Microscopy

What is Erne Abbe's formula for the diffraction limit?

(~300 nm)

What is Lattice Light-Sheet Microscopy?

-Very high speed imaging for live samples -Significant reduction of photobleaching and phototoxicity due to use of focal-plane illumination -Excellent optical sectioning capability -Volumetric field of view -Can image deep into large specimens

Where did biological dye chemistry evolve from?

19th century British and German textile dye chemistry

What is a dichroic mirror?

A dichroic mirror is a mirror with significantly different reflection or transmission properties at two different wavelengths.

What is the animation of the image acquisition process?

A lattice light sheet intersects a cell to produce fluorescence

What is the chromophore?

A light-generating hexapeptide fragment of GFP

Wide-Field Microscope

A widefield microscope is one type of Fluorescence Microscope. In a conventional widefield microscope (WFM), the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film.

Zacharias Janssen

Along with his father Hans, controversially thought to be the inventors of the first compound microscope in the late 16th century

What is an electron microscope?

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. It has a higher resolving power than a light microscope and can reveal the structure of smaller objects.

What is a confocal microscope?

An optical imaging technique for increasing optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light.

What does two-photon imaging and confocal imaging have in common?

Both use a laser to excite a fluorescent tag within a sample and detectors to measure the emitted light

Types of White Light Microscopy

Bright field, phase contrast, differential interference contrast

How does one tag/conjugate/couple a protein with GFP?

By adding the DNA sequence of GFP to the gene that codes for our protein of interest

Protein Dyes

Can be encoded by DNA (which can be custom-designed)

Robert Hooke

Coined the term "cell" to describe structural units of cork that were reminiscent of monastery cells

What are the steps of autocatalytic fluorescent protein chromophore formation?

Cyclization, Dehydration, oxidation

Who created PALM and STORM?

Eric Betzig (Nobel Prize for Chemistry 2014) Xiaowei Zhuang (Max Delbruck Prize 2010)

What is flow cytometry?

Flow cytometry is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.

What did Martin Chalfie do?

GFP can be put in any organism and still fluoresce when exposed to UV light

Organic Dyes

Have affinity to certain molecules, or can also be chemically conjugated to molecules

Transmission Electron Microscope (TEM)

Illumination: electrons Magnification: ~100,000x How it works: Detect electrons scattered as they move through the sample Image: Monotone (But may be color enhanced)

What is a Scanning Electron Microscope (SEM)?

Illumination: electrons Magnification: ~100,000x How it works: detect electrons back-scattered by the sample Image: Monotone

What is the difference in the views between 1-photon and 2-photon microscopes?

In 1-photon imaging, fluorescence is from out of focus planes. In 2-photon imaging, fluorescence is from focal spot only

What was the first natural dye to be synthetically duplicated?

In 1868, Alizarin component became the first natural dye to be synthetically duplicated by German chemists Carl Graebe and Carl Liebermann

What is Hoechst?

It has an affinity for DNA and it stains blue

What is anti-acetylated tubulin?

It has an affinity for antibodies.

What is phalloidin?

It stains red, it's fluorescent, and it has an affinity for actin

Where does GFP come from and who discovered it?

Jellyfish Aequorea victoria and Osamu Shimomura.

Ibn al-Haytham (Alhazen)

Known as the first scientist. Laid the foundations for modern physical optics, transforming the way in which light and vision were understood

Structure of a Light Microscope

Light source > Sample > Detector > Recording Device

What are the benefits of two-photon imaging?

Low phototoxicity: the long wavelengths used for two-photon microscopy are less damaging Low scattering: long excitation wavelengths do not scatter in the tissue like lower wavelength lights used for confocal Deep tissue penetration: the requirement for near simultaneous absorption is only achieved near the focal plane where the laser light is most concentrated

Scanning Electron Microscope (SEM)

Magnetic lens-focuses electron beam Scanning coils-for systematic scanning (left to right, then down) Backscattered electron detector- detects electrons that bounced off the film (i.e. food for imaging surfaces) Secondary Electron Detector- detects electrons emitted by the film

What did Roger Tsein do?

Modified GFP to enhance its photostability and engineered a whole palette of fluorescent proteins (derived from GFP) that cover all the colors of the rainbow)

What does using a compound objective/eyepiece combination allow for?

Much higher magnification, reduced chromatic aberration, exchangeable objective lenses to adjust the magnification

Who were the men awarded for their discoveries in GFP?

Osamu Shimomura, Martin Chalfie, Roger Tsien

What is Photo Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM)?

Photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit.

Bright Field Illumination

Sample contrast comes from absorbance of light in the sample.

Phase contrast illumination

Sample contrast comes from interference of different path lengths of light through the sample

Dark field illumination

Sample contrast comes from light scattered by the sample

Cross-Polarized Light Illumination

Sample contrast comes from rotation of polarized light through the sample

Confocal Imaging

Sample is exposed to laser light and light is emitted from fluorophores both within and outside focal point.

Stimulated Emission Depletion (STED)

Stimulated emission depletion (STED) microscopy is one of the techniques that make up Super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.

How does GFP emit light?

Strong light source stimulates electrons in the GFP to a higher energy state, and then electrons return to ground state and releases photon

Structured Illumination Microscopy (SIM)

Structured Illumination Microscopy (SIM) is a super-resolution fluorescence optical microscope imaging technique that increases resolution by exploiting interference patterns (moiré patterns) created when two grids are overlaid at an angle.

Super-resolution Microscopy

Super-resolution techniques allow images to be taken with a higher resolution than the diffraction limit.

What is total internal reflection fluorescence (TIRF) microscopy?

TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.

The Eyepiece of the Bright Field Microscope

The image from the objective lens is then magnified by a second lens or group of lenses that gives the viewer an enlarged inverted virtual image of the object.

Two-photon imaging

The lasers used in two-photon microscopy excite by using near simultaneous absorption of two long wavelength (~800 nm) photons. Sample is exposed to laser light throughout, but there is only sufficient photon density to excite the fluorophores at the focal point.

Objective Lens of the Bright Field Microscope

The lens close to the sample that collects light which focuses a real image of the object inside the microscope.

The Diffraction Limit

This is the smallest distance by which we can distinguish one cell from another. This is dependent on the wavelength we are trying to image.

What is single-molecule microscopy?

This method relies upon the possibility to turn the fluorescence of individual molecules on and off. Scientists image the same area multiple times, letting just a few interspersed molecules glow each time. Superimposing these images yields a dense super-image resolved at the nanolevel.

Types of Electron Microscopy

Transmission, scanning, reflection, scanning transmission

What is Stimulated Emission Depletion (STED) Microscopy?

Two laser beams are utilized; one stimulates fluorescent molecules to glow, another cancels out all fluorescence except for that in a nanometre-sized volume. Scanning over the sample, nanometre for nanometre, yields in image with a resolution better than Abbe's stipulated limit.

Types of Fluorescence Microscopy

Wide-field, confocal, two-photon, STED, PALM, STORM, SIM, light sheet

Who, when, and what was the first synthetic dye discovered?

William Perkin; 1856; Annilin Mauve