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FUNDAMENTALS OF FLUORESCENCE MICROSCOPY

James Jonkman james@aomf.caAdvanced Optical Microscopy Facility, Toronto, Canada

Optical Microscopy Users Group (O‐MUG)

11th AnnualCOMPREHENSIVE COURSE ON FLUORESCENCE MICROSCOPY

June 8‐12, 2015Register:  www.aomf.ca ($950/week)

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Robert Hooke’s Micrographia, 1664

Project Gutenberg eBook:www.gutenberg.org

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Observ. LIII. Of a Flea.

Project Gutenberg eBook:www.gutenberg.org

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Fundamentals of Microscopy

• Lenses and magnification• Contrasts in microscopy• Fluorescence and fluorophores• Resolution• Confocal microscopy• Four key elements of a

fluorescence microscope

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Lenses and Magnification

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Magnification by a lens

Microscopy Primer: Interactive Java Tutorials(Michael Davidson, Florida State University)

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The Compound MicroscopeCompound microscope (finite tube length)

Compound microscope (infinity corrected)

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Image formation: Inverted microscope

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Total Magnification ??

× 10x = 600x ??

• In the digital era, “total magnification” is an outdated concept.• Best to use a scalebar to denote the magnification.

60x

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Magnification isn’t everything!

• A microscope magnifiesthe features of your sample that interest you.

• Those features‐of‐interest only stand out from their surroundings if a certain contrast is achieved.

• It’s quite easy to magnify the sample beyond the resolution limit of the microscope system.

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Contrasts in Microscopy

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DICPhase contrast

Histological stain

Fluorescence microscopy

Contrasts in microscopy

Unstained sample

Brightfield microscopy

Unstained sample

Fluorescence labeling

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Brightfield microscopy, H&E staining

H&E stained rabbit brain section, imaged at 0.8x and 10x (inset) magnifications.

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Brightfield slide scanners

• Virtual microscopy

• Whole-slide analysis

• Telepathology

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Whole-slide 20x/40x scanning and quantification of stained histology slides

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Imaging unstained samples

• Unstained cells/tissues are typically too thin to absorb an appreciable amount of light, so most light passes through the sample without interacting

• Contrast techniques try to diminish the light that did not interact with the sample, and accentuate the tiny amount of light that did refract or scatter.

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Phase Contrast

• Illuminate with a ring of light

• Light that didn’t diffract off features in the sample is darkened and phase shifted by a second ring built into the objective lens

Fritz Zernike – Nobel prize (1953)

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Differential Interference Contrast (DIC)

• Polarizer and analyzer are cross‐polarized

• 1st prism splits the light into two beam‐paths through the sample

• 2nd prism recombines them

• If the beams traversed different distances through the sample, the interference rotates the polarization, allowing light through the analyzer

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Transmission complements fluorescence

Glioma cells, stained with Propidium Iodide (green) and Hoechst (blue), and imaged with a 63x / 1.4NA oil objective lens.

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Timelapse imaging of wound‐healing assay

Jonkman JEN, Cathcart JA, Xu F, Bartolini ME, Amon JE, Stevens KM, Colarusso P.  An Introduction to the wound healing assay using live‐cell microscopy.  Cell Adhesion & Migration 2015; 8: ‐

2

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Fluorescence and Fluorophores

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Why use fluorescence?

• Specific labeling of structures or molecules of interest

• Multiple probes don’t interfere

• Colourful?

• Photobleaching

• Labeling can cause artifacts

Why not?

DAPI FITC

Cy3 Composite

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Wave-like properties of light

400nm 500nm 600nm

Frequency (Hz)

Wavelength (m)

Lowenergy

Highenergy

E=hc/

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Light as a particle: Photons- Fluorescence is generated when a single photon of light interacts

with a single molecule of your fluorescent dye

- The energy of the photon (ie., the wavelength) must match an absorption energy level in the molecule

Jablonski Diagram

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Fluorophores: Fluoroscein isothiocyanate (FITC)

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Stokes shift

Fluorophores: 4',6-diamidino-2-phenylindole (DAPI)

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Choosing your Fluorophores

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Vital dyes: Hoechst and PI

Glioma cells, stained with Propidium Iodide (green) and Hoechst (blue), and imaged with a 63x / 1.4NA oil objective lens.

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Immunofluorescence

10 m

Confocal fluorescence image of an immuno-fluorescently labeled, fixed myoblast cell. Green: tubulin; Red: actin; Blue: myosin.

