What I will discuss <ul><li>how living/non-living, organic/inorganic specimens, absorb and subsequently re-radiate light. </li></ul><ul><li>We will learn that fluorescence microscopy is basically a method of studying material which can be made to fluoresce, either in its natural form or when treated with chemicals capable of fluorescing. </li></ul><ul><li>Some Examples </li></ul>
A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities. conventional microscope fluorescence microscope <ul><li>uses light to illuminate the sample and produce a magnified image of the sample. </li></ul><ul><li>uses a much higher intensity light to illuminate the sample </li></ul><ul><li>This light excites fluorescence species in the sample, which then emit light of a longer wavelength. </li></ul><ul><li>A fluorescent microscope also produces a magnified image of the sample, but the image is based on the second light source </li></ul>
<ul><li>general term applied to all forms of cool light </li></ul><ul><li>that does not derive energy from the temperature of the emitting body </li></ul>TV Screen Sonoluminescence is the emission of light by bubbles in a liquid excited by sound.
Fluorescence v/s Phosphorescence <ul><li>If the luminescence is caused by absorption of some form of radiant energy, such as ultraviolet radiation or X rays (or by some other form of energy, such as mechanical pressure), and ceases as soon as (or very shortly after) the radiation causing it ceases, then it is known as fluorescence . </li></ul><ul><ul><li>If the luminescence continues after the radiation causing it has stopped, then it is known as phosphorescence . . </li></ul></ul>The term phosphorescence is often incorrectly considered synonymous with luminescence
Basic Concepts <ul><li>let excitation light radiate the specimen </li></ul><ul><li>then sort out the much weaker emitted light to make up the image. </li></ul><ul><li>the fact that the emitted light is of lower energy and has a longer wavelength is used. </li></ul><ul><li>The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast </li></ul>
Why Prepare the Specimen <ul><li>Most Cells are colourless by nature </li></ul><ul><li>Cellular components do not fluoresce themselves. Fluorescent markers are therefore introduced. </li></ul>
Preparation of Specimen Tagging of Proteins Fluorescent Dyes Immunofluorescence Techniques Fluorescent Dyes Immunofluorescence Tagging of Proteins <ul><li>taken up by the cells </li></ul><ul><li>incorporated and concentrated in specific subcellular compartments </li></ul><ul><li>living cells are then mounted on a microscope slide and examined in a fluorescence microscope. </li></ul><ul><li>use of antibodies to which a fluorescent marker has been attached. </li></ul><ul><li>recognize and bind selectively to specific target molecules in the cell </li></ul><ul><li>modify cells so that they create their own fluorescing molecules </li></ul><ul><li>the location of that protein can be studied. It is also possible to watch the movements of the proteins and its interactions with other cellular components inside the cell </li></ul>
TYPES OF FLUOROPHORES USED <ul><li>fluorescein, </li></ul><ul><li>DAPI, </li></ul><ul><li>propidium iodide </li></ul><ul><li>green fluorescent protein (GFP) </li></ul><ul><li>Texas Red </li></ul>
<ul><li>irradiate the specimen with a desired and specific band of wavelengths </li></ul><ul><li>then to separate the much weaker emitted fluorescence from the excitation light </li></ul>
Cutaway diagram of a modern epi-fluorescence microscope
Working of the Fluorescence Microscope 1. Light source – epi-fluorescence lamphouse 2. Light of a specific wavelength (or defined band of wavelengths), is produced by passing multispectral light from an arc-discharge lamp through a wavelength selective excitation filter 3. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also termed a dichroic ) mirror or beamsplitter through the microscope objective to bathe the specimen with intense light
Working of the Fluorescence Microscope 4. If the specimen fluoresces, the emission light gathered by the objective passes back through the dichromatic mirror 5. It is Filtered by a barrier (or emission ) filter, which blocks the unwanted excitation wavelengths
Working in greater detail 1. Excitation light travels along the illuminator perpendicular to the optical axis of the microscope 2. The light then impinges upon the excitation filter where selection of the desired band and blockage of unwanted wavelength occurs.
3. Fluorescence emission produced by the illuminated specimen is gathered by the objective 4. Because the emitted light consists of longer wavelengths than the excitation illumination, it is able to pass through the dichromatic mirror and upward to the observation tubes or electronic detector. Working in greater detail
The Dichroic Mirror <ul><li>The excitation light reflects off the surface of the dichroic mirror into the objective. </li></ul><ul><li>The fluorescence emission passes through the dichroic to the eyepiece or detection system. </li></ul>dichroic, two color
The Dichroic Mirror dichroic, two color <ul><ul><li>Each dichroic mirror has a set wavelength value -- called the transition wavelength value -- which is the wavelength of 50% transmission. </li></ul></ul><ul><ul><li>reflects wavelengths of light below the transition wavelength value (90%) </li></ul></ul><ul><ul><li>transmits wavelengths above this value. (90%) </li></ul></ul><ul><ul><li>Ideally, the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission. </li></ul></ul>
Total Internal Reflection in Prism Same Principle used in Dichromatic beam splitter
Modern fluorescence microscopes are capable of accommodating between four and six fluorescence cubes. This is where the “turret’s” come into picture. The “cube” A specific combination of excitation filter, emission filter and dichroic mirror are needed
The Filters <ul><li>Excitation Filters </li></ul><ul><li>to select the excitation wavelength, an excitation filter is placed in the excitation path just prior to the dichroic mirror. </li></ul><ul><li>Emission Filters </li></ul><ul><li>In order to more specifically select the emission wavelength of the light emitted from the sample and to remove traces of excitation light </li></ul>Fig: Light path through the filter cube in a fluorescence microscope.
