contains a field lens which was the first optical advance about 1660. Only opaque objects can be viewed.
• Front: Guiseppe Campani, Rome -1690 - Campani was the leading Italian telescope and microscope maker in the late `17th century - he probably invented the screw focusing mechanism shown on this scope - the slide holder in the base allows transparent and opaque objects to be viewed
Screwbarrel Microscope - 1720
• Made by Charles Culpeper
The issues between simple and compound microscope
• Simple microscopes could attain around 2 micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration
• In the 1730s a barrister named Chester More Hallobserved that flint glass (newly made glass) dispersed colors much more than “crown glass” (older glass). He designed a system that used a concave lens next to a convex lens which could realign all the colors. This was the first achromatic lens.
Some 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 diffraction• 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
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
Refraction
But it is really here!!
He sees the fish here….
Absorption
Control
No blue/green lightred filter
B & G absorbed
Light absorption
white light blue light red light green light
B & G absorbedR & G absorbed B & R absorbed
Absorption ChartColor in white light Color of light absorbed
red
bluegreen
magenta
cyan
yellow
blue
blue
blue
blue
greengreen
green
green
redred
redredblack
gray green bluepink
The light spectrumWavelength ---- Frequency
Blue light488 nm
short wavelengthhigh frequencyhigh energy (2 times the red)
Red light650 nm
long wavelengthlow frequency
low energy
Photon as a wave
packet of energy
θ1
θ2
θ3 θ4
Principles of light microscopy-- a single lens
Optical propertiesrefraction
magnification
resolution
distortion
Van Leeuwenhoek’s microscope
Limitations of a simple microscope
magnification
illumination
difficult to use
Robert Hooke’s microscope
Principles of a compound microscope
Ocular lens
Objective lens
Condensor lens
Calculating magnification
Hooke never reported seeing bacteria
Why?
Distortion is compounded
Compound ? (a neat idea)Simple Compound
Lenses and the bending of light
• Lens is a collection of prisms• Convex lens focuses light to the focal point.• Refractive index is a measure of how much
a subs slows the velocity of light.• Direction and magnitude is determined by
the RI of the two media forming the interface.
Nikon Labophot(Question 8)
Field lens(collects light)
Brightness control dial
Nikon Labophot(Question 8)
Condenser(focuses light on specimen)
Nikon Labophot(Question 8)
StageSlide holder
Stage X/Y-axis travel knobs
Nikon Labophot(Question 8)
Focus knobs
Nikon Labophot(Question 8)
Eyepiece(2nd level of
magnification)
Objectives(1st level of
magnification)
Nosepiece
Microscope Features
Illuminator: light source + collectorlens
Substage condenser: focuses light on specimen
Stage: specimen
Objective: 1st level of magnification(10X, 40X, 100X)
Nosepiece: to move objective
Eyepiece: 2nd level of magnification(10X)
ALWAYS MOVE MICROSCOPEBY GRASPING THE STAND AND
SUPPORTING THE BASE.
Compound Light Microscope
Magnification(Question 9)
Real image projectedby objective
Virtual image projectedby eyepiece
Total magnification= objective x eyepiece
Ex: 40X x 10X = 400X
Theoretical limit = 1000X> 1000X:
Image enlarged withno additional resolution
Magnification• Magnification: Degree of enlargement. i.e.
number of times the L, B, D are multiplied. • Limit of useful magnification set by
resolving power or limit of resolution.• Overall magnifying power obtained by
multiplying MP of objective with eye piece• Inclination of microscope head increases
magnification power of objective
• Optical tube length (160mm)/FL of objective
• Objective with FL 2 mm has a magnifying power of 80
• Objective lenses: – Low power FL 16mm– High power FL 4mm– oil immersion FL 2mm
RESOLUTION• The ability of the lens to differentiate between two small
objects.• Resolving power of a microscope is determined by
d=0.5/nsin• : Wavelength of light• Better resolution, shorter the wavelength. Therefore blue
range better -450-500nm• Limit if resolution of unaided eye=200 microns• LR of bright field microscope= 0.2 micron
Numerical Aperture• Ratio of diameter of lens to its Focal length FL (nsin )• is half the angle of cone of light entering the objective or
half the angle of aperture which is formed by 2 most divergent rays of light from the center of the object.
