Exercise 3

SPRING 2015

Lets begin by doing some review questions!

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What We Can Do with Microscopy

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.

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
dont 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 Microscopy

Amoeba

Mysis zooplankton

Phase Contrast Microscopy

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!

Frits Zernike (1888-1966)

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
wavelength out of phase with the diffracted light as it
passes through the phase plate.

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Phase Contrast Microscopy

Brightfield

Phase Contrast

Human glial cells grown in culture

Fluorescence Microscopy

Untreated amoeba, observed

by fluorescence microscopy

Amoeba treated with a

fluorescently labeled antibody
against microtubules, observed
by fluorescence microscopy
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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

We need molecules that can fluoresce!

Excitation
Emission
Absorbance

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.

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)

Absorbed UV

Emitted light

365 nm

420 nm

Fluorophores used in this lab

Fluorophore

Absorbs

Emits

BLUE

>420 nm

Examine

Nucleic acids

DAPI

UV

365 nm

Texas-red

YellowRED

Green 552
570 nm

Actin

BODIPY FL

BLUE

495 nm

Tubulin

Green

525 nm

 The cytoskeleton is a network of fibers extending throughout

the cytoplasm

It organizes the cells structures and activities, anchoring many
organelles

It is composed of three types of molecular structures

Microtubules

Microfilaments

Intermediate filaments

10 m

Note in video below- how

microtubules are assembled in the
cell- dynamic!

Column of tubulin dimers

25 nm

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

Tubulin dimer

10 m

Actin subunit

7 nm

Fluorescent Labels

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



Some stain (bind to) cellular components directly.

Others can be attached (conjugated) to nonfluorescent molecules that bind to cellular
components.

DAPI

Directly binds to A-T rich regions in DNA

Direct fluorescence

Texas Red

Conjugated fluorophore- binds to another molecule

(which then binds to cellular component).

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!

Texas Red
conjugated to
Phalloidin

Phalloidin bound
to actin protein

Actin protein

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!

BODIPY FL

conjugated to either primary
or secondary antibody

Secondary
Antibody

Indirect

Primary
Antibody

Direct

Tubulin protein

Direct and Indirect

Immunofluorescence

Immunofluorescence Utilizes
Fluorescent Labels

Direct

Indirect
fluorophore

fluorophore

mouse anti-actin
primary antibody

Actin

goat anti-mouse
secondary antibody

mouse anti-actin
primary antibody

Actin

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Hank

29

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

Corn kernel (Zea mays)

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).

Fluorescence Microscopy

CCD
camera

Digital Camera:

Used to document images.

Associated software allows editing and
merging of acquired images.

Fluorescence
lamp

Fluorescence Light Sources:

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

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.

Fluorescence Microscopy

Setup in Principle

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Light Path Through a Epi-Fluorescence Microscope DAPI

Excitation: 365 nm----> Emission >420 nm

Quiz Question

What color of light do we want a DAPI
excitation filter to pass?

A.
B.
C.
D.

Blue only
Green only
Red only
UV only

Beware of Photobleaching!

Epidermis fibroblast cells after various times of exposure

0 min

2 min

4 min

6 min

8 min

10 min

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