Dr' Gisle Giorgi

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Advanced Microscopy

• Dr. Gisèle Giorgi
• Fall 2008

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Review, pt. 1
Recognition of microscopy techniques First
lecture from bio 35 Light paths (transmitted,
incident/reflected) Microscope components Properti
es of light (EM spectrum, amplitude,
wavelength) Light interacting with matter
reflection, absorption, refraction,
diffraction. Fluorescence Biotech primer
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Guess the technique!
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African Water Mongoose Skin Fibroblast Cells
micro.magnet.fsu.edu
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Blue-Green (Spirulina) Algae
www.microscopyu.com
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Blue-Green (Spirulina) Algae
www.microscopyu.com
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Mouse Embryonic Fibroblasts
Dr. Jan Schmoranzer
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Foot of a house fly

• www.mos.org

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Rattlesnake tail shaker muscle
Dr. Stan Lindstedt and Marilee Sellers www4.nau.ed
u
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Skeletal muscle (frog)
micro.magnet.fsu.edu
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Tobacco leaf

• UC Berkeley Imaging Facility

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Mouse sperm flagella
UC Berkeley Imaging Facility
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Vero cells (African Green Monkey Kidney
Epithelial Cells)
micro.magnet.fsu.edu
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Human Bone Osteosarcoma Cells
micro.magnet.fsu.edu
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Chinese Liver Fluke

• micro.magnet.fsu.edu

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• Embryonic Stem Cell
• (ignore the color)

ncmir.ucsd.edu
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Mascara Brush

• www.mos.org

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Optic Nerve
www.udel.edu/biology
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potato starch (ignore the color)

• www.microscopyu.com

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Jumping Spider (ignore the color)
David Scharf images at www.microscopy.info
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Cat testes(ignore the color)
www.microscopyu.com
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Testudinella sp.(ignore the color)
Image courtesy of www.microscopyu.edu
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Bean weevil emerging from a bean seed
(Acanthoscelides obtectus).Dennis Kunkel, PhD
(ignore the color)
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Dr. Sharon Minsuk
sea urchin embryo
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Original Lecture 1!(or see other ppt)
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Properties of light
transmitted
reflected
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Microscopy techniques
light source/objective
Transmitted brightfield phase DIC
Incident/epi-illumination fluorescence
(dissecting scope)
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Microscopy techniques
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Transmitted light
Techniques include - brightfield - phase - DIC
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Objects and transmitted light
light wave
amplitude object
seen as color
phase object
not seen
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Transmitted light
amplitude object - pigmented or stained samples
- e.g. histology specimens - seen with
brightfield microscopy
phase object - most biological samples! - seen
with phase or DIC microscopy
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CONTRAST!
Cells typically are - transparent (not
amplitude objects) - phase objects - low in
contrast

• Contrast-generating techniques such as DIC
  turn phase differences into intensity differences
  so we can see unstained cells using transmitted
  light!

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Contrast
We need contrast to be able to see an
object. Contrast can come from variations in -
intensity (DIC, phase) - color (brightfield,
fluorescence)
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Brightfield

• Uses transmitted light
• For regular brightfield, specimens must be
  amplitude objects naturally (due to pigments) or
  due to staining.
• Widely used in histology, pathology, botany
• NOTE Yes, technically, DIC, phase, etc. are
  also types of brightfield techniques! As in
  brightfield vs.fluorescence.

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Phase

• Uses transmitted light
• Specimens can be phase objects, . unstained
• - Phase haloes are typically visible
• - Note amplitude objects are also visible in
  phase!
• So dont be fooled by the presence of color!
• used in cell culture (to monitor growth of cells)
• relatively inexpensive and simple

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DIC

• a.k.a. Nomarski
• Uses transmitted (polarized) light
• Specimens can be phase objects, . unstained
• - Typical 3-D appearance (but is pseudo 3-D)
• - Note amplitude objects are also visible in
  DIC!
• So dont be fooled by the presence of color!
• widely used in biology
• somewhat expensive

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Polarized light microscopy

• Uses transmitted light
• Specimens must be anisotropic/birefringent
• - typical bright, multi-colored appearance
• especially used in geology
• relatively inexpensive

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Fluorescence

• Uses INCIDENT light
• aka epifluorescence
• Specimens are fluorescent (light emitting) either
  naturally (autofluorescence) or due to
  manipulation.
• Manipulation includes
• Fix, permeabilize and stain with antibodies
  linked to fluorophores
• Transfect with genes that contain GFP and other
  variants
• Live cell dyes
• Contrast is generated by presence/absence of
  (colored) light
• Includes
• Widefield epifluorescence (our current scopes)
  which collects all the light coming from the
  sample
• Confocal which sees only one plane of focus
  (sections) at a time.
• Also multiphoton, deconvolution, etc. etc.
  (later in the course!)
• widely used in biology
• somewhat expensive