• Narrow cone of light has poor resolution.• Angle depends upon RI of the medium (n).• RI of air is 1.• Max NA =1 when working in air• resolution increased by adding oil
BRIGHTFIELD MICROSCOPE
Speed of light in vacuumSpeed of light in medium
What were the future advances in light microscopy?
1) Oil immersion lens
2) Fused lenses
3) Advanced optics
How do and oil immersion fused lenses improve resolution?
Refractive index =
Borosilicate glass = 1.52Air = 1.00Water = 1.33Immersion oil = 1.52
Fused lenses combine different types of glass-- compensate for ‘diffraction’
Objectives
ObjectiveType
SphericalAberration
ChromaticAberration
FieldCurvature
Achromat 1 Color 2 Colors No
Plan Achromat 1 Color 2 Colors Yes
Fluorite 2-3 Colors 2-3 Colors No
Plan Fluorite 3-4 Colors 2-4 Colors Yes
Plan Apochromat 3-4 Colors 4-5 Colors Yes
Numerical Aperture (N.A)
Air objective N.A ~ 0.95Oil objective N.A ~ 1.4
Water objective N.A ~ 1.25
ABILITY OF AN OBJECTIVE TO RENDER THE OUTLINE OF AN IMAGE CLEAR AND DISTINCT.
• Spherical aberration: Rays passing the edge of a lens not brought to same focus as those passing the center.
• Chromatic aberration: White light traversing a lens is separated into its component colours of different wavelegth, which are refracted to diff extent.
• Aberrations corrected by combining lenses of dispersive qualities
ObjectivesFunctions
1. Gather light from specimen2. Resolving power (reconstitute
the light from specimen into a clear image
3. Project a magnified real image into the body tube
Problems1. Chromatic aberrations2. Spherical aberration3. Curvature of field
The famous patent of 1758• George Bass was the
lens-maker that actually made the lenses, but he did not divulge the secret until over 20 years later to John Dollond who copied the idea in 1757 and patented the achromatic lens in 1758.
© J.Paul Robinson
Secondary Microscopes• George Adams Sr. made many microscopes from about 1740-
1772 but he was predominantly just a good manufacturer not inventor (in fact it is thought he was more than a copier!)
“New Improved Compound Microscope, George Adams, 1790Adams described this instrument in his “Essays on the Microscope” in
1787. The mechanism allowed freedom of movement. The specimen could be viewed in direct light or in light reflected from a large mirror.
© J.Paul Robinson
George Adams Toymaker to Kings
• This microscope made by George Adams, Mathematical Instrument maker to King George III around 1763, It was probably intended for the Prince of Wales, the future King George IV. The instrument is based on the design of the “Universal Double Microscope" (London Museum of Science)
Giovanni Battista Amici• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He had previously (1813) designed “reflecting microscopes” using curved mirrors rather than lenses. He recognized the importance of coverslip thickness and developed the concept of “water immersion”
© J.Paul Robinson© J.Paul Robinson
Joseph Lister• In 1830, by Joseph Jackson Lister (father of Lord Joseph Lister) solved the
problem of Spherical Aberration - caused by light passing through different parts of the same lens. He solved it mathematically and published this in the Philosophical Transactions in 1830
© J.Paul Robinson
Joseph Lister
Pasteur - 1860
Louis Pasteur – his microscope was made in Paris by Nachet in about 1860 and was made of brass
Abbe & Zeiss • Ernst Abbe together with Carl Zeiss published a paper in 1877
defining the physical laws that determined resolving distance of an objective. Known as Abbe’s Law“minimum resolving distance (d) is related to the wavelength of light (lambda) divided by the Numeric Aperture, which is proportional to the angle of the light cone (theta)
formed by a point on the object, to the objective”.“The impetus for the emergence into the industrial age was given by Ernst
Abbe (appointed Associate Professor in 1870), who, while still in his early 30s, developed his theory of microscope image formation, which took into
consideration the familiar phenomenon of diffraction, and thus made the leap in microscope construction from trial and error to methodical design. He was given this commission by a university mechanic, Carl Zeiss, who had been
steadily perfecting the construction of optical equipment in his private workshops. Otto Schott, who received his doctorate at Jena in 1875, was the
third to enter into this alliance by founding, at Abbe’s urging, a "Laboratory for Glass Technology" in 1884, to produce the highly pure special lenses for
Zeiss’s microscopes and optical equipment. Humboldt’s pupil Matthias Jakob Schleiden, Professor of Botany and famous for his cell theory, encouraged --
and later benefited from -- this process, which was to prove exemplary in German economic history.”
http://www.uni-jena.de/History-lang-en.htmlAbbe
Abbe & Zeiss
• Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of glass. Thus they were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum resolution of visible light microscopes). Leitz was also making microscope at this time.
Zeiss student microscope 1880
Schott• Dr Otto Schott formulated glass lenses that color-corrected objectives
and produced the first “apochromatic” objectives in 1886.
Henri Hureau de Sénarmont (1808-1862)• Sénarmont was a professor of mineralogy and director of studies at the
École des Mines in Paris, especially distinguished for his research on polarization and his studies on the artificial formation of minerals. He also contributed to the Geological Survey of France by preparing geological maps and essays.
• Perhaps the most significant contribution made by de Sénarmont to optics was the polarized light retardation compensator bearing his name, which is still widely utilized today
Historical Figures
William Hyde Wollaston
William Hyde Wollaston (1766-1828) - Although formally trained as a physician, Wollaston studied and made advances in
many scientific fields, including chemistry, physics, botany, crystallography, optics, astronomy and mineralogy. He is
particularly noted for originating several inventions in optics, including the Wollaston prism that is fundamentally important to interferometry and differential interference (DIC) contrast
microscopy.
Georges Nomarski
• Georges Nomarski (1919-1997) - A Polish born physicist and optics theoretician, Georges Nomarski adopted France as his home after World War II. Nomarski is credited with numerous inventions and patents, including a major contribution to the well-known differential interference contrast (DIC) microscopy technique. Also referred to as Nomarski interference contrast (NIC), the method is widely used to study live biological specimens and unstained tissues.
Additional Information and Image at right from:ttp://micro.magnet.fsu.edu/optics/timeline/people/nomarski.html
Robert Day Allen
• Robert Day Allen (1927-1986) - Robert Day Allen was a renowned microscopist, a prominent researcher of cell motility processes, and a co-developer of video-enhanced contrast microscopy ((VEC)), which is a modification of the traditional form of differential interference contrast (DIC) microscopy. Along with Georges Nomarski and G. B. David, Allen assisted the Carl Zeiss Optical Company in developing a Nomarski differential interference microscope for transmitted light applications. In a hallmark paper published in Zeitschrift für wissenschaftliche Mikroskopie und mikroskopische Technik, Allen and his colleagues defined the basic principles of the DIC technique and the interpretation of images.
• Rebhun LI. Robert Day Allen (1927-1986): an appreciation. Cell Motil Cytoskeleton. 1986;6(3):249-55
More information at: (Image reproduced from below URL)ttp://micro.magnet.fsu.edu/optics/timeline/people/dayallen.html
Modern Microscopes
• Early 20th Century Professor Köhler developed the method of illumination still called “Köhler Illumination”
• Köhler recognized that using shorter wavelength light (UV) could improve resolution
Köhler
• Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light
• Two conjugate image planes are formed– one contains an image of the specimen and the
other the filament from the light
Köhler Illumination
Specimen Field stopField iris
Conjugate planes for illuminating rays
Specimen Field stopField iris
Conjugate planes for image-forming rays
condenser eyepiece
retina
Magnification• An object can be focussed 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
Magnification1000mm
35 mm slide24x35 mm
M = 1000 mm35 mm
= 28
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 d h fi ld f i b f f 10
Some Principles
• Rule of thumb 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
Basic Microscopy
• Bright field illumination does not reveal differences in brightness between structural details - i.e. no contrast
• Structural details emerge via phase differences and by staining of components
• The edge effects (diffraction, refraction, reflection) produce contrast and detail
© J.Paul Robinson
Microscope Basics• Originally conformed to the
German DIN standard• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm– object to image distance set to 195
mm
• Currently we use the ISO standard
Focal lengthof objective= 45 mm
Mechanicaltube length= 160 mm
Object toImage Distance = 195 mm
The Conventional Microscope
Focal lengthof objective= 45 mm
Object toImage Distance = 195 mm
Mechanicaltube length= 160 mm
Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press
