leeuwenhoek the simple microscope .leeuwenhoek microscope (circa late 1600s ) upright microscope

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  • The simple microscope Leeuwenhoek Microscope

    (circa late 1600s)

  • Upright microscope .

    Inverted microscope

    Transmitted and Fluorescence Illumination

  • The Objective

    The Microscopes 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 / (NA objective +NA condenser)

    Kohler Illumination: Condenser and objective focused at the same plane

  • Resolution versus Contrast

    d = 0.61/NA

    =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

    LEDs

    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

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