Leeuwenhoek
The simple microscope
Microscope
(circa late
1600s)
Transmitted and Fluorescence Illumination
Upright microscope
.
Inverted microscope
The Microscope’s Most Important Component
The Objective
http://www.microscopyu.com/articles/optics/objectiveintro.html
http://zeiss-campus.magnet.fsu.edu
The second most important component…
The Condenser
Condenser maximizes resolution
dmin = 1.22
λ / (NA objective +NA condenser)
Kohler Illumination: Condenser and objective
focused at the same plane
Resolution versus Contrast
•  d = 0.61λ/NA
•  λ=wavelength; NA=Numerical Apeture
0 Units
50 Units
100 Units
C ONTRAST
50 Units
50
0
-0.33
1
50 – 50 / 50 + 50 =
50 – 0 / 50 + 0 =
50 – 100 / 50 + 100 =
Brightness of Specimen - Brightness of Background
Brightness of Specimen + Brightness of Background
50
Electromagnetic Spectrum
Higher
Resolution
Longer
Illumination Techniques - Overview
Transmitted Light
Incident Light
•
•
•
•
•  Darkfield
•  Polarized Light
•  Fluorescence (Epi)
•  Brightfield
•  Oblique
Darkfield
Phase Contrast
Polarized Light
DIC (Differential Interference
Contrast)
•  Brightfield
•  Oblique
DIC (Nomarski)
"   High Contrast and high resolution
"   Full Control of condenser aperture
"   3-D Image appearance
"   Color DIC by adding a wave plate
"   Selectable contrast / resolution via different
DIC sliders
"   Orientation-specific > orient fine details
perpendicular to DIC prism
DIC (Differential Interference Contrast) after
Nomarski
Observing local differences in phase retardation
9 Image
8 Tube lens
7 Analyzer (7a with Wave Plate)
6 Wollaston Prism behind objective
5 Objective
4 Specimen
3 Condenser
2 Wollaston Prism before condenser
1 Polarizer
Required Components for DIC:
•  Nosepiece with DIC receptacles
•  Polarizer
•  Low Strain Condenser and Objective
•  DIC Prisms for Condenser (#I orII orIII)
•  Specific DIC Slider for each objective
•  Analyzer
Fluorescence
•  Easy to set up > Objective = Condenser
•  Highly specific technique, wide selection of markers
•  Detection and Identification of Proteins, Bacteria,
Viruses
•  Basics for
–
–
–
–
–
Special Techniques eg. TIRF, FRET, FRAP etc.
3-D imaging
Deconvolution
Structured Illumination
Confocal Techniques
Where does energy go?
Green light emitted
Blue light absorbed
Quantum Yield = light out/light in
490nm
Stokes Shift
520nm
Q ~ 0.8 fluorescein
~ 0.3 rhodamine
Light Sources
•  Mercury (Hg)
•  Xenon, Hg/Xe
Combination
•  Laser
•  LED’s
•  Tungsten Halogen
Epi - Fluorescence
(Specimen containing green
fluorescing Fluorochrome)
Observation port
Excitation Filter
FL
Light
Source
Specimen containing green
fluorescing Fluorochrome
Emission Filter
Dichromatic Mirror
How to improve Fluorescence
Imaging in a major way:
• Optical Sectioning
Overview of
Optical sectioning Methods
1.  Confocal and Multi-photon
Laser Scanning Microscopy
–
–
Pinhole prevents out-of-focus light getting to the
sensor(s) (PMT - Photomultiplier)
Multi Photon does not require pinhole
2.  Spinning disk systems
–
–
A large number of pinholes (used for excitation and
emission) is used to prevent out-of-focus light getting to
the camera
E.g. Perkin Elmer, Solamere
3. Deconvolution
–
–
Point-Spread function (PSF) information is used to
calculate light back to its origin
Post processing of an image stack
Laser Scanning Confocal Microscopes (LSCM)
Zeiss LSM710 with Two-photon laser
Chameleon Ultra II Laser
Leica SP5 Spectral High Speed
Confocal Microscopy just a form of
Fluorescence Microscopy
www.olympusfluoview.com
Optical Sectioning: Increased Contrast and Sharpness.