Upright Scope
BrightfieldSource
Epi-illumination
Source
Image from Nikonpromotional materials
Inverted Microscope
BrightfieldSource
Epi-illumination
Source
Image from Nikonpromotional materials
Image from Nikonpromotional materials
Typical Inverted Microscope
Conventional Finite Optics
Sample being imaged
Intermediate Image
Telan Optics
Objective
Other optics
Ocular
45 mm
160 mm195 mm
Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press
Infinity Optics
Sample being imaged
Primary Image Plane
Objective
Other optics
Ocular
Other optics
Tube Lens
InfiniteImage
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
Images reproduced from:
http://micro.magnet.fsu.edu/
Advanced optics only improve contrast
Theoretical limit of resolution= 0.2 uM
Dark fieldmanipulates the path of light
Phase contrastand
Nomarskimanipulate phase of light
Dark field
Bright field
Phase contrast Nomarski
Phase contrast
• Unstained living organisms• Good contrast• High resolution• Objects dark grey against a light background.• Direct and diffracted rays from the objects which differ in their wave
phase, recombine so they interfere with each other. Hence reduce light intensity in the area of the image.
• Unstained bacteria have alternate areas of different refractive indices. These act as diffraction gratings. Differences are maximized by causing the direct and diffracted rays to pass through different thicknesses of glass in a phase plate.
• Phase plate retards diffracted rays one quarter of a wavelength with respect to direct rays.
• Condenser has an annular stop. Opaque disc with transparent ring.
Phase contrast
Phase contrast• Hollow cone of light produced.• When this cone passes thru a cell, some light rays are bent
due variations in density and RI and are retarded by 1/4 of wavelength
• Direct rays go through the phase ring in the phase plate which is present in the objective. Deviated rays miss the ring.
• Difference between the two is half the wavelength. If they recombine, they cancel each other.
• Useful for detecting bacterial components.• Also used in studying eukaryotes.
Darkground Microscope
• Bacteria brightly lit against a dark background.• Can observe living unstained organisms• Specimen illuminated by rays of light so oblique that unless scattered
by objects, they do not enter the objective.• Darkground condenser- The special condenser incorporates concentric
reflecting mirrors. A central one prevents light rays from passing directly up and passes them onto peripheral mirrors. The latter reflects rays Optical system enhances the contrast of unstained bodies.
• inwards onto specimen at a very oblique angle.• High intensity lamp• Funnel stop which reduces the numerical aperture to less than 1. It
consists of a funnel shaped piece of metal or palstic and is fitted into the objective.
• Particularly useful in observing smaller spirochetes
Schematics of the ray paths in a dark-field contrastmicroscope. Rays fall into the objective lens only if theincoming light is scattered at the specimen in the focal plane
Dark-field contrast
Immunofluorescenceand
Confocal Microscopy
Part I: Immunofluorescence
Principle of Fluorescence Microcopy
Principle of Fluorescence Microcopy
Exciter filter
Principle of Fluorescence Microcopy
Exciter filter Barrier filter
In fluorescence microscopy, electrons in labeling agent molecules(here for simplicity drawn as single atoms with electron shells) ofthe fluorescence species are excited by light with shortwavelength to higher energetic molecular orbitals. The excitedelectrons loose their energy by emission of fluorescence light withlonger wavelength compared to the excitation radiation. Theeminent light is used for image production in the microscope.
Fluorescence microscopy
Fluorescence microscope
• Light source is UV mercury vapor lamp• UV light is filtered to select excitation light
to pass through• Excitation light is reflected by a dichroic
mirror to strike on the specimen • Emission light passes through the dichroic
mirror
Fluorescence microscope
• Barrier filter blocks the excitation light amid the light path to visualization
• Fluorescent labels are visulized against a dark background
Fluorescence microscope
• The combination of exciter filter, dichroic mirror and barrier filter should be selected according to the fluorochrome label
• The 3 components are usually built into a single module called the filter block
Epi-fluorescence microscope
Transmitted light flurorescence microscope
The basic principle of immunofluorescence
• To use a fluorescent compound (usually fluorescein) to detect the binding of antigenand antibody.
