Phase Contrast Dark Field Fluorescence

o Cells are made to fluoresce

by illuminating them with
light of a single color.
o Light does not pass through
the specimen (directed o Glow in a black
o Cells differ in refractive from the sides). background
index from surrounding. o Specimen appears light on o Naturally fluorescent:
a dark background chlorophyll
o Phase ring: amplifies this
difference o Stained with a fluorescent
o Often has better resolution
dye: DAPI - bright blue, it
o Obtain dark image on a than light
complexes with the cell’s
light background o Visualize microbial DNA
motility (bundles of o Visualize cells in their
flagella) natural habitats, such as
soil, water, food or in
clinical diagnosis.

Differential Interference Contrast (DIC) Confocal Scanning Laser (CSLM)

o Form of light microscopy
o Polarizer (in condenser) → Polarized light o Computer-controlled (laser + fluorescence
→ Prism → 2 beams* → Specimen →
microscope) – 3D
Objective lens → 1 beam o Access several planes of focus in the
*2 beams have different refractive indexes, specimen (1 layer in focus at a time)
combined beams are not in phase – interference ex: Bacterial biofilm
→ 3D perspective, which enhances subtle
o Light microscopy: only cells on the surface
differences in cell structure. are observed
o Visualize cellular structures: nucleus, o CSLM: various layers are observed
endospores, vacuoles, more 3D than other
forms of light microscopy + used on o Visualize thick specimens.
unstained cells.
Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM)
o Examine cell structure at very high
magnification and resolution.
o Visualize structures at the molecular level
(proteins, nucleic acid). o Specimen is coated with a thin film of a
𝝀 electrons <<< 𝝀 visible light heavy metal (gold).
o Resolving power of light microscope: 0.2 o Electrons scattered from metal are collected
μm and projected to produce 3D image.
o Resolving power of TEM: 0.2 nm o Wide range of magnifications can be
obtained (15 up to about 100,000)
o Thin sections of the cell are needed + treated
with stains (osmic acid, permanganate, o Only visualize the surface of an object
uranium…) scatter electrons → contrast ↑
o Intact cells or cell components can be
observed by negative staining.