Light Microscopy

What are some features of GFP?

- GFP absorbs light at ~470nm and emits light at 510nm. - GFP fusion construct prepared and transformed into target cell. - No/minimal interference with function of tagged proteins. - Talin-GFP fusion protein shows hairs from an Arabidopsis leaf. - GFP-histone shows what happens during the cell cycle of a Drosophila embryo.

What are the features of DIC (Differential Interface Contrast - Normarski)?

- Polarized light passes through the beam splitter prism, generating 2 light beams travelling very close together. - If one of the beams is diffracted by the specimen, when they are re-combined by the top Wollaston prism, they will interfere and generate contrast ranging from black to white. - If the beams are not diffracted by the specimen, they don't interfere when re-combined and produce a pale grey.

How do phase contrast light microscopes work?

- The annular stop in the condenser and the phase plate in the objective lens are aligned. - Light passes through the annular stop, and forms a hollow cone focused on the specimen. - Light, which is undeviated by the specimen goes through the phase ring in the phase plate and is advanced by a 1/4 of a wavelength. - Light diffracted by the specimen is retarded a 1/4 of a wavelength and passes through the phase plate without going through the ring. - The diffracted and undiffracted rays give rise to a change in brightness. This creates contrast.

How do fluorescence microscopes work when blue incident light (<500nm) is absorbed and >520nm green light is emitted?

- The first barrier filter lets through only blue light, with a wavelength between 450 and 490nm. - The beam splitting mirror reflects light below 510 nm but transmits light above 510 nm. - A second barrier filter cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm.

What are the applications of fluorescent light microscopes?

- You can visualize organelles within cells without looking at the whole cell, by just looking at the fluorescence that comes from the probe. All other diffracted light is removed. - You can determine the relative localisation of molecules to one another within tissue sections (multiple probes) as they show up as a colour.

What are the 5 types of light microscopy?

1) Bright field 2) Phase contrast 3) DIC 4) Fluorescence 5) Confocal

What's an annular ring?

A blacked out ring with a hollow circular centre.

What is a prism?

A block made of 2 quartz wedges that split a beam of polarized light into 2 beams travelling very close together.

How do confocal microscopes work?

A fluorescent specimen is illuminated with a focus point of laser light from a pin hole. Emitted fluorescent light from in-focus point is focused at pinhole and reaches the detector. Emitted light from the out of focus point is out of focus at the pin hole, and is largely excluded from the detector. Fluorescence comes from above and below the plane of focus.

What is the method for making a fluorescent probe?

A primary (rabbit) antibody is directed against an immobilized antigen A. Secondary antibodies (marker-coupled antibodies) are directed at the rabbit antibodies. This creates a marker.

What did old light microscopes look like?

An oil lamp (light source) was placed behind a water flask (that acted as a condenser lens to focus the beam of light onto the specimen). In 1866, Zeiss made lenses allowing resolution at the theoretical limits.

What does the selective barrier filter out?

Anything other than light.

What can be viewed with a light microscope?

Between 0.2mm and 0.2µm.

What can be viewed with an electron microscope?

Between 100µm and 0.2nm.

What is confocal microscopy used for?

Co-localisation, intra-cellular studies, examination of thick specimens, and studies where the 3D structure of the sample is important.

What is confocal microscopy?

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM), is 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 are the other engineered types of GFP?

Cyan, green, yellow and red derivatives are available.

How can enzyme substrates reveal the site of activity?

Enzyme substrates can be linked to coloured products, revealing the site of activity. E.g. an artificial substrate reveals protease activity from a pathogenic fungus infecting insect cuticle with an orange product.

How are samples prepared?

It depends on the material. Single cells can be viewed direct or after staining. Tissues may need to be sectioned. Cellular compartments, cell types and cell viability can be viewed using dyes, chromogenic enzyme substrates or dyes-included/excluded.

How can dyes show living or dead cells?

Neutral red is taken up by living cells and accumulates in the vacuole. Dead cells have a damaged vacuolar membrane, so the dye isn't retained. Living cells show that the vacuoles have taken up the dye.

Why do confocal microscopy images have high resolution?

Only the plane that is in focus is visible. This is relative to standard fluorescence microscopy.

What are the features of phase contrast light microscopy?

Optics translate minute variation in place to changes in amplitude; appears as contrast. There is a grey background, so you can see the image better. Below the stage, there's an annular ring. Instead of the light being a solid cone of light, it's a hollow cone of light. This is focused on the specimen. Any light that doesn't hit the specimen passes through, creating the contrast.

What determines the resolution of each particular microscope?

Resolution is determined by wavelength of radiation source used for illumination - electrons vs light.

Who was the first person to come up with the idea of a cell?

Robert Hooke (1635-1763) was the first person to observe plant tissue with a microscope. He came up with the idea of a cell.

How can auto-fluorescence reveal certain cell components/types?

Some compounds have the ability to auto-fluoresce if you excite them with the right wavelength, e.g. lignin is found in a structural secondary walls of plants (root epidermis and hypodermis) is bright white and Avenacin (an antifungal compound in the epidermis of oat roots) is blue.

What is vital staining?

Staining that determines whether a cell is alive or dead (e.g. the stain is a different colour if the cell is dead).

Describe the optical pathway in a compound optical microscope.

The lamp field stop and the condenser work together to control the light beam. These must be aligned to focus a cone shaped beam of light onto the specimen in order to see a sharp image. Light is collected by the objective lens to create the image, which is reflected onto the eyepiece which directs the light into your retina in the eye so you can see the image.

What kind of things can be viewed with the naked eye?

The naked eye (with good eye site) can see just less than 1mm and above.

What is fluorescence?

The property of absorbing light of a particular wavelength and then emitting light of a different colour and wavelength.

What is the main difference between fluorescence microscopy and bright field light microscopy?

The use of selective filters and directing the excitation light through the objective lens. - The light source in fluorescence microscopes is to the side. - A mirror would reflect the beam downwards through the objective lens, to excite the specimen to make it fluoresce. - This is then reflected down the epi-fluorescence light path.

What are the features of confocal microscopy?

There's a focal plane in the specimen. The light that is collected is called the image volume (this is deeper than the focal plane). The microscope uses the laser to scan across the image, creating optical sections. Each of the images are collected on a computer. Each of the optical sections can be put together to create a 3D image.

What are histo/cyto-chemical dyes?

These stains are used to selectively stain cells and cellular components used to characterize tissues, e.g. plant root cells can be stained for phenolic compounds, and lignin can be identified in cell walls of root epidermis and hypodermis.

What do fluorescent probes do?

They can be attached to antibodies for detecting specific proteins within cells.

What do light microscopes look like now?

They have all the same components of the original light microscope - they're just more sophisticated. The quality of the lens has improved. The boltons (the add ons) have got much better.

How can cytoplasmic calcium be measured in transgenic plants expressing aequorin?

Using calcium sensitive fluorescent protein. The amount of fluorescence is proportional to the amount of Ca ions. Lots of fluorescence means there is lots of calcium present.

What are the features of bright field light microscopy?

You don't get much contrast as there's too much light density. The background is white. They show poor images of living cells.