H-BP Microscopes

History

• Hans and Zacharias Janssen - “first” compound microscope

• Joseph Jackson Lister → Solved spherical aberration - caused by light passing through different parts of the same lens

• Ernst Abbe - Numerical Aperture of Condensor

• Carl Zeiss & Ernst Abbe - Objective of microscope

Resolution

• Between LM, CM, SEM, TEM; TEM is the best.

• Resolving power: the smallest distance between two particles at which they can be seen as seperate objects

• In a compound microscope wavelength limits resolution.

• Due to diffraction, the limit resolution is 0.2 micrometers.

Magnification

Ocular lens x objective lens

Mechanical System

Optical System

• Transmitted illumination: light is directed through the specimen from the base

• Vertical or Reflected Illumination: Light comes from above and reflects off the specimen

• Condenser: Lens system under the microscope stage that focuses light onto the specimen

Optical Principles

• The objective lens is a very high powered magnifying glass with a very short focal length → virtual, inverted and enlarged image

• Eyepiece is usually a compound lens → focus between the two lenses

• The image is viewed with eyes focused at infinity

Bright Field Microscopy

• When to use:

  • Viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms

  • only dark or strongly refracting objects

• Objects absorb light partially or completely

• Disadvantages:

  • Low optical resolution.

  • Diffraction limits resolution to approximately 0.2 micron.

  • Out of focus light from point outside the focal plane reduce image clarity

• How to enhance:

  • Oil immersion objective

  • Staining

  • Filters on the light source

Dark Field Microscope

• An opaque disc is placed under the condenser so that only light scattered by objects on the slide can reach the eye

• Used with low magnification (up to x100)

• Used in examining:

  • Urine for crystals (uric acid, oxalate)

  • Spirochetes (bacteria) (Trepenoma pallidum → syphilis)

  • suspensions of cells such as mushrooms (yeast), bacteria, small protists

  • cell and tissue fractions (cheek epithelial cells, blood cells)

  • determination of motility in cultures (moving independently using metabolic energy)

• Live and unstained samples, increase the contrast

• Must be strongly illuminated → damaging the sample

Phase Contrast Microscpy

• Preferable to BFM when high magnification is needed (400×, 1000x) but the specimen is colorless or the details are too fine → not enough contrast

• Uses:

  • Cilia and flagella

  • Amobea

• No need to stain

• How it works:

  • The change in phase can be increased to half a wavelength by a transparent phase-plate in the microscope and thereby causing a difference in brightness → object shines out in contrast to the surroundings

Differential Interferance Microscopy

• Produces an apparently 3D image of living cells and tissues

• Resembles phase-contrast but higher resolution.

• Uses polarizing lenses like the polarizing microscope → can be quantitative

• Halojen light beam is polarized, split by a beam splitter, and passed through the specimen

Invert Microscope

• Modified BFM for special uses

• Allow viewing of cells in flasks, welled-plates, or other deep containers

• Light source above specimen, objectives beneath stage

Stereo (Dissecting) Microscope

• Two compound microscopes which focus on the same point from slightly different angles

• Specimen viewed in 3 dimensions

• The image upright and laterally correct

• Both eyes can see the image

• Used for:

  • Surfaces of solid specimens

  • Sorting, dissection, microsurgery, watch-making, small circuit board manufacture or inspection

• Great working distance and depth of field! (higher the NA, the smaller the depth of field and working distance)

Fluorecence Microscope

• The specimen is illuminated with a specific wavelength of light which is absorbed by the fluorophores (fluorescent dyes that absorb excitation light at given wavelength), then emits light with a longer wavelength.

• UV → Visible light

• Brilliant, shiny particles on dark background

• The microscope has a strong UV light source, and special filters that eliminate UV light coming to the eye

• Photoluminence: Light energy, or photons, stimulate the emission of a photon

• Fluorescence: Type of photoluminescence where light raises an electron to an excited state. The excited state undergoes rapid thermal energy loss to the environment through vibrations, and then a photon is emitted.

Confocal Microscope

• A laser is focused at a plane in the specimen and scans the specimen in a horizontal plane

• Only light from the plane of focus reaches the detector

• The scanned image is digitally recorded, computer compiles images created from each point to generate a 3D image

• Images from consecutive focal planes can be recorded

• A very thin section within the tissue

• Used for:

  • Live cells

  • Fixed cells

  • Localize/measure enzyme activity

• Advantages:

  • Reduced blurring

  • Optical sectioning

  • highly sensitive photomultipliers → improved signal to noise ratio

  • 3D

  • Magnification can be adjusted digitally

• Disadvantages:

  • Slow scan

  • Limited use in dynamic tracking

  • Photobleaching or damage to living cells by laser

  • Lower resolution than camera detection

Transmission Electron Microscopy (TEM)

• Fixed, dehydrated specimens are embedded in a resin, hardened, sectioned, stained with heavy metals such uranium and lead, and inserted into electron column

• The electron beam is absorbed or deflected by the heavy metal stains and shadows are cast onto film or a phosphorescent plate (image is a shadow) at the bottom of the column

• Features:

  • 2D image

  • reveals internal structure

  • high resolution, high magnification

  • electron beam is focused by magnetic field

Scanning Electron Microscope

• Fixed, dehydrated specimens are mounted stubs and surface-coated with gold, palladium or rhodium.

• The specimen is placed in a vacuum and an electron beam scans back and forth over it

• Electrons that bounce off the metal-coated specimen surface are collected, converted to a digital image and displayed on a monitor.

• 3D image

• Electron beam is focused using a magnetic field

• Gives information about external topography of specimen

• Higher resolution and magnification than LM

• Can be used to observe individual atoms

Light VS Electron Microscope

Scanning Tunneling Microscopy

• An extremely fine conducting probe is held about an atom’s diameter from the sample

• Electrons tunnel between the surface and the tip, producing an electrical signal

• While it slowly scans across the surface, the tip is raised and lowered in order to keep the signal constant and maintain the distance

• This enables it to follow even the smallest details of the surface it is scanning.

Atomic Force Microsopy

• AFM consists of cantilever with a sharp tip at its end that is used to scan the specimen surface

• The cantilever is typically silicon or silicon nitride with tip radius of curvature on the order of nanometers

• When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever