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 identifythe 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 microscopeinvented 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 wavelengthof the radiation used for visualization. Wavelength of visible light ranges from 400 – 700 nm and thus thespatial 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

Cell membrane thickn.: 7 – 10 nm

Synapse diameter: 0.1 – 0.5 μm

Bacterium diameter: 0.2 – 2 μm

DNA strand diameter: 2 nm

Proteins diameter: 4 - 20 nm

Synaptic vesicle diameter: 50 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20 years ago light microscopy seemed to be outdated…..Since then it has gained tremendous popularity again,mainly due to several technical innovations: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 lowerenergy after absorption of a quantum of light.The emitted light has a longer wavelength than the light usedfor excitation.

What is epifluorescence microscopy?

In an epifluorescent microscope, broad spectrum excitation light isdirected 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 – areexperimentally attached to the molecules or structures under study. The main approaches used for this today are:

Attaching fluorphoresto fixed tissue

Expressing fluorphoresin living tissue

Fluoresceine (FITC)

Hoechst 33258

Alexa Fluor 488

GFP

How is specificity achieved in fluorescent microscopy?Some fluorophores have a high affinity to certain molecules (Hoechst33258 selectively intercalates into DNA). Usually fluorophores are attached to other molecules that specificallybind to certain molecules. Such ‘carrier’ molecules are antibodies(bind to peptides and proteins), lectins (bind to carbohydrates) andtoxins (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 toconventional light microscopy:Structures/molecules are located with high sensitivity and specificity.The location of different structures/molecules can be correlated byusing fluorphores with different excitation and emission spectra.Fluorescence microscopy has evolved into confocal and multiphotonmicroscopy….

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 ofsensory hairs called aesthetascs on theouter branch of the 1st antenna.

The brain contains a large areadevoted to the processing of olfactory input from the ‘nose’

What do we study?We are specifically interested in understanding the generation of newneurons during adulthood (= adult neurogenesis). Adult neurogenesispersists 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 spinylobster labeled with an antibody against a synaptic protein (anti-synapsin) coupled to the fluorophore CY3 (red) and the nuclearmarker Hoechst 33258 (blue)..

….and some more epifluorescent micrographsThese micrographs show another section through the brain of the spinylobster 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 markerHoechst 33258 (blue)..

Introduction to confocal microscopyA confocal microscope is an epifluorescence microscope in which asmall pinhole in front of a light detector only lets pass the light emittedfrom the focal plane. Point-wise illumination by a laser achieves highexcitation energy without heating up (boiling) the specimen (section).

Laser: Argon and Helium-Neon lasersare used providing distinct excitationlines at 458, 488, 543, 633 nm, etc.

Scanner: galvanometer-driven mirrorsprovide point-wise deflection of thelaser beam – up to 2048x2048 points

z-Control: a piezo drive allowing tochange the ‘hight’ of the section insmall increments (sub μm) thusallowing the generation of stacks ofconfocal sections

Advantages of confocal microscopyThe main advantage of confocal microscopy is that each ‘opticalsection’ 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 withthe neuronal stem cell in the lateral soma cluster of the olfactorymidbrain collected with the LSM 510 confocal microscope (Zeiss)

3-D reconstruction of confocal stacksOne possibility to obtain a 3-D visualization of a stack of opticalsections is to look at it from different angles and combine theseviews into a short movie. These images are from tegumental glandsassociated with the olfactory sensilla of the spiny lobster.

Introduction to multiphoton microscopy

The latest development in the field of epifluorescence microscopy ismultiphoton microscopy. Utilizing non-linear quantum physics, fluorescence is elicited by absorption of 2 or more quanta of long-wavelength light provided by ultrashort (fs) laser pulses.

Advantages of multiphoton microscopy

One advantage of multiphoton microscopy is that fluorescence is onlyelicited from the focal plane eliminating the need of a confocal pinhole.The main advantage is that long-wavelength excitation light causesless photo damage and penetrates deeper into tissue. Thus multiphotonmicroscopy can be used in living organisms.

Examples of multiphoton microscopy

Examples for the current use of multiphoton microscopy from otherlabs. The first example demonstrates the motility of microglial cellsin the mouse brain. The second example shows the differentiationof 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

http://probes.invitrogen.com/resources/education/

about fluorescence and fluorophores

http://micro.magnet.fsu.edu/primer/index.html

about fluorescence microscopy

http://www.expasy.org/tools/pathways/

about biochemical pathways in cells

Some interesting links for further information