Transcript Essentials of Light Microscopy

Microscopy
Nikitchenko Maxim
Baktash Babadi
Boot Camp 2009
2009/08/25
Plan of the lecture
•
•
•
•
•
•
•
Basic properties of light
Light/matter interaction
Wide-field microscopy
Scanning microscopy
EM
Ultra-high resolution microscopy
Dyes
Part 1
Part 2
Part 3
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Corpuscular/wave dualism
www.olympusmicro.com
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Diffraction
Fluorescent Microscopy
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Basic electromagnetic wave
properties
E

E
kz


t)
0sin(
•
•
•
•
•
Amplitude
Wavelength
Frequency
Phase
Polarization
hyperphysics.phy-astr.gsu.edu
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Polarization
Fluorescent Microscopy
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Light/matter interaction
• Particles point of view:
Absorption
Emission
Scattering
• Waves point of view:
Refraction
Reflection
Absorption
Diffraction (Change of Phase
and Polarization)
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Optics of a thin lens (1)
Focus
d
Thin Lens:  0
F
C
C=2F
F
d
F
F
C
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Optics of a thin lens (2)
• Three different scenarios:
2F
F
F
2F
2F
F
F
2F
2F
F
F
2F
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Optics of a thin lens (3)
f
C
F
F
p
C
q
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Compound Microscope
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Basic optical structure of a
microscope
specimen
objective
eyepiece
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Specimen Illumination System
• Parts
– Specimen plane
– Condenser
– Diaphragm
– Light Source
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope Illumination
Conditions:
• Critical illumination
– The condenser focuses
the light onto the
specimen plane
Filament image effect
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Köhler illumination
• The specimen is
illuminated homogenously
• The specimen and the
images of the light source
are in different planes
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Types of Microscopy
•
•
•
•
Bright Field
(absorption)
Dark Field
(scattering)
Phase-contrast (phase change)
Polarization (scattering by birefringent
specimen)
• Differential interference contrast (DIC)
(gradients of optical thickness)
• Fluorescent (frequency change as a result
of absorption/emission by fluorophores)
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Dark field Microscopy
uses the difference in
scattering abilities
 block out the central light
rays (leave oblique only)
Result: only highly diffractive
and scattering structures
are seen

Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Dark-Field example
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Phase Contrast Microscopy




uses the λ/4 phase change when
light passes through thin
structures
Similar oblique illumination to the
Dark Field method
The specimen diffracts some of the
light that passes through it and
introduces phase lagging λ/4
A phase difference (λ/2) is
introduced between background
and diffracted light (using phase
plate) → destructive interference
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Phase Contrast Microscopy
• Suitable for unstained specimens
Human glial cells
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Polarization Microscopy
Uses polarization property of
light and birefringence
• Polarizer polarizes light
• Analyzer passes only the light
with polarization perpendicular
to the source light

Birefringent material introduces
2 perpendicularly polarized
components, propagating at
different speed in the specimen

→ Δφ
• Constructive interference
following analyzer is possible
only for phase shifted light
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Polarized microscopy example
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Differential Interference Contrast
(DIC) (Nomarski optics)
• Addon to the polarization
microscopy
• Wollaston prism generates 2
|| beams, π/4 polarized to
polarizer and laterally
displaced (this is the
difference to polarization
microsc., endowing optical
density gradient sensitivity)
• The rest is similar to pol. Micr.
(except for 2nd Wollaston
prism)
• Result: good for edge detection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Nomarski optics principle
polarizer
Beam
Splitter
shear
specimen
condenser
combiner
objective
intensity
analyzer
Basic Properties of Light
Thin lenses
Wide-field Microscopy
DIC example
Fluorescent Microscopy
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Fluorescent Microscopy (1)
• Fluorescence
– Emission light has longer wavelengths than
the excitation light: Stokes shift.
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Fluorescent Microscopy (2)
• Types of Fluorescence
– Auto-Fluorescence (Plants, Fungi,
Semiconductors, etc)
– Fluorescent dyes
• Fluorochromes (Flurescein, Acredine Orange,
Eosin, Chlorophyll A, … )
• Genetically coded (GFP, YFP,…)
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Fluorescent Microscopy (3)
• The basic task of the fluorescence
microscope:
– Illuminate the specimen with excitation light
– Separate the much weaker emission light
from the brighter excitation light.
– Only allow the emission light to reach the
eye or other detector.
– The background is dark, the fluorescent
objects are bright
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Epi-Fluorescent Illumination
• The emission light does not pass
through the specimen
• The objective acts as the condenser
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Fluorescent Microscopic images
(1)
Human cortical neurons
Human brain glioma cells
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Fluorescent Microscopic images
Fluorescence/DIC
combination, cat brain
tissue infected with
Cryptococcus
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Brainbow
Fluorescent Microscopy
Microscope selection
Basic Properties of Light
Thin lenses
Wide-field Microscopy
Fluorescent Microscopy
Microscope selection
Selection of the microscope
no
Is it thin? (<50 microns)
Is it reflective?
e.g. gold, silver
yes
no
Is it fluorescent?
yes
Epi-fluorecence
Is it fluorescent?
no
yes
Is it reflective?
e.g. gold, Silver
yes
Dark field
Confocal
no
Is it colored, densely
contrasted or stained?
yes
Bright field
no
Is it transparent?
yes
Phase contrast, DIC
no
Is it birefringent?
yes
Polarization
Rubbi, C.P., 1994