View
219
Download
2
Category
Tags:
Preview:
Citation preview
Slide 1 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Lecture 1
The Principles of Microscopy
• BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively for material and ideas to support the class.
UPDATED January, 2000
Slide 2 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Introduction to the Course• Microscopy• Fluorescence• Basic Optics• Confocal Microscopes
Evaluation
• Basic Image Analysis• 3D image analysis• Live Cell Studies• Advanced Applications
• End of term quiz - 100% grade
Slide 3 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Introduction to Lecture 1
• Early Microscopes• Modern Microscopes• Magnification• Nature of Light• Optical Designs
Slide 4 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Microscopes
• Upright
• Inverted
• Köhler Illumination
• Fluorescence Illumination
"Microscope" was first coined by members of the first "Academia dei Lincei" a scientific society which included Galileo
Slide 5 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Earliest Microscopes• 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscopes• 1660 - Marcello Malpighi circa 1660, was one of the first great microscopists, considered the father
embryology and early histology - observed capillaries in 1660
• 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. 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.”
Slide 6 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Overview of discovery
Slide 7 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Early Microscopes (Hooke)
1665
Slide 8 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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 Transactions of 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
Slide 9 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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!)
• 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 names Chester More Hall observed 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. 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 Dolland who copied the idea in 1759 and patented the achromatic lens.
• 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”
Slide 10 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 11 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Abbe, Zeiss & Schott• 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”. • 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.
• Dr Otto Schott formulated glass lenses that color-corrected objectives and produced the first “apochromatic” objectives in 1886.
Slide 12 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 13 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 14 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Köhler Illumination
Specimen Field stopField iris
Conjugate planes for illuminating rays
Specimen Field stopField iris
Conjugate planes for image-forming rays
condenser eyepiece
retina
Slide 15 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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 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
Slide 16 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 17 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Refraction
But it is really here!!
He sees the fish here….
Slide 18 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Absorption
Control
No blue/green light red filter
Slide 19 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Light absorption
white light blue light red light green light
Slide 20 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Absorption Chart
Color in white lightColor in white light Color of light absorbedColor of light absorbed
red
blue
green
magenta
cyan
yellow
blue
blue
blue
blue
green
green
green
green
red
red
red
redblack
gray green bluepink
Slide 21 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
The light spectrumWavelength ---- Frequency
Blue light
488 nm
short wavelength
high frequency
high energy (2 times the red)
Red light
650 nm
long wavelength
low frequency
low energy
Photon as a wave packet of energy
Slide 22 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 23 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Magnification1000mm
35 mm slide24x35 mm
M = 1000 mm35 mm
= 28
p 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.
Slide 24 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Some Principles
• 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
Slide 25 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 26 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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 lengthFocal lengthof objectiveof objective= 45 mm= 45 mm
Mechanicaltube length= 160 mm
Object toImage Distance = 195 mm
Slide 27 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
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
Slide 28 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Upright Scope
BrightfieldSource
Epi-illuminationSource
Slide 29 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Inverted Microscope
BrightfieldSource
Epi-illuminationSource
Slide 30 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Conventional Finite Opticswith Telan system
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
Slide 31 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Infinity Optics
Sample being imaged
Primary Image Plane
Objective
Other optics
Ocular
Other optics
Tube Lens
InfiniteImageDistance
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
Slide 32 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Summary Lecture 1• Simple versus compound microscopes
• Achromatic aberration
• Spherical aberration
• Köhler illumination
• Refraction, Absorption, dispersion, diffraction
• Magnification
• Upright and inverted microscopes
• Optical Designs - 160 mm and Infinity optics
Recommended