Microscopes and Study of Microbes: L2

Units remember conversions. CM, MM, UM, NM

1cm = 10mm

1mm = 1000um

1um = 100nm

Magnification

The use of lens to enlarge an object.

As magnification increases we are enlarging the object and looking at it more closely.

Resolution

The ability to see two items as separate and distinct.

A microscope can theoretically have unlimited magnification but has a limited resolution. Needs both in a microscope.

The limit to resolution is due wavelengths of light having to be able to fit between two objects in order to differentiate them.

Contrast

The ability to see something as different from its surroundings
It depends on how the amount of light being transmitted by the object compared to its surroundings.

What are the 3 types of microscopes

1. Light microscope

2. Confocal

3. Electron

Light microscope

Its a compound microscope: Bright field microscope is one of them

Illuminator is the light source and the light goes through the CONDENSOR where the light rays gets directed through the specimen.

The light rays then go through the objective lens and the image is magnified by the ocular lens

What are the techniques to obtain optimal images on a compound microscope.

1. Kohler illumination

2. Immersion Oil

3. Staining

Kohler illumination

To obtain an even illumination of a specimen which increases CONTRAST. Basically the light is evenly spread among the specimens.

Immersion oil

This technique maximized RESOLUTION.

Since the oil has the same refractive index as glass, the oil fills up the space between the lens and the glass to prevent light rays from bending away from the lens.

Staining

Staining increases CONTRAST while maintaining high resolution.

Sometimes used to isolate parts of microbes to detect certain types.

Gram Stain

Gram staining was historically one of the first classification methods used with bacteria. It distinguishes Gram-positive (G+) and Gram-negative (G−) bacteria based on differences in their cell wall structures.

What are techniques used to increase contrast on Live cells

Bright-field microscopy

Phase-contrast

Microscope for contrast on Live cells

A Bright-field microscopy (this is a compound microscope) can see that some cells are larger and pigmented and this makes them easier to see

Phase contrast

The specimen and the background have different refractive indexes, which mean light passes through them differently.

A phase ring amplifies the differences and makes it more visible to see the difference between the background and the specimen.

How to increase contrast on Dead cells

Fluorescence microscopy

Fluorescence microscopy

Adding fluorescent molecules that bind to cells.

Confocal microscope

Specimens are dyed with fluorochrome dyes that bind to the cells. Then only small portions of the field are illuminated with light at a time. This produces a three dimensional image up to 100um.

What is electron microscopes

Its a microscope that gives a high resolution of the specimen and its a beam of electrons (Shorter wavelength).

Transmission Electron Microscopy (TEM)

A beam of electrons passes through the specimen then a electromagnetic lens films it.

In TEM, electrons pass through the specimen. Denser areas scatter more electrons, producing darker areas in the image.

Scanning Electron Microscopy (SEM)

A electron gun produces a beam of electrons that scans the surface of a whole specimen.

Secondary electrons being emitted from the specimen produce the image specifically a 3D image.