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:

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 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: 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Introduction to fluorescence microscopy 20 years ago light microscopy seemed to be outdated…..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 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) Hoechst Alexa Fluor 488 GFP

How is specificity achieved in fluorescent microscopy? Some fluorophores have a high affinity to certain molecules (Hoechst 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 antibodyD. 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 1 st 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 (anti- synapsin) coupled to the fluorophore CY3 (red) and the nuclear marker Hoechst (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 (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 long- wavelength 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: , 2005 From: Mizrahi, Nat.Neurosci. 10: , 2007

about fluorescence and fluorophores about fluorescence microscopy about biochemical pathways in cells Some interesting links for further information