Announcements, Agenda Week 3

• Reading for today: Ch. 1, 2 in Hibbs, Zucker 2006

• Start up your computers – you will need them for some in-class exercises.

• Open today’s Power point slides and Internet Explorer

I. Lecture: Intro to Confocal, optics

II. Paper discussion: Zucker 2006

III. TBA: Collect Z-series of Artemia samples

IV. Assignment due Jan. 29

TBA times with Dr. Hertzler: Spring 2007

Time Tuesday Wednesday Thursday Friday

8

9 SEM Cell Biology TEM Cell Biology

10 Group 1 Office Group 2

11 Amy, Lauren, Rachel

Hours Andrea, Emily, Molly

12

1 403 Group 3 Lab meeting

2 students Becky, Ellen, Katie

Group 4

3 UCC Amanda, Brittaney, Joe

4 Faculty Meeting

Seminar

Outline: Understanding Microscopy

A. Introduction to Confocal Microscopy1. Confocal versus conventional (widefield) fluorescence2. Optical sectioning3. Imaging modes and applications4. Advantages, limitations of confocal

B. Essential Optics1. Wave/particle nature of Light2. Diffraction3. Numerical aperture4. Lateral resolution5. Axial resolution

Useful resource: Molecular Expression Microscopy Primer:• http://micro.magnet.fsu.edu/primer/index.html

Laser Scanning Confocal Microscope Components

Laser

Scan Head

Microscope

Controllerbox Computer, display

1. Conventional versus confocal fluorescence

Conventional epifluorescence Confocal epifluorescence

Sea urchin eggs (100 μm diameter)stained with antibody to tubulin.

Human brain slice Rabbit muscle fibers Sunflower pollen grain

Widefield

Confocal

Wide-field fluorescence: dichroic (dichromatic) mirror

Confocal Light Path• Confocal means “having

the same focus.”

• Basis of optical sectioning: coherent light emitted by the laser system (excitation source) passes through a pinhole aperture that is situated in a conjugate plane (confocal) with a scanning point on the specimen and a second pinhole aperture positioned in front of the detector (a photomultiplier tube).

2. Optical slicing

3. Imaging Capabilities

1. XY fluorescence imaging

a) Singleb) Doublec) Single or Double +

transmitted (not confocal)

d) 3-channel (need 3 lasers)

2. XYZ imaging, 3-D reconstruction

3. Time-lapse• Including 4D

Applications

• Immunolabelling• Organelle ID• Protein trafficking• Locating genes on chromosomes• Analysis of molecular mobility• Multiple labeling• Live cell imaging• Transmission imaging• Measurement of subcellular functions and ion

concentrations

4. Advantages, limitations of confocal microscopy

• Optical sectioning ability– Can image cells/tissues internally

• 3D reconstruction– Improved spatial relationships of

structures• Excellent resolution

– Close to theoretical limit of LM: 0.2 μm

• Improved multiple labeling– Since specific wavelengths of

light used by lasers• Very high sensitivity

– Capable of collecting single fluorescent molecule

• Easy manipulation and merging of images

– Since they are digital• Computer controlled

– Complex settings can be programmed and recalled.

• Expensive to buy and maintain.– $250,000 +

• Difficult to operate.– Fixed material easy, live difficult.

• Fluorescent tag usually required.– May be bulky or toxic

• Objects smaller than 0.2 not resolved

– Need to use EM.• Damaging high intensity laser

– Need to minimize exposure, especially in live cells.

• Digital images are easily mishandled.

– Honesty in imaging very important.

B. Basic Optics1. The nature of light

• Light behaves as both a particle and a wave.

• Can bounce (reflect) and bend (diffract or refract)

• Has wave properties– Amplitude– Wavelength: visible is

between 400-700 nm• White light carries all

visible wavelengths

– Frequency– Direction of travel– Direction of vibration

Relation between Wavelength, Frequency, Energy

Blue light

488 nm

short wavelength

high frequency

high energy (2 times the red)

Red light

650 nm

long wavelength

low frequency

low energy

Photon as a wave packet of energy

Light-Matter Interactions

• Absorption• Reflection• Refraction: bending of light as it passes, at an

angle, from one material to another • Diffraction: bending of light as it passes an

edge• Fluorescence: spontaneous emission of light

after excitation• Polarization• Dispersion

2. Diffraction: Bending of light as it passes an edge

λ < d λ > d

See: Microscopy primer,

One long continuous wave,unlike light from a lampor the sun.

Diffraction Pattern from SlitResults from Interference

Java Tutorial: Diffraction Patterns

• http://micro.magnet.fsu.edu/primer/java/diffraction/basicdiffraction/index.html

• How does the width of the central maximum vary with the wavelength?

Diffraction Through a Circular Aperture creates an Airy Disk

• The radius of the Airy disk is the distance r from the center to the first dark ring, given by the resolution equation.

Increasing resolution of lens

Resolution and Airy disk patterns

Java Tutorial: Airy Pattern Basics

• http://micro.magnet.fsu.edu/primer/java/imageformation/airydiskbasics/index.html– How does resolution vary with wavelength and

numerical aperture?

