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# MICROSCOPY

"micro" (small)
"scopeo" (to watch)
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
MICROSCOPY
MICROSCOPY

1590 2018
PARTS OF A LIGHT MICROSCOPE
THE LIGHT

## Light: electromagnetic radiation of a wavelength that is visible to the human

Light can exhibit properties of both waves and particles (photons). This
property is referred to as wave–particle duality.

## Complex light: mixture of light rays with more different wavelengths.

THE LIGHT

Characteristic parameters
of light waves:
Wavelength: the distance between repeating units of a
propagating wave of a given frequency. (λ, expressed in nm).
Frequency: The number of oscillations within a minute.
Amplitude: distance from the center y position to the peak
COMPONENTS OF VISIBLE LIGHT
Major Imaging Functions of the Microscope

• Magnification

• Resolution

## • Capture and Display Images

Lenses work by refraction

Incident light

focus

Focal
length f
Light travels more slowly in matter
The speed ratio is the Index of Refraction, n

v = c/n

## n=1 n>1 n=1

Refractive Index Examples
• Vacuum 1
• Air 1.0003

• Water 1.333
• Cytoplasm 1.35–1.38 ?
• Glycerol 1.475 (anhydrous)
• Immersion oil 1.515

## • Fused silica 1.46

• Optical glasses 1.5–1.9

• Diamond 2.417

## Depends on wavelength and temperature

Working Distance
In an infinity-corrected optical system, a light beam emitted from a specimen passes
through the objective lens which does not form an image and enters as an infinity
parallel beam in the tube lens which forms an intermediate image. In a finite correction
optical system, the objective lens forms an intermediate image by itself.

## In general, high NA lenses have

short working distances
However, extra-long working
distance objectives do exist

Some examples:
10x/0.3 WD = 15.2mm
20x/0.75 WD = 1.0mm
100x/1.4 WD = 0.13mm
MAGNIFICATION
MAGNIFICATION
Total visual magnification of the microscope is derived by multiplying
the magnification values of the objective and the eyepiece.

## Ocular: 5-30 x magnification

Objective:
4-100 x magnification
Maximum
magnification:
3000X
The resolution ~0.2 to .25 mm

## Resolution: Ability of a lens to separate or

distinguish small objects that are close together.
(The resolution of our optical equipment is better
as closer points can be seen as separate ones.)

## •wavelength of light used is major factor in resolution

shorter wavelength  greater resolution
The resolution limit of a light microscope
The resolution of the microscope, () :

 wavelength
() = =
2 A numeric aperture

Distance objective
Higher is not good
Lower is good object

A = n·sinα
The letter n is the refraction index of the media between the cover slip
and the objective (air n=1, distilled water n=1.33, cedar oil n=1.51).

α labels the angle closed by the main optical axis and the outermost light
beam (half angle of the objective)
Possibilities of resolution improvement

## (i)Reduction of the numerator, i.e. application

of light beam with a shorter wavelength.
With UV light the resolution can be reduced to
0.1 μm, but special quartz lenses and UV-light
detector are needed, therefore the light
microscope with UV light source is only a
theoretical possibility.
Possibilities of resolution improvement
(ii) To increase the value of the numeric aperture (A=
n·sinα)
Increasing n (refraction index) :
The resolution of the microscope can be enhanced by dropping
a solution with higher refractive index between the front lens
and the coverslip. Those lenses (mainly objectives with 100x
magnification) are named as immersion objectives.
The immersion liquid mentioned above is cedar oil, thus these
lenses are objectives with oil immersion (they are labeled with
HI).

Increasing α:
The half angle of a lens can be increased only until 72°, since
at larger angle than this the light beams became totally
reflected.

## 1. Phase contrast microscopy

2. Fluorescence microscopy

## 3. Electron microscopy (SEM and TEM)

Phase contrast microscopy Fritz Zernike Nobel prize 1953
A large spectrum of living biological specimens are virtually
transparent when observed in the optical microscope under
brightfield illumination.

