NIKON SINGAPORE PTE LTD

Instruments Department

Basic Microscopy Techniques

Clement Khaw, Ph.D.

 From Genome to Human
DNA

Human
Genome
m-RNA

Ribosome Microscope
Protein

Proteome
Protein
Tissue
Cellome

Functional Protein
 What is a Microscope?

The microscope performs three basic tasks

• Produce a magnified image of the specimen

• Separate the details of the magnified image

• Render the details visible to the eye or camera

 Optical Techniques

• Brightfield
• Darkfield
• Phase
• DIC
• Epifluorescence
BRIGHTFIELD
 Brightfield
• Most common of all applications
• Amplitude objects usually exhibit high natural
absorption, reflection & contrast.
– Biological materials are stained to produce these properties
– Not recommended for unstained biological specimens or
transparent materials
Darkfield
 Darkfield
• a simple and popular method for making unstained
transparent specimens clearly visible. Such objects
often have refractive indices very close in value to that
of their surroundings and are difficult to image in
conventional brightfield microscopy.

• For instance, many small aquatic organisms have a

refractive index ranging from 1.2 to 1.4, resulting in a
negligible optical difference from the surrounding
aqueous medium. These are ideal candidates for
darkfield illumination.
Brightfield Darkfield
Phase Contrast
 Phase Contrast

• Why can’t we see living cells

in brightfield?
– Little contrast (2-5%)
• Very low light absorption
– RI of cells similar to
background
– RI within the cells between
cytoplasm and nucleus is
similar
 Phase Contrast
• Phase Objects- Transparent materials that absorb little light
but produce a phase change in the light as it passes through
the sample. The human eye cannot detect phase differences.
– Phase changes are primarily due to thickness and RI
variations in the specimen structure
– Live cells

• Contrast-Ability to distinguish specimen detail when compared

to the background or adjacent features
– Measured by the comparison between the highest and
lowest intensity in an image
– Positive contrast: Specimen darker than background
– Negative contrast: Specimen brighter than background
 Phase Contrast
• The specimen information is there but we cannot
see it.

• Two main obstacles to overcome

– Specimen information (higher orders of diffraction) is too
weak when compared to undiffracted signal
(background, 0 order)
– Convert small phase shift in specimen detail to large
changes in intensity to visualize
 Phase Contrast
Converts minute changes in phase to large changes in
amplitude (grey values) which are viewed as
differences in contrast.
Differential Interference Contrast
(DIC)
 DIC

• Observation of transparent or low contrast specimens

• Does not produce halo and has almost no affect on
objective performance in non DIC applications
• Phase Contrast Issues
– Halo obscures fine detail
– Phase plate reduces non phase specimen intensity by ~ 20-
30% depending on plate composition
– Image sharpness with phase objectives is compromised in
non phase applications
Phase Contrast DIC
 DIC
• DIC is a beam shearing interference system that
produces a shadow-cast 3D image.
• The shadow effect and contrast levels can be controlled
by rotation of the polarizer or position of objective prism
• DIC allows optical sectioning of the specimen
• The 3D image is an optical illusion and is primarily based
on refractive index and path difference
• Two types of prisms are used
– Nomarski (Wollaston variation-Nikon system) & Wollaston
– The shear is normally just below the resolution limit of the obj.
• Does not work with plastic dishes or wells
Phase Contrast DIC
Fluorescence
 Why use Fluorescence?
• Increased sensitivity
• Improved signal-to-noise
• Specificity
– Allows for imaging of single molecules
– Allows for labeling biological specific structures, proteins or
even genes
– Allows for tagging of multiple structures in one cell
• Viability
– Can be used in live cells and tissues
• Quantification
– Determine concentrations of specific proteins, calcium, or
measure pH
 Fluorescence
Def: The process by which a molecule absorbs the energy from
photons of a certain wavelength and then emits photons of
light with a lower energy (longer wavelength).
- Higher energy (UV) to lower energy (IR).
- Shorter wavelength (488nm) to longer wavelength (520nm)
- Higher frequency to lower frequency
> Emitted light is several orders weaker than the excitation energy
> Stokes shift - the difference between excitation and emission
peaks