• http://micro.magnet.fsu.edu/primer/java/imageformation/airyna/index.html– What is the effect of higher NA?

• http://micro.magnet.fsu.edu/primer/java/imageformation/rayleighdisks/index.html– What is the Rayleigh criterion?

3. Numerical aperture (NA)NA = n sin

where n = refractive index and = the collecting angle.nair = 1.00 and noil = 1.515.

W.D.

Maximum theoretical NA

• Maximum collecting angle is 90o

• sin 90o = 1.00.• For dry objective, max. NA = (1.00)(1.00) = 1.0

– In practice, it is 0.95.– All dry objectives have NA < 1.00

• For oil objective, max NA = (1.515)(1.00) = 1.5.– In practice, it is 1.4.– All oil objectives have NA > 1.00

4. Lateral Resolution (XY or rlateral)

• The smallest distance two objects can be imaged as two. Depends on wavelength and NA.

objobjlateral

condobjlateral

NANA

λ.r

NANA

λ.r

61.0

2

221

thenNANA If

aperture. numerical isNA h, wavelengtis Where

221

condobj

Optimal Resolution for LM

• Visible light ranges from 400-700 nm• Best NA lens is 1.4• Calculate best theoretical resolution using 520

nm emission of fluorescein:

• (Footnote: for confocal, the resolution equation is slightly better: rlateral = 0.4λ/NA so best resolution is closer to 0.15 μm).

μm 0.2nm 2621.4

nm 5200.61r

XY under- and over-sampling

• Optimal zoom settings (for full xy resolution) for 512 X 512 pixel box are given for various lenses on p. 126.– You don’t need to operate at these settings unless

you want to push the resolution limit.

• Rules of thumb for 1024 X 1024 box:– 60X 1.4 NA: 4X max zoom– 40X 0.75 NA: 5X max zoom– 20X 0.7 NA: 6X max zoom

• Zooming higher than this creates empty magnification.

No Zoom 2X Zoom

Zooming for maximum XY resolution

Java Tutorial: 3D Airy disk is the Point Spread Function

• http://micro.magnet.fsu.edu/primer/java/imageformation/depthoffield/index.html

This Z step will not resolve the objects in Z axis.

This Z step will resolve the objects in Z axis.

5. Axial Resolution (Z or raxial)• Minimum distance between the 3D diffraction patterns of two points along the Z axis that can still be seen as two.• Depends on wavelength and NAobj as follows:

• Rule of thumb: step size = ½ Z resolution. See also http://www2.bitplane.com/sampling/index.cfm and http://www.cemedigital.com/clients/brand_aic_lrg/support/presentation04.shtml

•

μm 2nm 2165

0.5625

nm 1218

(0.75)

nm)(1.5) 1.4(580

)(

4.1

:lensNA 0.75 40XFor

2

2

axial

axial

objaxial

r

r

NA

nr

μm 0.6nm 621

1.96

nm 1218

(1.4)

nm)(1.47) 2(580

)(

2

:lensNA 1.4 60XFor

2

2

axial

axial

objaxial

r

r

NA

nr

Ideal step sizes

Ideal step size(higher Z resolution, e.g. NA=1.4)

Ideal step size(lower Z resolution, e.g. NA=0.7)

Z axis under- and over-sampling

UndersampledToo few sections for full Z

resolutionBut: full Z resolution may

not be needed.

Oversampled:Overlapping sections add no

additional information since full Z resolution is realized;

just makes a bigger file.

XY and Z resolutions (μm)

10X

0.4 NA

20X

0.7 NA

40X

0.75 NA

60X

1.4 NA

rlateralfluorescein

488/5180.790 0.451 0.421 0.226

rlateralrhodamine

543/5800.885 0.505 0.471 0.253

raxial

step

fluorescein

488/5186.80 2.22

1.1

1.93

1

0.555

0.275

raxial

step

rhodamine

543/5807.61 2.49

1.25

2.17

1

0.621

0.3

The bottom line on optimal step size

• The Nyquist Sampling Theorem states that the pixel size should be 2.3X smaller than the resolution limit of the microscope (p. 126).– So 1.4 NA objective with rlateral = 0.2 μm

requires xy pixel size of 0.08 μm, optimal zoom of 3.7X at 512 X 512.

– Step size should be 3X xy pixel size = 0.24 μm for 1.4 NA objective with raxial = 0.6 μm

Week 3 TBA

• Assignment (each person):– Collect Z-series of one of your Artemia samples,

using the 20X lens and a step size of 1 or 2 um.– Display the sections in tile mode.– Save (as a normal TIFFs) extended focus images in

black and white, showing (a) every section of the Z-series, (b) the top 1/3, (c) the middle 1/3, and d) the bottom 1/3.

• Always include a scale bar on your images.• Save in the BIO553 file on the imaging computer.

– Turn in a description of your images using the form available on Blackboard.

Paper discussion

• Today, Jan. 22: Zucker 2006 (Hertzler)

• Jan. 29: (Hertzler)

• Feb. 5:

• Feb. 12:

• Feb. 19:

• Feb. 26: