The simple microscope Leeuwenhoek Microscope

(circa late 1600s)

Upright microscope .

Inverted microscope

Transmitted and Fluorescence Illumination

The Objective

The Microscope’s Most Important Component

http://zeiss-campus.magnet.fsu.edu http://www.microscopyu.com/articles/optics/objectiveintro.html

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

C ONTRAST

50 – 0 / 50 + 0 = 1

50 – 100 / 50 + 100 = -0.33

50 – 50 / 50 + 50 = 0

Background of BrightnessSpecimen of BrightnessBackground of Brightness-Specimen of Brightness

+

50 Units 0 Units 100 Units

50 Units 50 50

Electromagnetic Spectrum

Longer

Higher Resolution

Transmitted Light •  Brightfield •  Oblique

•  Darkfield •  Phase Contrast •  Polarized Light •  DIC (Differential Interference

Contrast)

Incident Light •  Brightfield •  Oblique

•  Darkfield •  Polarized Light •  Fluorescence (Epi)

Illumination Techniques - Overview

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

Blue light absorbed

490nm 520nm

Green light emitted

Stokes Shift

Where does energy go?

Quantum Yield = light out/light in

Q ~ 0.8 fluorescein

~ 0.3 rhodamine

•  Mercury (Hg)

•  Xenon, Hg/Xe Combination

•  Laser

•  LED’s

•  Tungsten Halogen

Light Sources

Epi - Fluorescence (Specimen containing green fluorescing Fluorochrome)

Dichromatic Mirror

Emission Filter

Excitation Filter

Observation port

FL

Light Source

Specimen containing green fluorescing Fluorochrome

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 out-of-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

Confocal Speed - 90 fps

Crista Cilia Labeled in vivo with FM1-43

4nsec

Two-Photon Excited Fluorescence (Jablonski diagram)

0.8 emitted Excitation from coincident absorption of two photons

Two-Photon microscopy

Optical sectioning by non-linear absorbance --> broad excitation maxima

0

0.1

0.2

0.3

0.4

0.5

450 500 550 600nanometers

norm

aliz

ed in

tens

ityYFPCFPDilGFPEtBrRFP

TPLSM excitation at 900nm excites multiple dyes and GFP variants

Two-photon microscopy is somewhat color-blind

Two Photon Microscopy

•  No need for pinhole

•  No bleaching beyond focal plane

•  Potentially more sensitive

•  IR goes deeper into tissue

•  Laser $$$

•  Samples with melanin

•  Samples with multiple fluorescent labels

Advantages Disadvantages

Super-Resolution Confocal Imaging: Below the wavelength of light

•  STED: STimulated Emission Depletion (Deterministic)

•  PALM: PhotoActivated Localization Microscopy (Stochastic)

•  STORM: STochastic Optical Reconstruction Microscopy

http://zeiss-campus.magnet.fsu.edu

“True” Super-resolution

“Functional” Super-resolution

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

Shuo Lin, UCLA Caryl Forristall, University of Redlands Rudi Rottenfusser, Carl Zeiss Carlos Alonso, Leica Supported by NIH and NIDCD

Olivier Bricaud Aldo Castillo Aicha Castillo Frank Stellabotte Kalpana Desai

Bill Dempsey Periklis Pantazis

• Caltech Scott Fraser

Sung-Hee Kil Erik Waldman

Le Trinh

Field aperture

Condenser 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