DIC, fluorescence and confocal microscopy

Department of MechatronicsGIST

Yong-Gu Lee

References: 1. “Fundamentals of light microscopy and electronic

imaging,” Chapter 10,11,12, Douglas B. Murphy, Wiley-Liss, 2001

2. http://www.bio.unc.edu/courses/2005Spring/Biol188/

A DIC microscope is a polarizing microscope with condenser and

objective DIC Prisms

E-W

N-SAnalyzer

Compensator

Rotatable

Stage

Polarizer

Condenser

Objective

DIC Prism

DIC Prism

DIC (Differential Interference Contrast)

How does DIC differ from phase and polarizing?

Comparison of phase contrast to DIC for cheek cell

What are 3 major features of a DIC image?

• Contrast is directional: maximum in one direction and minimum in the orthogonal direction

• Contrast highlights edges; uniform areas have brightness of background

• In direction of contrast, one edge is brighter, the other darker than the background

The DIC microscope is a dual-

beam interferometer made with polarization

optics(shear =

0.15-0.6nm depending

on NA)

Shear

The DIC microscope is a dual-beam interferometer made with polarization

optics

The condenser DIC prism splits illumination light into 2 divergent

orthogonal polarized beams

Prism is oriented with the optic axes at 45o to polarizer.why?

Divergent beams from condenser prism pass through specimen as parallel

beams

Image intensity for

test specimen with no

compensation

Image intensity for

test specimen with plus

compensation

Image intensity for

test specimen with minus

compensation

Comparison of DIC image intensity for

test specimen with no, plus and minus

compensation

Physical basis of fluorescence

• Molecules that are capable of fluorescing are called fluorescent molecules, fluorescent dyes, or fluorochromes. If a fluorochrome is conjugated to a large macromolecule (through a chemical reaction or by simple adsorption), the tagged macromolecule is said to contain a fluorophore, the chemical moiety capable of producing fluorescence. Fluorochromes exhibit distinct excitation and emission spectra that depend on their atomic structure and electron resonance properties.

Basic concept of absorption and emission

Jablonski diagram showing energy levels occupied by an excited electron within

fluorescent molecule (chlorophyll a)

• Chlorophyll a is unique in absorbing blue and red wavelengths of the visual spectrum. Blue photons are excited to a higher energy level than are red ones (straight upward arrows, left), but the collapse to the ground state by an electron excited by either wavelength can occur through any of the following three pathways: Chlorophyll can give off a photon (fluorescence emission, straight downward pointing arrow); it can release vibrational energy as heat without photon emission (internal conversion, wavy downward pointing arrows); or its electron can enter an excited triplet state (intersystem crossing, dotted downward arrow), which can make the molecule chemically reactive. Electrons in the triplet excited state can return to the ground state through internal conversion or by emission of phosphorescence. Refer to the text for details.

Stokes shift

Properties of fluorescent dyes

• An important criterion for dye selection is the molar extinction coefficient, which describes the potential of a fluorochrome to absorb photon quanta, and is given in units of absorbance (optical density) at a reference wavelength (usually the absorption maximum) under specified conditions. The quantum efficiency (QE) of fluorescence emission is the fraction of absorbed photon quanta that is re-emitted by a fluorochrome as fluorescent photons. QE varies greatly between different fluorochromes and for a single fluorochrome under different conditions. For soluble fluorescein dye at alkaline pH, the quantum efficiency can be as high as 0.9

Properties of fluorescent dyes cont’d

• Quenching and photobleaching reduce the amount of fluorescence and are of great practical significance to the microscopist.

• Quenching reduces the quantum yield of a fluorochrome without changing its fluorescence emission spectrum and is caused by interactions with other molecules including other fluorochromes. Conjugation of fluorescein to a protein usually causes a significant reduction in the quantum yield because of charge-transfer interactions with nearby aromatic amino acids. Proteins such as IgG or albumin that are conjugated with 5 or more fluorescein molecules, for example, fluoresce less than when bound to 2–3 molecules, because energy is transferred to nonfluorescent fluorescein dimers. Photobleaching refers to the permanent loss of fluorescence by a dye due to photon-induced chemical damage and covalent modification. As previously discussed, photobleaching occurs when a dye molecule, excited to one of its electronic singlet states, transits to a triplet excited state. Molecules in this state are able to undergo complex reactions with other molecules. Reactions with molecular oxygen permanently destroy the fluorochrome and produce singlet oxygen species (free radicals) that can chemically modify other molecules in the cell. Once the fluorochrome is destroyed, it usually does not recover. The rate of photobleaching can be reduced by reducing the excitation or lowering the oxygen concentration.

Basic concept of epi-fluorescence microscopy

Ploem-type epi-illuminator

Filter cubes

Basic design features

Exciter and barrier filters are designed to separate emission

light from excitation light

Problems in filter Design: example absorption and

emission Spectra

The dichromatic mirror further isolates the emission light from

the excitation light

Combined Transimttance

A

C

B

10%

90%

500400

10%

90%

500400

10%

90%

500400

A: Trasmittance

B: Trasmittance

C: Trasmittance

D

10%

90%

500400

D: Trasmittance

500 lambda

500

lam

bda

)(10

9)(

10

9)(

10

1)(

10

1)(

10

1)(

10

9)(

10

1

)(10

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10

1)(

10

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1

ABCDCBA

BCDCBA

Transmission profiles of filters in a

fluorescence filter set

Confocal laser

scanning microscop

y

Optical pathway in a confocal scan head

• EX and EM indicate the paths taken by the excitation and fluorescence emission wavelengths.

• Photomultiplier tube (PMT) detects different fluorescent wavelengths

Pinhole apertureThe heart of confocal optics is the pinhole aperture, which accepts fluorescent photons from the illuminated focused spot in the raster, but largely excludes fluorescence signals from objects above and below the focal plane, which, being out of focus, are focused on the pinhole as disks of much larger diameter. Because the size of the disk of an out-of-focus object is spread out over such a large area, only a fraction of light from out-of-focus objects passes through the pinhole. The pinhole also eliminates much of the stray light in the optical system. Examine the figure carefully to see how the pinhole blocks out-of-focal-plane signals.

Spatial filter

Tandem scanning confocal microscopy

• The Yokogawa design features two disks each with > 20,000 pinholes that rotate as a single unified piece around a central axis. The upper disk is fitted with microlenses that focus incident rays on a pinhole in the second disk. The pinholes of the disk are confocal with the specimen and the surface of an electronic imager such as a charge-coupled device (CCD) camera. A fixed dichroic mirror positioned in between the rotating disks transmits excitatory wavelengths from the laser while reflecting fluorescent wavelengths to the camera