Introduction to light microscopy
All living organisms consist of cells.
Cells contain thousands of proteins and other molecules partitioned
into various compartments (organelles).
In each compartment these molecules form a complex ‘mess’
and are constantly interacting with each other.
from: http://www.expasy.org/tools/pathways/
Introduction to light microscopy
To understand how organisms or cells function it is critical to identify
the cell type under study and to know which cells or molecules are
where (morphological organization) and when (temporal dynamic)
they are there.
Until now the main instrument to study this, is the light microscope
invented by Antony van Leeuwenhoek (1632 – 1723).
Introduction to light microscopy
Since these days light microscope have evolved a lot….
Introduction to light microscopy
Light microscopy is still limited by the same physical principles.
Most importantly, spatial resolution is determined by the wavelength
of the radiation used for visualization.
Wavelength of visible light ranges from 400 – 700 nm and thus the
spatial resolution of any light microscope is about 0.3 μm.
Introduction to light microscopy
How does the spatial resolution of a light microscope (ca. 0.3 μm)
compare with interesting biological structures?
Cell diameter: 5 – 100 μm
Bacterium diameter: 0.2 – 2 μm
Synapse diameter: 0.1 – 0.5 μm
Synaptic vesicle diameter: 50 nm
Cell membrane thickn.: 7 – 10 nm
Proteins diameter: 4 - 20 nm
DNA strand diameter: 2 nm
Introduction to fluorescence microscopy
20 years ago light microscopy seemed to be outdated…..
Since then it has gained tremendous popularity again,
mainly due to several technical innovations:
Epifluorescence microscopy
Confocal microscopy
Multiphoton microscopy
What is fluorescence?
Fluorescence is the ability of molecules to emit a photon of lower
energy after absorption of a quantum of light.
The emitted light has a longer wavelength than the light used
for excitation.
What is epifluorescence microscopy?
In an epifluorescent microscope, broad spectrum excitation light is
directed onto the specimen through the same light path (objective)
used to image the fluorescence emitted by the specimen.
Excitation and emission light are separated by a dichroic mirror.
How do biological structures become fluorescent?
In few cases molecules are naturally fluorescent (autofluorescence).
Usually fluorescent molecules – also called fluorophores – are
experimentally attached to the molecules or structures under study.
The main approaches used for this today are:
Attaching fluorphores
to fixed tissue
Expressing fluorphores
in living tissue
Fluoresceine (FITC)
Alexa Fluor 488
Hoechst 33258
GFP
How is specificity achieved in fluorescent microscopy?
Some fluorophores have a high affinity to certain molecules (Hoechst
33258 selectively intercalates into DNA).
Usually fluorophores are attached to other molecules that specifically
bind to certain molecules. Such ‘carrier’ molecules are antibodies
(bind to peptides and proteins), lectins (bind to carbohydrates) and
toxins (bind to diverse targets – e.g. phalloidin binds to f-actin) .
Mammalian antibody
D. biflorus seed lectin
What is the advantage of fluorescence microscopy?
Fluorescence microscopy has several key advantages compared to
conventional light microscopy:
Structures/molecules are located with high sensitivity and specificity.
The location of different structures/molecules can be correlated by
using fluorphores with different excitation and emission spectra.
Fluorescence microscopy has evolved into confocal and multiphoton
microscopy….
What do we study?
Our main experimental animal is the Caribbean spiny lobster
Panulirus argus. We study its sense of smell (= olfaction).
The ‘nose’ is represented by a tuft of
sensory hairs called aesthetascs on the
outer branch of the 1st antenna.
The brain contains a large area
devoted to the processing of
olfactory input from the ‘nose’
What do we study?
We are specifically interested in understanding the generation of new
neurons during adulthood (= adult neurogenesis). Adult neurogenesis
persists in the ‘nose’ and the olfactory midbrain of the spiny lobster.
Olfactory sensilla on ‘nose’
Olfactory midbrain
Let’s look at some epifluorescent micrographs
These micrographs show a section through the brain of the spiny
lobster labeled with an antibody against a synaptic protein (antisynapsin) coupled to the fluorophore CY3 (red) and the nuclear
marker Hoechst 33258 (blue)..
….and some more epifluorescent micrographs
These micrographs show another section through the brain of the spiny
lobster labeled with an antibody against a neuropeptide (anti-orcokinin)
coupled to the fluorophore CY3 (red), a lectin (wheat germ agglutinin)
coupled to the fluorophore Alexa-488 (green) and the nuclear marker
Hoechst 33258 (blue)..
Introduction to confocal microscopy
A confocal microscope is an epifluorescence microscope in which a
small pinhole in front of a light detector only lets pass the light emitted
from the focal plane. Point-wise illumination by a laser achieves high
excitation energy without heating up (boiling) the specimen (section).
Laser: Argon and Helium-Neon lasers
are used providing distinct excitation
lines at 458, 488, 543, 633 nm, etc.
Scanner: galvanometer-driven mirrors
provide point-wise deflection of the
laser beam – up to 2048x2048 points
z-Control: a piezo drive allowing to
change the ‘hight’ of the section in
small increments (sub μm) thus
allowing the generation of stacks of
confocal sections
Advantages of confocal microscopy
The main advantage of confocal microscopy is that each ‘optical
section’ only contains light from the focal plane – it is in focus.
The second advantage is that registered stacks of ‘optical sections’
can be generated allowing 3-D reconstruction of the imaged section.
Example of confocal stack – original data set
Stack of optical sections through clump of cells associated with
the neuronal stem cell in the lateral soma cluster of the olfactory
midbrain collected with the LSM 510 confocal microscope (Zeiss)
3-D reconstruction of confocal stacks
One possibility to obtain a 3-D visualization of a stack of optical
sections is to look at it from different angles and combine these
views into a short movie. These images are from tegumental glands
associated with the olfactory sensilla of the spiny lobster.
Introduction to multiphoton microscopy
The latest development in the field of epifluorescence microscopy is
multiphoton microscopy. Utilizing non-linear quantum physics,
fluorescence is elicited by absorption of 2 or more quanta of longwavelength light provided by ultrashort (fs) laser pulses.
Advantages of multiphoton microscopy
One advantage of multiphoton microscopy is that fluorescence is only
elicited from the focal plane eliminating the need of a confocal pinhole.
The main advantage is that long-wavelength excitation light causes
less photo damage and penetrates deeper into tissue. Thus multiphoton
microscopy can be used in living organisms.
Examples of multiphoton microscopy
Examples for the current use of multiphoton microscopy from other
labs. The first example demonstrates the motility of microglial cells
in the mouse brain. The second example shows the differentiation
of newborn neurons in the olfactory bulb of adult mice.
From: Davalos et al., Nat.Neurosci. 8:752-758, 2005
From: Mizrahi, Nat.Neurosci. 10:444-452, 2007
Some interesting links for further information
about biochemical pathways in cells
http://www.expasy.org/tools/pathways/
about fluorescence microscopy
http://micro.magnet.fsu.edu/primer/index.html
about fluorescence and fluorophores
http://probes.invitrogen.com/resources/education/