5 um

Repair molecules are recruited to sites of DNA damage in irradiated nuclei. Blue: DAPI; Green: FITC-H2AX

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Genetic labeling: Fluorescent proteins

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Common FluorophoresFluorophore Excitation Emission

DAPI Ultraviolet (UV) Blue

Hoechst Ultraviolet (UV) Blue

FITC Blue Green

Alexa 488 Blue Green

GFP Blue Green

Cy3 Green Red

TRITC Green Red

Rhodamine Green Red

Texas Red Green Red

Alexa 594 Green Red

Cy5 Red Infrared (IR)

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Choosing your FluorophoresFluorophore Excitation max Emission max

DAPI 352 nm 460 nm

Hoechst

FITC

Alexa 488 488 nm 520 nm

GFP

Cy3

TRITC

Rhodamine

Texas Red

Alexa 594

Cy5

Consider the entire spectra

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Spectral overlap

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Fluorophore stability

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Fluorophore Brightness

Brightness depends on:– Fluorescence Quantum Yield (φ)

• The ratio of photons absorbed to photons emitted

– Extinction Coefficient (ε)• Capacity of a fluorophore to absorb photons

– Number of fluorophores per molecule

– Fluorescence quenching

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Fluorescent Protein Properties

Nathan Christopher Shaner, Chapter 6 - Fluorescent proteins for quantitative microscopy: Important properties and practical evaluation, In: Jennifer C. Waters and Torsten Wittman, Editor(s), Methods in Cell Biology, Academic Press, Vol 123, Pages 95-111 (2014)

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How do you choose fluorophores?

• Use bright, stable fluorophores

• Shorter wavelengths give you better resolution

• Longer wavelengths give you less phototoxicity/photobleaching

• Minimize spectral overlap

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Resolution

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The Point Spread Function (PSF)

• A point-like object (such as a single fluorescent molecule, or a nanometer-size bead), is not invisible under the microscope; rather, the point-like object appears to be bigger than it really is.

Airy disk

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The size of your PSF depends on the wavelength of the fluorescence emission

Point-spread function (PSF)

Lower wavelengths give you better resolution

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The size of your PSF depends on the Numerical Aperture (NA) of your objective lens

Numerical Aperture

NA = nsin

Point-spread function (PSF)

Low-NA objective

High-NA objective

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The Rayleigh Criterion for Resolution is related to contrast

Resolution is the minimum separation between two point objects such that they can still be distinguished.

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Resolution in optical microscopy

1.22

2xyrNA

200xyr nm

500

1.4

nm

NA

(GFP)

(63x, oil)

Rayleigh Criterion (resolution rule‐of‐thumb)

You need at least twice as many pixels as your smallest resolution element.

100 nm / pixel0.1 m / pixel

Nyquist Theorem(sampling suggestion)

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Super-resolution, or “nanoscopy”

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Stimulated Emission Depletion (STED) Microscopy

The red STED beam squeezes the green excitation laser beam to create a tighter focal spot

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Confocal Microscopy

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Widefield microscopes work well for thin fluorescence specimens

Thin (5um) Intermediate (15um) Thick (35‐50um)Molecular Probes Slide #1Cultured BPAE cells, fixed

Molecular Probes Slide #3Fixed kidney slice

3D Culture of mammary epithelial cells

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A confocal microscope allows you to do optical sectioning in thick specimens

Widefield Confocal

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The laser-scanning confocal principle

Objective Lens

Laser

Specimen

Beamsplitter

Excitation Emission

Beamsplitter

Pinhole

Detector

Pinhole Lens

Laser

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The laser-scanning confocal principle

Scanning mirrors deflect the beam laterally (x-y directions)

Stage moves specimen through the focus axially (z-direction)

x

y

z

Different fluorophores may be imaged simultaneously or sequentially

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Laser-scanning confocal performance - 50um thick sample

Widefield Laser‐scanning confocal

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Image deeper with Two-Photon microscopy

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Image faster with spinning-disk and resonant-scanning confocals

GFP-Tubulin micro-injected into smooth muscle cells; imaged on Yokogawa spinning-disk confocal

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Four key elements of a fluorescence microscope

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Four key elements of a fluorescence microscope

Lamp / Laser

Camera / Detector

Filter sets

Objectivelenses

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Light sources for fluorescence: lamps• Powerful (100W for widefield illumination)

• Uniform illumination

• Stable

• White (contains the entire visible spectrum)

• Long life, easy to align, low cost

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Objective lenses

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Numerical Aperture (NA)

2x NA = 4x sensitivity

High-NA objective

Low-NA objective

Resolution WorkingDistance

Sensitivity

sin

Increase your NA by using:•Oil (n = 1.518)•Water (n = 1.33)

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Refractive index mismatch

2 m

63x / 1.2NA water immersion 63x / 1.4NA oil immersion

Water lens Oil lens

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Filter sets for fluorescence

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Filter sets for fluorescence

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Cameras for fluorescence

• Sensitive (high “quantum efficiency”)

• Millions of pixels???

• Monochrome (use filters to choose colour)

• Cooled to reduce thermal noise

• CCD, sCMOS, EMCCD ?

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Four key elements of a fluorescence microscope

Lamp / Laser

Camera / Detector

Filter sets

Objectivelenses

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FUNDAMENTALS OF FLUORESCENCE MICROSCOPY

James Jonkman james@aomf.caAdvanced Optical Microscopy Facility, Toronto, Canada