Exercise 3

SPRING 2015

Let’s begin by doing some review questions! Log into www.labclicker.com/lab

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Microscopy allows us to examine very small structures in various conditions within a living cell. in vivo: The structure or process of interest is studied within the living cell. in vitro: The structure of interest is separated from the cell. in situ: The structure of interest is studied while in the same relative position within the cell as it was when the cell was alive.

What We Can Do with Microscopy

Learning Objectives

•  Perform different types of Microscopy –  Brightfield (what you did last week) –  Darkfield –  Phase contrast –  Fluorescence

•  Advantages/disadvantages of different types of microscopy •  Identify cellular components of cells •  Acquire digital images

Procedures

•  Procedures 3.1 to 3.3: Cheek cells –  Brightfield without stain –  Brightfield with stain –  Then using fluorescent stains

•  DAPI •  Texas Red- Phalloidin

•  Procedure 3.4: Bovine cells (BPAE) –  First without fluorescence excitation light –  Then using fluorescent stains

•  DAPI •  Texas Red® -phalloidin •  BODIPY® FL

Procedures- Optical Microscope

•  Procedures 3.5 to 3.7: Diatoms –  Brightfield –  Darkfield –  Phase Contrast

How can you improve the contrast of a biological specimen viewed through an optical microscope?��� ���A. By staining the specimen���B. By changing how the microscope interacts with the light that passes through the specimen C. By increasing magnification D.  A and B E.  A,B and C

Staining can kill…. What if you want contrast and want to keep your sample alive?

Problem with stains •  Specimens prepared with stains usually are

killed. •  Stains may add artifacts to cell structures that

don’t normally exist in the live cells.

Darkfield Microscopy

Darkfield: The Light approaches the specimen at a very wide angle. Image: Formed only by light that has been scattered by the specimen.

Darkfield ring

Amoeba Mysis zooplankton

Darkfield Microscopy

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Phase Contrast Microscopy

Frits Zernike (1888-1966)

Small phase shifts of the light waves passing through a transparent specimen are converted into amplitude (i.e., contrast) changes in the image. No staining necessary to view the specimen, which facilitates live (i.e., in vivo) imaging!

Condenser: A transparent ring allows only a hollow cone of light to reach the specimen. Objective: A semi-opaque phase plate retards light that did not interact with the specimen. Image: Formed by light that has been diffracted by the specimen and by background light that is now ½ wavelength out of phase with the diffracted light as it passes through the phase plate.

Human glial cells grown in culture

Brightfield Phase Contrast

Phase Contrast Microscopy

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Untreated amoeba, observed by fluorescence microscopy

Amoeba treated with a fluorescently labeled antibody against microtubules, observed by fluorescence microscopy

Fluorescence Microscopy

Another way of staining a cell…

•  A fluorophore is a molecule which has the property of fluorescence.

•  A fluorophore absorbs electromagnetic radiation of some specific wave length and then emits radiation of some slightly longer, lower energy wave length.

•  A fluorophore can mark the location of the structure of interest.

Fluorescence

Excitation

Emission

For fluorescence to occur , a molecule must be capable of absorbing light of a relatively short exciting wavelength and emitting some of this energy as longer wavelength fluorescent light.

We need molecules that can fluoresce!

Absorbance

Fluorescent Labels

Fluorophores (or fluorochromes) are used as stains in fluorescent microscopy. These are molecules that “fluoresce”, i.e. they absorb a relatively short wavelength of light, become excited, and decay to the ground state by emitting a slightly longer wavelength of light.

One way it can lose this energy is to emit a photon of slightly lower energy (i.e., longer wave length)light.