• The Ab is labeled with the fluorescent compound and its presence is detected using a fluorescence microscope.
• Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurred.
Exciter filter
The basic principle of immunofluorescence
• To use a fluorescent compound (usually fluorescein) to detect the binding of antigenand antibody
• The Ab is labelled with the fluorescent compound
• Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurs
Using the fluorescence microscope
• Select the correct filter block for the fluorescent compound
• Fluorescence fades quickly under UV light; try to limit the time of exposure to UV as much as possible
• Use high speed films for photography
Direct Immunofluorescence
• The aim is to identify the presence and location of an antigen by the use of a fluorescent labelled specific antibody
One step Direct Immunofluorescence
Two step Direct Immunofluorescence
Medical applications of direct IF
• Renal diseases for evidence of immune deposition
• Skin diseases for evidence of immune deposition
• Detection of specific antigens, especially those of infective organisms
Application in renal diseases
IgG
• A section of kidney is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
• The presence of fluorescence in the glomeruli indicates that IgG was deposited prior to the biopsy
• IgG is deposited in granular clumps along the capillary walls, enabling a diagnosis of membranous glomerulonephritis in this case
• A section of kidney is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
• The presence of fluorescence in the glomeruli indicates that IgG was deposited prior to the biopsy
• IgG is deposited in granular clumps along the capillary walls, enabling a diagnosis of membranous glomerulonephritis in this case
Direct Fluorescent Antibody Test for the Presence of Immunoglobulin Deposits in Skin
IgG
• A section of skin is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
• The presence of fluorescence in the upper layers of the epithelium indicates that IgG was deposited in this skin (prior to the biopsy)
• The presence of immunoglobulins deposited around keratinocytes is consistent with a diagnosis of pemphigus
• A section of skin is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away
• The presence of fluorescence in the upper layers of the epithelium indicates that IgG was deposited in this skin (prior to the biopsy)
• The presence of immunoglobulins deposited around keratinocytes is consistent with a diagnosis of pemphigus
Double labelling
Lymphoid tissue:the two Ig light chains are separately labelled.
Indirect Immunofluorescence
Indirect Immunofluorescence
• The aim is to identify the presence of antigen specific antibodies in serum. The method is also be used to compare concentration of the antibodies in sera.
Indirect Immunofluorescence
• A known antigen is placed on a slide; the patient's serum is added, then rinsed away.
• A fluorescein-labeled antiglobulin is added, then rinsed away.
• The presence of fluorescence over the antigen indicates the presence of antibodiesto this antigen in the patient.
Diagnosis of Bacterial Diseases
• Clostridial diseases (direct) • Brucella canis (indirect) • Afipia catei, cat scratch disease (indirect) • Borrelia burgdorferi (indirect) • Coxiella burnetii, Q Fever (indirect) • Rickettsia rickettsiae, Rocky Mountain
Spotted Fever (indirect)
Diagnosis of Viral Diseases
• rabies virus (direct)• bovine immunodeficiency-like virus (indirect)• canine coronavirus (indirect)• canine distemper (indirect)• feline infectious peritonitis (corona-) virus
(direct)• porcine respiratory and reproductive syndrome
(indirect)
Diagnosis of Protozoal Diseases
• Babesia species (indirect)• Ehrlichia species (indirect)• Toxoplasma gondii (indirect)• Trypanosoma cruzi (indirect)• Cryptosporidia/Giardia (direct)• Encephalitozoon cuniculi (indirect)• Neosporum caninum (direct, indirect)
Some examples
Indirect Immunofluorescence
Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
• Toxoplasma organisms are killed and placed on the slide; the patient’s serum is added, then washed away.
• A fluorescein-labeled antiglobulin is added, then washed away.
• The presence of the green fluorescence outlining the T. gondii organisms indicates the presence of antibodies in the patient's serum.
Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii
Immune-Mediated Disorders
• antinuclear antibody (ANA) test (for diagnosis of systemic lupus erythematosus)
• Direct fluorescent antibody test for deposition of Abs in tissues, e.g. kidney, skin
Indirect Fluorescent Antibody Test for Antinuclear Antibodies
Indirect Fluorescent Antibody Test for Antinuclear Antibodies
• Cells from a cultured cell line are placed on a slide; the patient's serum is added, then rinsed away.
• A fluorescein-labeled antiglobulin is added, then rinsed away.
• The presence of fluorescence in the nucleus of these cells indicates the presence of antibodies to nuclear antigens in the patient.
Indirect Fluorescent Antibody Test for Antinuclear Antibodies
Advantage over Immunoperoxidase
• Technically easier (fewer steps)• More sensitive results
Drawbacks
• Microscope is more costly• Frozen sections preferred• Preparations need refrigeration• Preparations cannot be kept for too long• Quick fading of fluorescence under
illumination (bleaching effect)
Part II: Confocal microscopy
Principles of confocal microscopy
• a focused laser beam serves as a high intensity point source
• light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane
Principles of confocal microscopy
• a sensitive detector (photomultipler) behind a pinhole to measure the intensity of light
• the laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor
The confocalconcept
Modes of scanning
• Mechanical scanning stage• Beam scanning (by means of mirror)• Combined stage and beam scanning• Slit may be used instead of a pinhole
– Shortens time for scanning an area– Direct vision of real color image made possible– At the expense of a lower resolution
Use of confocal microscope
• Performs optical sectioning of thick samples• Three dimensional image reconstruction• Detects very weak fluorescent signals• Selective photobleaching• Cell ablation
Image modalities
• Autofluorescence• Single, double or treble fluorescent
labeling:– immunofluorescence, in-situ hybridization
• Image formed by reflectance intensified with metallic coating e.g. AgNOR, immunogold labeling
Application in biomedical science
• Growth of small organisms, cells, embryos• Movement of intracellular structures• Change in membrane permeability• 3 dimensional reconstruction• Image analysis
BIO-RAD MRC-1000
• beam scanning LSM• Zeiss "Axioskop" upright microscope for ordinary
bright field transmitted light and epi-fluorescence• argon/krypton laser, 15 mW, wavelengths at 488
nm (e.g. FITC), 568 and 647 nm• reflectance mode• fluorescence mode:
– simultaneous recording of 2 channels, ie. For double labels
BIO-RAD MRC-1000
• upgradable to 3 simultaneous channels in fluorescence mode
• computer-controlled stage motor for vertical motion at 0.5µm per step
• performs frame scan, line scan, vertical scan• maximum image size 1024 x 1024 pixels• scanning speed at maximum of 16 frames/s
at 768 x 30 pixels, and 1 frame/s at 512 lines
BIO-RAD MRC-1000
• beam park function for photobleaching, ablation
• computer programs for morphometric analysis, 3-D visualization
IgG
IgG
Confocal
Confocal microscope gives a clearer image andcleaner background overconventional fluorescent
microscope
Microphotographyfor weak signalsis much easierwith a confocal
microscope
Microphotographyfor weak signalsis much easierwith a confocal
microscopeExcellent for picking up weak signals
Fluorescence microscope
How do electron microscopes yield higher resolution?
Resolution ~ 0.2 nM (0.0002 uM)- light microscope ~ 0.2 uM- eye ~ 0.2 mm
Electrons have very small wavelength
Wavelength of visible light ~ 550 nm… of electrons ~ 0.005 nm
What are the two types of EM?
Scanning vs Transmission
False coloration common
.
Optical principles of (A) confocal laser scanning microscopy (CLSM) and (B) multi-photon excitation microscopy (MPEM).
Method of use microscope
• Eye piece in level with observer’s eye.• Check posture• Objectives and Eye pieces should be free of dirt.• Use only Benzol or Xylol to clean lenses.• View specimen in low power, then move to next objective.• Use fine focus only when image is in focus.• Adjust eye pieces so single field is seen.• If can’t focus, check for dirt or oil on lenses.• Slide has a film of oil?• Slide upside down. • Coverslip too thick