Examples: Zebrafish images, Inner ear
• Bit Depth
• 8 bits = 256
• 12 ” = 4,096
• 16 ” = 65,536
• Maximize Histogram
3-D Reconstruction Zebrafish Cranial Ganglia
A
P
M
L
Neural Gata-2 Promoter GFP-Transgenic; Shuo Lin, UCLA
Spectral or Lambda Scanning
•  Separate very similar colored fluorophores
–  fluorescein and green fluorescent protein (GFP).
•  Could be used to eliminate non-specific background
fluorescence that has different emission spectra.
•  Different technologies for spectrum detection
–  Sequentially (Leica SP)
–  Simultaneously (Zeiss QUASAR)
Lambda Stack
Lambda Stack
Lambda Stack
In vivo Hair Cell Dye, FM1-43 Spectra
High Speed Confocal Microscopy
1.  Spinning disk systems
–
A large number of pinholes with microlenses (used
for excitation and emission) is used to prevent outof-focus light getting to the camera
–
E.g. Perkin Elmer, Solamere
2.  Resonance Scanner (Leica, Nikon)
3.  Double your scanning speed (Bidirectional)
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
Crista Cilia Labeled in vivo with FM1-43
Confocal Speed - 90 fps
Two-Photon Excited Fluorescence
(Jablonski diagram)
4nsec
Excitation from
coincident absorption of
two photons
0.8 emitted
Two-Photon microscopy
Optical sectioning by non-linear absorbance
--> broad excitation maxima
Two-photon microscopy is somewhat colorblind
normalized intensity
0.5
YFP
CFP
Dil
GFP
EtBr
RFP
0.4
0.3
0.2
0.1
0
450
500
550
600
nanometers
TPLSM excitation at 900nm excites multiple dyes and GFP variants
Two Photon Microscopy
Advantages
Disadvantages
•  No need for pinhole
•  Laser $$$
•  No bleaching beyond
focal plane
•  Samples with melanin
•  Potentially more sensitive
•  IR goes deeper into
tissue
•  Samples with multiple
fluorescent labels
Super-Resolution Confocal Imaging:
Below the wavelength of light
•  STED: STimulated Emission Depletion (Deterministic)
•  PALM: PhotoActivated Localization Microscopy (Stochastic)
•  STORM: STochastic Optical Reconstruction Microscopy
“True” Super-resolution
“Functional” Super-resolution
http://zeiss-campus.magnet.fsu.edu
STED: STimulated Emission Depletion
http://zeiss-campus.magnet.fsu.edu
PALM: PhotoActivated Localization Microscopy
http://zeiss-campus.magnet.fsu.edu
Microscopy Resources on the Web
•  http://www.olympusmicro.com
–  Olympus
•  http://www.microscopyu.com
–  Nikon
•  http://zeiss-campus.magnet.fsu.edu
–  Zeiss
Acknowledgements
Olivier Bricaud
Aldo Castillo
Aicha Castillo
Frank Stellabotte
Kalpana Desai
Sung-Hee Kil
Erik Waldman
Le Trinh
Bill Dempsey
Periklis Pantazis
Scott Fraser
• Caltech
Shuo Lin, UCLA
Caryl Forristall, University of Redlands
Rudi Rottenfusser, Carl Zeiss
Carlos Alonso, Leica
Supported by NIH and NIDCD
Condenser aperture
Field aperture
Kohler Step 1: Close field aperture
Move condenser up-down to focus image of the
field aperture
Kohler Step 2: Center image of field aperture
Move condenser adjustment
centered
Kohler Illumination gives best resolution
Set Condenser aperture so
NAcondenser = 0.9 x NAobjective
Open field aperture to fill view