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Transmission electron microscopy

• Uses electrons rather than photons of light
• aka TEM or EM
• Specimens are fixed, thin, highly prepared
• Increased resolution (much below the resolution
  of light), so we can view smaller details
• widely used in biology, quality control
• somewhat expensive
• requires extensive training

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Scanning electronmicroscopy

• Uses electrons rather than photons of light
• aka SEM
• Specimens are fixed, relatively quickly prepared
  (as compared to TEM)
• Increased resolution (much below the resolution
  of light), so we can view smaller details
• widely used in biology, quality control
• somewhat expensive
• requires extensive training (but less than TEM)

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End of original lecture 1
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• Light paths

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Components
Light source Condenser Stage Objective Oculars/de
tector
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Light path
Light -gt condenser -gt specimen -gt objective -gt
ocular
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upright vs. inverted scopes
Upright light -gt condenser -gt specimen -gt
objective -gt ocular Inverted light -gt
condenser -gt specimen -gt objective -gt
ocular
stage
stage
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Inverted
Upright
condenser
objective
objective
condenser
Images from Nikon promotional materials
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Light path
Transmitted light Light -gt condenser -gt specimen
-gt objective -gt ocular Epifluorescence Light -gt
objective -gt specimen -gt objective again -gt
ocular
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Upright Scope
Epi- illumination Source
B
A1
Brightfield Source
A2
Light paths Fluorescence A1 -gt B Transmitted
A2-gt B
modifications by GG
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Inverted
Light paths Fluorescence A2 -gt B Transmitted
A1 -gt B
modifications by GG
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• MICROSCOPE
• COMPONENTS

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Our Olympus CKX41
Arc lamp first ON/last OFF leave on for at
least 30 min. Fluorescence filters slider
manual. ND filters slider no ND filter, use as
shutter. Phase slider match it to
objective Trinocular head slider to
camera. Camera QCAM, black and white.
Computer Qcapture is QCAM imaging software.
(Image J?)
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Cell structures
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Light
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Visible light
Visible light 400-700 nm
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Light as wave

• ? wavelength
• - measured from peak to peak, in nanometers (nm)
• - seen as color
• Amplitude
• 1/2 from peak to valley
• seen as intensity/brightness

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What is White Light?
A combination of all wavelengths originating
from the source
Pl.note that wavelength relationship exceeds
visible range
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Visible light
400 nm
700 nm
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Description of light, p.18

• Light can be described both as particles and as
  waves.
• Can be referred to as
• photons
• waves
• rays/beams

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Description of light

• Quanta particles called photons which are
  packets of energy.
• - CCD cameras measure photons

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Description of light

• Waves electrical (E) and magnetic (B) fields
  oscillate as sine waves.
• E and B are perpendicular to each other
• E and B have the same phase and amplitude

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Description of light

• Waves usually just the E field is shown.
• Amplitude strength of field, shown on Y axis
• Wavelength time or distance of one cycle,
  shown on X axis
• Note often this drawing is used to represent an
  entire beam of light, not just one color.

x
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Description of light

• Rays or beams as a straight line (or arrow)
• - used in geometrical optics (i.e. light going
  through lenses)

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Qualities of light , p.20

• Monochromatic or Polychromatic
• Polarized or Nonpolarized
• Coherent or Noncoherent
• Collimated or Divergent

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Qualities of light

• Monochromatic waves with same wavelengths (and
  therefore one color).
• Polarized waves with E vectors in parallel
  planes. (Drawn as )
• Coherent waves of a given wavelength that
  maintain the same phase relationship with each
  other while traveling.
• Collimated waves propagating in the same
  direction (coaxial).

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Qualities of light
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Polarized light
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Light and matter
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Interaction of Light with MatterEM SPECTRUM
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Light interacting with matter

• When light hits matter it can be
• Reflected
• Partially absorbed
• Refracted transmitted
• Diffracted

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Reflection
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Reflection
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Reflection
?
?
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?(angle of incidence) ?(angle of reflection)
When a beam of light strikes a surface at an
angle measured from a line perpendicular to that
surface, it is reflected in the opposite
direction at an angle equal in size
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Absorption

• When light hits matter it can be
• Absorbed reduction of amplitude of one or more
  wavelengths.
• - whatever isnt absorbed goes through the
  specimen and is transmitted.
• Optical filters absorb certain wavelenghts of
  light, and transmit others.
• In fluorescence the absorbed light excites
  electrons, leading to emission of fluorescence

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Absorbed light chemistry

• Light that was absorbed can be
• - very rapidly re-emitted as light of a longer
  wavelength (fluorescence)
• - rapidly re-emitted as light of a longer
  wavelength (phosphorescence)
• - slowly re-radiated as infrared waves (heat)
• - transformed into chemical energy (e.g. breaking
  chemical bonds)

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Refraction

• When light hits matter it can be
• Refracted bends as it passes from one material
  to another.
• The refractive index (n) of material is important
  in microscopy.

