Microscopy flourescent staining techniques

Flourescent microscopy

1. 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

2. 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

3. 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

4. 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

5. 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.”

6. Slide 6 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Overview of discovery

7. Slide 7 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Early Microscopes (Hooke)
1665

8. 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

9. 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”

10. 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

11. 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.

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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….

18. Slide 18 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Absorption
Control
No blue/green light
red filter

19. Slide 19 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Light absorption
white lightblue lightred light green light

20. Slide 20 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Absorption Chart
Color in white lightColor of light absorbed
red
blue
green
magenta
cyan
yellow
blue
blue
blue
blue
green
green
green
green
red
red
red
redblack
gray green bluepink

21. Slide 21 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
The light spectrum
Wavelength ---- 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

22. 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

23. Slide 23 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Magnification
1000mm
35 mm slide
24x35 mm
M =
1000 mm
35 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.

24. 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

25. 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

26. 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 length
of objectiveof objective
= 45 mm= 45 mm
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm

27. Slide 27 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
The Conventional Microscope
Focal length
of objective
= 45 mm
Object to
Image
Distance
= 195 mm
Mechanical
tube length
= 160 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press

28. Slide 28 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Upright Scope
Brightfield
Source
Epi-
illumination
Source

29. Slide 29 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Inverted Microscope
Brightfield
Source
Epi-
illumination
Source

30. Slide 30 t:/classes/BMS524/524lect1.ppt 2000 J.Paul Robinson - Purdue University Cytometry Laboratories
Conventional Finite Optics
with Telan system
Sample being imaged
Intermediate Image
Telan Optics
Objective
Other optics
Ocular
45 mm
160 mm
195 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press

31. 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
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

32. 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