## Phase contrast microscopy improves contrast in unstained biological

specimens without significant loss in resolution, and is widely
utilized to examine dynamic events in living cells.
Phase contrast microscopy
In a phase-contrast microscope, the annular rings in the objective lens
and the condenser separate the light.
The light that passes through the central part of the light path is
recombined with the light that travels around the periphery of the
specimen.
The interference produced by these two paths produces images in
which the dense structures appear darker than the background.
Phase contrast microscopy
Fluorescence microscopy/
Laser scanning confocal microscopy
Fluorescence Microscopy

## Helps in the analysis of intracellular distribution of specific

macromolecules in sub-cellular assemblies, such as the nucleus,
membranes, cytoskeletal filaments, mitochondria, Golgi
apparatus, and endoplasmic reticulum.

## It is also useful to probe intracellular dynamics and the

interactions between various macromolecules, including diffusion,
binding constants, enzymatic reaction rates, and a variety of
reaction mechanisms, in time-resolved measurements.

## For example, fluorescent probes have been employed to monitor

intracellular pH and the localized concentration of important ions.
Fluorescence

Fluorescence - The process by which a suitable atom or molecule, which is transiently excited by
absorption of external radiation at the proper energy level (usually ultraviolet or visible light), releases the
absorbed energy as a photon having a wavelength longer than the absorbed energy. The fluorescence
excitation and emission processes usually occur in less than a nanosecond.
Stokes’
shift
Fluorochromes
Organelle/DNA/Ca2+ probes
Organelle probes:

## a fluorochrome nucleus attached to a target-specific

moiety that assists in localizing the fluorophore

## DNA: Acridine orange, Propidium iodide, DAPI,

Hoechst
Bovine pulmonary artery endothelial cell labeled
with probes to visualize mitochondria,
peroxisomes, and the nucleus. MitoTracker Red
CMXRos reagent. Peroxisomal labeling was
Ca2+: fura-2 and indo-1, fluo-3, fura red
achieved with a primary antibody directed
against PMP70, visualized using green-
fluorescent Alexa Fluor 488 goat anti–rabbit IgG.
The nucleus was stained with blue-fluorescent
DAPI.
Fluorescence Filter Cube Structure

## Exciter filter: permits only selected wavelengths from the illuminator to

pass through on the way toward the specimen.
Barrier filter: blocks the excitation wavelengths and permit only selected
emission wavelengths to pass toward the eye or other detector.
Dichromatic beam splitter (dichroic mirror): reflects excitation
wavelengths and passes emission wavelengths.
Fluorescence Microscope Structure

## Epi-illuminators usually consist of a mercury or xenon

lamphouse (or laser system) stationed in a port at the rear of
the microscope frame.

## Fluorescence illumination from the arc lamp passes through a

collector lens and into a cube that contains a set of interference
filters, including a dichroic mirror, barrier filter, and
excitation filter.
Fluorescence Microscope Structure
 Chromatic aberration
• Different colors get focused to different planes
• Not good…
Microscopy: the past and present

## intestine cross section,

haematoxilin/eosin staining

## intestine cross section,

multicolor fluorescence image
Microscopy: the past and present

## testis cross section,

haematoxilin/eosin staining

## testis cross section, multicolor

fluorescence image
How early viral DNA enters in cell nucleus? (IFA)
BrdU IFI16 DAPI KSHV/IFI16 Enlarged
Uninfected
KSHV 15 min
KSHV 30 min

44

## Ansari et al. PLOS Pathogen, 2015

Light reflected from the dichroic mirror is restricted in
wavelength by the excitation filter and enters the objective
(now acting as a condenser) to bathe the specimen with a cone
of illumination.

## Secondary fluorescence, emitted by the specimen, returns

through the objective, dichroic mirror and barrier filter before
being routed through the microscope optical train. The
microscope presented above contains a binocular observation
tube that is equipped with a port and extension tube for mounting
a traditional or CCD camera system (a Peltier-cooled CCD
camera is illustrated).
Immunofluorescence Microscopy
A targeted molecular species (protein, nucleic acid, membrane, etc.) in a specimen is labeled with
a highly specific fluorescent antibody. After the labeled antibodies have been excited by a selected
region of wavelengths, secondary fluorescence emission is gathered by the objective to form an
image of the specimen. Antibodies are labeled either by coupling directly with a fluorochrome
(fluorescent dye; termed direct immunofluorescence), or with a second fluorescent antibody that
recognizes epitopes on the primary antibody (indirect immunofluorescence).
Immunofluorescence Microscopy
Immunofluorescence Microscopy

MT
DNS
CS
Fluorescent Proteins
Green Fluorescent Protein (GFP) - A naturally occurring protein fluorescent probe derived from the
jellyfish Aequorea victoria, which is commonly employed to determine the location, concentration,
interactions, and dynamics of a target protein in living cells and tissues. The excitation and emission
spectra of enhanced GFP (a genetic derivative) have maxima at 489 nanometers and 508 nanometers,
respectively. In order to incorporate the GFP (or any of its genetic derivatives) into a cell, the DNA
sequence for the gene is ligated to the DNA encoding the protein of interest. After cultured cells have been
transfected with the modified DNA, they are able to express chimeric fluorescent proteins for observation
in the microscope. There are genetically modified variants of GFP such as blue fluorescent protein (BFP),
cyan fluorescent protein (CFP), yellow fluorescent protein (YFP)

## DsRed fluorescent protein

GFP-FUSION PROTEINS
gene of interest plasmid with GFP gene

fusion protein
emits green light
upon excitation
transfection by blue light,
intracellular
localization
determined

GFP

transcription,
translation
Light sources for fluorescence/confocal microscopy

As opposed to traditional arc-discharge lamps used with the shortest range (10-20 nanometers) bandpass
interference filters in widefield fluorescence microscopy, the laser systems used for fluorophore
excitation in scanning confocal microscopy restrict excitation to specific laser spectral lines that
encompass only a few nanometers.
Z-sectioning
Z-sectioning

FISH

## Fluorescence in situ Hybridization

(FISH) - The fluorescence FISH
technique is based on hybridization
between target sequences of
chromosomal DNA with fluorescently
labeled single-stranded
complementary sequences (termed
cDNA) to ascertain the location of
specific genetic sequences. In medical
practice, the most important
application of FISH is the prenatal
diagnosis of chromosome number
abnormalities (and other chromosomal
mutations).
Proximity Ligation Assay (PLA)
1. Highly sensitive fluorescence microscopy assay to study protein- protein interactions and
protein modifications
2. Will give signal only when the epitopes recognized are in the vicinity of <40 nm.
3. PLA detects endogenous levels of proteins, provides a method for the detection of weak or
transient interactions and gives the spatial distribution and localization of a single or multiple
proteins.

55
D
PLA [IFI16 M and R PLA [IFI16 M and R
abs]+KSHV+ DAPI abs]+KSHV Enlarged
Uninfected
Untreated
KSHV (30 DNA copy/cell)
C-646
Electron Microscope

## The limit of resolution of Light microscopy is

2000Å or 200nm, which is insufficient to
visualize cell organelles, viruses and
macromolecules of current interest.

## All these are possible with electron

microscopy.
COMPARISON
Electron Generation

## • Thermionic Electron Gun  Field Emission Gun

– Heated filament produces  A very strong electric field is
electrons used to extract electrons from a
• Typically made of Tungsten metal filament
or Lanthanum hexaboride  Filament typically a single
– Electrons drawn towards an tungsten crystal
anode  Requires a vacuum
– An aperture in the anode  Similar anode setup
creates a beam
ELECTRON MICROSCOPY
Elecron ray source (Electron gun)
Filament

Heat
Wehnelt cap
- -
(negative potential)
- -
- -
- -
- -
- e- -
e-
Space charge e-
e-

+++++++++++ +++++++++++
Anode (positive potential)
Transmission electron microscope (TEM)
Electron ray source

Condenser lenses

Condenser aperture

Sample
OBJECTIVE
Objective apertures

Intermediate lenses

PROJECTOR

Detector
Transmission electron microscope (TEM)
Scanning Electron Microscope (SEM)

## 1st condenser lens

Condenser aperture
2nd condenser lens

Objective aperture

Scan coils
Amplifier/Detector
OBJECTIVE

Backscattered
electrons 2nd electrons
Sample
Scanning Electron Microscope (SEM)