Light Microscope

Dark Field Microscope

• What

• What part is different

  • Why

• How does it create an image

• Maximum magnification

• Resolution

• Advantages

• Ideal for

What: Specimen is brightly illuminated against dark background

Part is different: Condensor

• Why: Prevents the parallel and oblique rays from entering which makes the microscopic field dark

Creates an image by: In presence of specimen has a different refractive index

• Resulting in the oblique rays to scatter by reflection and refraction and enter the objective making the specimen brightly illuminated

Maximum magnification: 1500x

Resolution: 0.1 - 0.2µ

Advantages: Makes it easy to obtain the correct focal plane at low magnification

Ideal for: Small, low contrast specimens

What are the 5 uses of dark field microscope

• Morphology and motility of microorganisms

• Initial examination of cell suspensions

• Initial survey and observation at low powers of pond water samples

• Examination of lightly prepared slides

• Determination of motility in cultures

Brightfield vs Darkfield Microscopy

Darkfield has scattered light and central aperture

Dissecting / Inverted Microscope

• Designed for

• Uses what kind of light rays

• Magnification power

• Produces what kind of image

  • How

• How many light sources

Designed for: Low magnification observation of a specimen for dissection and parasitology

Uses: Reflected light rays from specimen surface

• Instead of transmitted light rays

Magnification power: Ranges from 5-80x

Produces: 3D image

• How: Because it contains 2 objective lens and eyepiece

  • This creates 2 separate optical paths for each eye

How many light sources: 2

• 1 from upper portion of specimen

  • Reflected in eyepiece

• Other from below portion of sample

  • For illumination through thinner samples

What is the difference between reflected light rays and transmitted light rays

• Sample type

• Light path

• Application

• Illumination method

	Transmitted Light Rays	Reflected Light Rays
Sample Type	Opaque	Transparent or semi-transparent
Light Path	Reflects off the surface	Passes through the sample
Application	Surface features	Internal structures
Illumination Method	Episcopic (reflected) illumination	Diascopic (transmitted) illumination

Phase Contrast Microscope

• What

• How

• Colour of background

• Advantages

• Disadvantages

What: Used to observe transparent and unstained specimens

How: By using the fact that structures have different refractive indexes causing them to bend light differently creating a contrast

• A phase plate slows down the highly refracted light rays putting them “out of phase” creating a contrast between the cell and background

Color of background: Dark

Advantages:

• Alive specimens can be used

• Can observe motility and stimuli responses

• No staining needed

Disadvantages:

• More expensive than bright field

• Must be properly aligned

UV Microscope

• What length of wavelengths lead to better resolution

• UV rays cannot pass through glass, so what lenses are used

• UV are invisible so how is an image displayed

• What other type of microscopes uses these principles

Better resolution: Shorter wavelengths

Lenses used: Quartz lenses

Image displayed by: Photographic plates or special filters

Other microscopes: Florescence

What is the phenomenon fluorescence?

When certain chemicals absorb light and reemit part of the radiant energy as light of longer wavelength

How does fluorescence microscope work?

• Light source

• Excitation filter

• Directing light to specimen

• Specimen staining

• Emission of fluorescent light

• Barrier filter

• Observation

• UV light option

1.Light source: High intensity mercury lamp which emits white light (containing all colors of spectrum)

2.Excitation filter: White light passes through the exciter filter, removing all colors except blue light

3.Directing light to specimen: Blue light is reflected downward onto specimen by dichroic mirror

4.Specimen staining: Stained with flourscent dye

• Parts of specimen retain dye causing it to

  • Absorb blue light

  • Emit green light

5.Emission of fluorescent light: Stained areas that emit green light travels back through microscope

6. Barrier filter: Emitted light passes through barrier filter which blocks unwanted blue wavelengths, only allowing the reemitted green light to reach observer

7. Observation: Sees the green fluorescence against a dark background.

• Unstained parts = remain invisible, providing high contrast.

8. UV light option: Sometimes UV light is used instead of visible light to excite fluorescent molecules

When is fluorescence microscope used?

In immunology to observe reactions of antigens and antibodies

Confocal Laser Scanning Microscope

• What is the light source and illumination

• Works in what mode

  • Meaning

• Spreading laser beam:

• Reflection by dichroic mirror:

• Fluorescence and pinhole aperture:

• Function of pinhole aperture:

Light source and illumination: Laser

Mode: Epi-illumination

• Meaning: Laser comes from above and shines directly onto sample

Spreading laser beam: Laser beam is widened using special lens to fill back part of the objective lens

• Allows light to focus as a small point onto sample

Reflection by dichroic mirror: A dichroic mirror is a mirror that reflects certain wavelengths but lets other pass through

• It directs the laser light to the sample while allowing fluorescent light to emit by the sample to pass through detector

Fluorescence and pinhole aperture:

• Sample absorbs laser light and re-emits it as fluorescence

• This passes back through objective lens

• Goes to detector through small pinhole aperture

Function of pinhole aperture: Ensures the only light from focused spot on sample is collected, eliminating out of focus blur (photo bleaching) that is normally generated by fluoresce microscope