Trends in Biotechnology TB4 Technological Advances in Microscopes 2016 1.

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Presentation transcript:

Trends in Biotechnology TB4 Technological Advances in Microscopes

Introduction This only looks at some aspects of the development of microscopy. To see more advances look at Nature Milestones in Light Microscopy /milelight/index.html /milelight/index.html 2

The reason people couldn’t understand the processes which are now used in biotechnology was because these processes often occur at the molecular level. 3

It was only in the early 1800’s that chemists realized that living things were made up of the same chemicals as non- living material. 4

It was very useful to have some way of examining biology at the smallest possible levels. Microscopy has developed and is still developing. This lets us study the processes of cells in more and more detail. 5

1595 Invention of the microscope We saw a brief history of the microscope in the last lesson. 6

1858, First histological stain, Synthesis of fluorescein When we use a microscope we are really studying the interaction of light with matter. 7

When colored chemicals were added to the specimen some things may be colored more than others. This is differential staining. Different types of stain would color some things more than others. Eg carmine - nucleus and nuclear granules 8

New colored chemicals were being produced because of a big interest in synthetic chemistry. Some stains were acidic and some stains were basic. 9

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The first fluorescent dye, fluorescein, was developed in

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Diffraction limit theory Researchers wanted to study living cells in the tiniest molecular detail. In 1873, Ernst Abbe showed a physical limit for the maximum resolution of traditional optical microscopes. The resolution could never be better than 0.2 micrometers. 16

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Compound Light Microscopes Uses light Has two lenses Magnification limited to 2000x 18

Monocular Compound Microscope at-gifs/products-small/ms- 351.jpg Binocular Compound Microscope com/Rev3.jpg 19

Electron Microscope Developed in the 1930s The electron microscope allowed for higher magnification Used electron beams (instead of light) and focused with an electromagnet (no lenses) 20

Electron Microscope The light microscope produces magnifications up to 2000X The electron microscope produces images that are magnified up to X or higher The electron microscope allowed scientists to see better quality images at higher magnification 21

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Scanning Electron Microscope (SEM) Electrons are reflected from the surface of the specimen Produces a 3-d image Good for the thicker specimens Lacks the magnification and resolution of the transmission electron microscope 23

Electron Microscope allaboratory/CM120/CM120.html Termite Head: amos/SIP.html 24

Spider

Transmission Electron Microscope (TEM) Uses beams of electrons Magnification of x Has two limitations: Good only for thin specimens Only dead cells can be observed 26

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1911, 1929, First fluorescence microscope, First epifluorescence microscope, The dichroic mirror The development of dyes was very useful to study the parts of cells. But these required the absorption of light which provide problems with measurement. Fluorescent dyes overcome this problem because we just measure the intensity of the emitted light. The intensity is directly proportional to the amount of excited dye. 28

Fluorescence Epiflourescence is useful because most of the excitation light is transmitted through the specimen. Only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal-to-noise ratio (you mostly see the light you want not, not much unwanted light). 29

Schematic of a fluorescent microscope 30

Source of fluorescence There are several methods of creating a fluorescent sample; the main techniques are labeling with fluorescent stains or, in the case of biological samples, expression of a fluorescent protein. Alternatively the natural fluorescence of a sample (i.e.,autofluorescence) can be used. 31

Source of fluorescence In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins or other molecules of interest. As a result there is a diverse range of techniques for fluorescent staining of biological samples. 32

Biological fluorescent stains Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules which are fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains which bind the minor groove of DNA, thus labeling the nuclei of cells. 33

Ethidium bromide 34

Biological fluorescent stains Other fluorescent stains are drugs or toxins which bind specific cellular structures and have been made with a fluorescent reporter. A major example of this class of fluorescent stain is phalloidin which is used to stain actin fibres in mammalian cells. 35

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Previous page Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial cells 37

Biological fluorescent stains There are many fluorescent molecules called fluorophores or fluorochromes which can be chemically linked to a different molecule which binds the target of interest within the sample. Eg. Fluorescently labeled antibodies. 38

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Immunofluorescence This technique uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. 40

Immunofluorescence A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. 41

Two interphase cells with immunofluorescence labeling of actin filaments (purple), microtubules (yellow), and nuclei (green). 42

a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody can be used. 43

Indirect immuno-cytochemistry This detection method is very sensitive because the primary antibody is recognized by many molecules of the secondary antibody. The secondary antibody is linked to a marker molecule that makes it detectable. 44

1972, Fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) FCS lets us see and measure fluorescence- tagged molecules in the biochemical pathway in intact living cells. This "in situ or in vivo biochemistry" means we can trace the biochemical pathway in intact cells and organs. 45

FRAP lets us see and measure the movement of proteins within and between cellular membranes, regions of the cytoplasm, mitotic spindle, nucleus, or another cellular structure, at micrometre-scale resolution. 46

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1994 – Green Fluorescent Protein (GFP) 48

GFP is a protein which is fluorescent. The gene encoding GFP can be cloned. GFP fluoresces when expressed in living organisms. It is useful as an in vivo tag for proteins. 49

GFP can be expressed from the promoter of a gene. We can see the time and place of the gene’s expression gene-expression patterns. 50

Engineered native GFP to become brighter, more photostable and excitable at a good wavelength. 51

Development of GFP variants to produce blue, cyan and yellow fluorescent proteins follow multiple tagged proteins in cells and organisms 52

Brainbow 53

Breaking the diffraction limit: STED, 2006 – Breaking the diffraction limit: PALM/STORM 54

Remember that Abbe showed a physical limit for the maximum resolution of traditional optical microscopes. The resolution could never be better than 0.2 micrometers. 55

But Abbe did not consider at least two things: Fluorescence and Computers 56

Stimulated emission depletion (STED) microscopy Developed by Stefan Hell in Two laser beams are used; one stimulates fluorescent molecules to glow, another cancels out all fluorescence except for that in a nanometre-sized volume. 57

Stimulated emission depletion (STED) microscopy Scanning over the sample, nanometre for nanometre, gives an image with a resolution better than Abbe's limit. 58

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Single-molecule microscopy Eric Betzig and William Moerner. The fluorescence of individual molecules are turned on and off. Scientists image the same area multiple times, letting just a few molecules glow each time. Adding these images gives a dense super- image resolved at the nanolevel. 60

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The following image is a 3-D version of PALM showing microtubules in a cell from a fruit fly. The tubules are labeled for depth, with red lower and blue and violet higher. A conventional optical microscopy image of the same cell (left) is shown for comparison. 62

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There are now several methods which can provide super-resolution images. Super-resolution techniques are revolutionizing our understanding of cell biology. 64

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Previous slide Nucleus of a bone cancer cell: normal high resolution fluorescence microscopy, cannot show details of its structure (left). two Color Localization Microscopy 2CLM (image on the right) shows 70,000 histone molecules (red) and 50,000 chromatin remodeling proteins (green) in a field of view of 470 µm 2 with an optical depth of 600 nm. 66

Further developments Now we can also see live cell processes at excellent resolution. l-videos-pushing-the-limits-of-live-cell- microscopy/ 67

This video shows actin fibers (orange- red), which are key components of the cell’s cytoskeleton, slowly pulling clathrin-coated pits (green), which are basket-like structures containing molecular cargo, away from the cell’s external membrane and deeper within the cell. 68