Sir George Stokes

 How Fluorophores work

• A photon’s energy is rapidly absorbed (10-15 sec), and

shifts one of the fluorophore’s electrons from a ground
state to an excited state

• The electron then loses some of the gained energy

through smaller vibrational states (in the form of heat)

• Electron hangs in the excited state for 10-9 sec and

then emits a single lower energy photon
Excitation and Emission
Excitation Effect on Emission
 Fluorescence Phenomenon
• First discovered in materials like Quinine, Fluorspar and other
minerals (natural or “auto-fluorescence”).
• Seen in plant material (pollen grains, privet leaf)

• Certain molecules have fluorescence properties (Fluorophores)

• DAPI, FITC, TRITC, Alexa Fluors, Cy Dyes and can act as either
stains or be tagged to cellular structures.

• Fluorescence proteins (GFP) were discovered to occur in

jellyfish species in the 1950s but not utilized as a marker for
gene expression until the 1990s.
• Through molecular biology these proteins can be used to study gene
expression in non-fluorescence organisms.
• Recently fluorescence proteins have also been found in coral species
as well.
 The Eyes of Science

Fluorescent Protein
History
– 1962：
Dr. Shimomura isolated GFP from Aequorea victoria
Journal of Cellular and Comparative Physiology. 1962 Jun;59:223-39.

01
 The Eyes of Science

Fluorescent Protein
History
– 1992：
Dr. Douglas Prasher showed gene sequences of wild GFP (238 a.a.
∼ 26.9kDa). Gene 111 (2): 229-33, 1992.

01
 The Eyes of Science

Fluorescent Protein
History
– Martin Chalfie ～ 1994
Expression in heterogeneous cells.
It has become a common Fluorescence technique.

01
Fluorescent Protein

Roger Tsien
 The Eyes of Science

Fluorescent Protein

HEK 293 cell expressing GFP, YFP, CFP and RFP

Jean Livet et al., 2007, Nature 450:56-62
01
 The Eyes of Science

Fluorescent Protein

Agar Plate of Fluorescent Bacteria Colonies expressing various fluorescent proteins.

Roger Tsien’s Lab, UCSD 01

 The Enemy:
Photo-bleaching

Decrease in emission intensity after exposure

Exciting a molecule once has a probability Qb of killing it

Each molecule will emit only a finite number of photons

 Photo-bleaching
Photo-stability varies between dyes
 What to do about photo-bleaching?
• Select fade-resistant dyes
• Label densely
• Decrease bleaching by anti-fade mounting media
• Glycerol
• Oxygen scavengers
• Free-radical scavengers
Note: some anti-fade agents quench some dyes.
• Budget the photons you have
• Only expose when observing
• Minimize exposure time & excitation power
• Use efficient filter combinations
• Use highly QE, low noise camera
• Use simple light path
 Fluorescent Labeling of Cells

Small Molecule Dyes

– Fluorescent molecules which will bind to certain structures
in cells or other targets (e.g. DNA, Proteins, Lipid
Membranes) due to native structure or linkers
Immunofluorescence
- Fluorescence molecules which are bound to antibodies that
attach to specific proteins in the cells. (Alexa 488-Anti
Goat)
Fluorescence Proteins
- By cloning the genetic code to produce FPs into the cell it
will add the fluorescence molecule to the amino acid chain
of your protein of interest so it can be located in the cell.
Brainbow
Fluorescent Proteins – pros/cons

• Pros
– Can be easily introduced into live cells
– Minimally perturbative
– Photoactivatible/photoconvertible versions exist
– Avoids fixing / staining

• Cons
– Require genetically tractable system
– Folding and maturation can be slow
– Some are pH and Cl- sensitive
– Some have very complicated photophysics (strange
photoactivation / photobleaching behavior)
When it all works!
www.nikonimagingcentre.com.sg
http://www.microscopyu.com/

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NIKON SINGAPORE PTE LTD