Stoke’s Shift <ul><ul><li>Excitation 495 nm </li></ul></ul><ul><ul><li>Emission: 520 nm </li></ul></ul><ul><li>The emission spectrum of an excited fluorophore is usually shifted to longer wavelengths when compared to the absorption or excitation spectrum </li></ul><ul><li>The intensity of the fluorescence is very weak in comparison with the excitation light (10 -3 to 10 -5 ). </li></ul><ul><li>The emitted light re-radiates spherically in all directions. </li></ul><ul><li>Dark background is required to enhance resolution. </li></ul>
Stoke’s Shift <ul><li>As Stokes' shift values increase, it becomes easier to separate excitation from emission light through the use of fluorescence filter combinations. </li></ul>Remember Dichoric Mirror ???
Data for Alexa Fluor 555 <ul><li>absorbs light in the yellow-green region </li></ul><ul><li>produces yellow-orange emission </li></ul><ul><li>to achieve maximum fluorescence intensity </li></ul><ul><ul><li>a fluorophore is usually excited at wavelengths near or at the peak of the excitation curve, </li></ul></ul><ul><ul><li>And detected at widest possible range of emission wavelengths that include the emission peak </li></ul></ul>
The radiation collides with the atoms in the specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. Principle of Fluorescence 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing.
Visualizing The Cytoskeleton using Fluorescence Microscopy An Example of Fluorescent Dyes
Two cytoskeletal elements examined: Why use Fluorescence Microscopy ? <ul><li>can visualize and localize individual proteins </li></ul><ul><li>within a cell. </li></ul>Actin Microtubules
Let us test the effects of different drugs on the cytoskeleton and cell shape Nocodazole prevents microtubule polymerization. Nocodazole Taxol binds and stabilizes microtubules, Taxol Latrunculin prevents actin polymerization. Latrunculin TPA/PMA causes a dramatic rearrangement of actin filaments TPA/PMA
Visualizing the cytoskeleton using fluorescence microscopy 1) Prepare samples: Fixation - kills and immobilizes cells A. aldehydes - cross-link amino groups in proteins (formaldehyde, glutaraldehyde) B. alcohols - denature proteins, precipitate in place (methanol) Permeabilization - detergents make proteins accessible to staining reagents (Triton X100)
2) Staining Actin - phalloidin covalently linked to rhodamine (red) - binds to filamentous actin only Microtubules - immunofluorescence 1 o ab: rabbit anti-tubulin; 2 o ab: fluorescein anti-rabbit
3) Fluorescence microscopy excitation emission fluorescent molecule wavelength ex em intensity
Microtubules = green DNA = blue interphase mitosis
Green Fluorescent Protein (GFP) An Example of tagging proteins
Green Fluorescent Protein (GFP) <ul><li>is found in a jellyfish </li></ul><ul><li>Why important - because it allows us to look directly into the inner workings of cells </li></ul><ul><li>It is easy to find out where GFP is at any given time: we just have to shine ultraviolet light, and any GFP will glow bright green </li></ul><ul><li>So, we can attach the GFP to any object that you are interested in watching say </li></ul><ul><ul><li>a virus. Then, as the virus spreads through the host, we can watch the spread by following the green glow. </li></ul></ul><ul><li>engineer the cell with the genetic instructions for building the GFP protein, and GFP folds up by itself and starts to glow. </li></ul>
These transgenic mice express enhanced green fluorescent protein under the control of a chicken beta-actin promoter and cytomegalovirus enhancer
Why do this ?? developing transgenic mice to identify critical neuronal subpopulations and target them for electrophysiological recordings and biochemical analyses.
Cotton A cross section of cotton stained with Rhodamine B. Mammalian Cells Fluorescence double-labeling of mammalian cells. The DNA in the cell nuclei are shown in blue. Cytoplasmic fiber structures (microfilaments) are shown in green. Photo: Petra Björk, Stockholm University
Researchers tag proteins with fluorophores to study the motion of these molecules. However, this creates an extremely complex motion picture (for example, in this image different colored particles move independently)
http://nobelprize.org/physics/educational/microscopes/fluorescence/fm.html Control of a fluorescence microscope
Figure 3: Problems with Fluorescence microscopy
Summary <ul><li>sample you want to study is itself the light source </li></ul><ul><li>study specimens, which can be made to fluoresce. </li></ul><ul><li>The sample can either be fluorescing in its natural form like chlorophyll and some minerals, or treated with fluorescing chemicals. </li></ul>
Future <ul><li>rapid expanding technique </li></ul><ul><li>in the medical and biological sciences. </li></ul><ul><li>certain antibodies and disease conditions or impurities in inorganic material can be studied with the fluorescence microscopy. </li></ul>
Fluoresence of chlorophyll-protein complex – Cytochrome b 6 f <ul><li>a protein containing a single chlorophyll a molecule is excited with polarized light </li></ul><ul><li>fluorescence is detected under 90O, with a polarizer either parallel or perpendicular to the original polarization direction. </li></ul>
Resources Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657. Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.