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Refraction
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- the bending of light
as it passes from one material to
another
Refraction
Snells Law n1 sin b1 n2 sin b2
b1
Normal (perpendicular to interface of different
materials)
b2
n1
n2
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Light beam through a plane-parallel glass plate
b1
b2
??
n1
n2
n1
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Light beam through a plane-parallel glass plate
b1
b2
b1
n1
n2
n1
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Refraction (Marching Band Analogy)
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Refraction (Marching Band Analogy)
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Refraction (Marching Band Analogy)
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Refraction (Marching Band Analogy)
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Diffraction

• When light hits matter it can be
• Diffracted bends as it passes an edge
  (including that of a small aperture).
• - Abbes theory of microscopy shows how
  diffraction is critical for image formation.

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Diffraction of waves
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Dispersion

• When light hits matter it can be
• Dispersed refraction and diffraction are
  wavelength dependant, so white light gets
  separated into its constituent colors when it is
  refracted and/or diffracted.
• - leads to blue/red shift (and corrections for
  it in confocal optics).

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Dispersion
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Dispersion in a plane-parallel glass plate
(e.g. slide, cover slip, window of a vessel)
White Light
Which expression is commonly used for unwanted
dispersion? Chromatic Aberration
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Light interacting with matter

• Scatter a combination of various effects (or
  mostly diffraction or reflection in many
  directions).
• (Why is the sky blue? Blue light is scattered in
  all directions by the molecules of the air, so no
  matter in what direction we look, we see blue
  skies.)
• Scatter in a microscope light wandering off
  from the desired path.

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Transmitted light

• Transmitted light, as it passes through an
  object, can be
• refracted
• diffracted by edges of opaque portions and by
  structures nearly as small as the wavelengths of
  the light.
• This diffraction allows us to use microscopes to
  see small structures. Its not just about
  magnification!

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Object names, p. 20

• Opaque objects absorb light.
• Transparent objects transmit light.
• Reflective objects reflect light.
• Scattering objects diffract light.

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Object names

• Objects usually have a combo of qualities we
  refer to them by the predominant or most relevant
  quality.
• Amplitude objects (a microscopy term) are
  somewhat opaque.
• Phase objects (a microscopy term) are fairly
  transparent.

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Light and matter putting it together.

• Light hitting a cell will be somewhat reflected,
  transmitted, refracted, absorbed, dispersed and
  diffracted.
• This leads to phase shifts and changes in
  polarity which are used by the microscope to form
  an image.
• We cant see phase differences or polarity
  differences with our own eyes, but the microscope
  can use them to generate contrast in an image.
  This is the basis of DIC and phase microscopy.
• Light is also absorbed and can cause fluorescence.

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Light interacting with matter

• When light hits matter it can
• Be absorbed and cause fluorescence.
• Be diffracted/refracted and change phase.
• Become polarized (or change polarization).

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• FLUORESCENCE

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Fluorescence

• light -gt fluorophore -gt light

EMISSION
EXCITATION
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Fluorescence
specimen
EXCITATION
EMISSION
light source/objective
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Jablonski diagram
www.molecularprobes.com/handbook/figures/0664.html
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Jablonski diagram
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Optical filters
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Filter cube
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Filter cube
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Epi - Fluorescence
(Specimen containing green fluorescing
Fluorochrome)
Observation port
Excitation Filter
Emission Filter
FL Light Source
Dichromatic Mirror



Specimen containing green fluorescing Fluorochrome
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• BIOTECH

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Biotech

• Biotech vs. pharma
• Genetic engineering recombinant DNA
• Genes as potential.
• Gene expression.
• Signal transduction.
• Vector. Plasmid. Bacteria.
• DNA or Protein electrophoresis.
• Western/Southern/Northern blotts.
• Sequencing.
• Microarrays.
• Bioinformatics.
• Genomics.
• PCR.

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Biotech

• Cells culture.
• Antibodies.
• IHC

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Review, pt. 2 and 3
Filters Fluorophores/spectra Bleedthrough Bleachin
g GFP NA Refractive index Diffraction Airy
disk/psf Resolution
Optical sectioning Geometric optics Dynamic
range, saturation, histogram Axiovision Image
J Koehlering Conjugate planes DIC Confocal