DAPI (4',6-diamidino-2-phenylindole)

Emitted light

420 nm

Absorbed UV

365 nm

Fluorophores used in this lab

Fluorophore Absorbs Emits Examine

DAPI UV 365 nm

BLUE >420 nm

Nucleic acids

Texas-red

Yellow- Green 552

RED 570 nm

Actin

BODIPY® FL

BLUE 495 nm

Green 525 nm

Tubulin

•  The cytoskeleton is a network of fibers extending throughout the cytoplasm

•  It organizes the cell’s structures and activities, anchoring many organelles

•  It is composed of three types of molecular structures –  Microtubules –  Microfilaments –  Intermediate filaments

Tubulin dimer

25 nm

α β

Column of tubulin dimers

10 µm

http://www.youtube.com/watch?v=5rqbmLiSkpk&feature=fvsr

Note in video below- how microtubules are assembled in the cell- dynamic!

10 µm

Actin subunit

7 nm

How fluorophores are used to mark location of component of interest:

•  Some stain (bind to) cellular components directly. •  Others can be attached (conjugated) to non-

fluorescent molecules that bind to cellular components.

Fluorescent Labels

DAPI

Directly binds to A-T rich regions in DNA

Direct fluorescence

Conjugated fluorophore- binds to another molecule (which then binds to cellular component).

Texas Red ®

Phalloidin • A peptide isolated from the deadly Amanita phalloides (Death Cap Toadstool).

• Binds specifically to actin.

• It is NOT fluorescent.

• Can be conjugated with Texas Red!

Phalloidin is colored as red because it is conjugated to Texas Red!

Actin protein

Phalloidin bound to actin protein

Texas Red conjugated to Phalloidin

Immunofluorescence: Antibodies

Antibodies are proteins which are produced the body in response to a foreign substance, known as an antigen. An antibody recognizes the antigen and binds to it. Scientists can produce antibodies to target any macromolecule-like tubulin and even other antibodies!

Tubulin protein

Primary Antibody

BODIPY® FL conjugated to either primary or secondary antibody

Secondary Antibody

Direct and Indirect Immunofluorescence

Direct

Indirect

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Immunofluorescence Utilizes Fluorescent Labels

fluorophore

Direct

mouse anti-actin primary antibody

Indirect

fluorophore

mouse anti-actin primary antibody

goat anti-mouse secondary antibody

Actin Actin

Hank

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Advantages of direct •  shorter sample staining times •  simpler dual and triple labeling

procedures. Disadvantages of direct •  lower signal •  generally higher cost •  less flexibility and difficulties with

the labeling procedure.

Advantages of indirect •  greater sensitivity/amplification of the

signal (more than one secondary antibody can attach to each primary)

•  secondary antibodies are relatively inexpensive, available in an array of colors, and quality controlled.

Disadvantages of indirect Potential for cross-reactivity and the need to find primary antibodies that are not raised in the same species.

Autofluorescence

Endogenous, or autofluorescence in tissues can arise from a variety of biomolecules (including lignins, chlorophyll, carotene, and xanthophyll). For instance, chlorophyll has an absorption band in the blue and green excitation regions (around 450 nm) and emits red light (around 680nm).

Corn kernel (Zea mays)

Fluorescence Microscopy

Fluorescence Light Sources: To generate sufficient excitation energy, powerful light sources, such as high-energy short arc-discharge lamps, are used.

Fluorescence lamp

Digital Camera: Used to document images. Associated software allows editing and merging of acquired images.

CCD camera

Fluorescence Microscope •  The epi-fluorescence microscopes we will use

expose slide with light of specific wave lengths (UV, Blue, Green) from above.

•  Light that excites fluorophores enters specimen via objective.

•  Molecules in cells absorb the light and re-emit the light at a longer, lower energy wave length.

•  What is observed is just the fluorescence. Everything else remains black.

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Fluorescence Microscopy

Setup in Principle

Light Path Through a Epi-Fluorescence Microscope DAPI���Excitation: 365 nm----> Emission >420 nm

What color of light do we want a DAPI excitation filter to pass?������A. Blue only���B. Green only���C. Red only���D. UV only

Quiz Question

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Beware of Photobleaching!

0 min

Epidermis fibroblast cells after various times of exposure

2 min 4 min

6 min 8 min 10 min