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Lecture 3:

Digital AcquisitionIlan Davis November 2006

Advanced topics to be covered

Light path for digital acquisition -multiwavelength, multi-Z sections - e.g.DeltaVision microscopy

OMX microscope light path

Kohler illumination / critical illumination / fibre optic delivery

Field diaphragm, Aperture diaphragm, neutral density filters, stops.

Filter wheels, motorized XYZ stages, piezoelectric focus drives.

High resolution DIC and how to image fluorescence and DIC at the same time

The use of fluorescent beads to test lens and microscope performance

Determination of resolution of the microscope (full width half Max measurement)

Airy rings (out of focus light)Point spread function (PSF) and optical transfer function (OTF)

The concept of convolution

Deconvolution

New methods of deconvolution in development: Pupal function, 3D variations in psf

in a specimen

CCD cameras including Intensified CCD and EMCCD.

TIRF and high resolution DIC

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Last lecture

How do fluorescence microscopes work ?

Mercury arc

Light sourcespecimen

Objective lens

Dichroic

mirror

Excitation

filter

Emmision

Filter

Eye piece

camera

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to use DIC and Fluorescence without loss

signal by polarizing filter?

Mercury arc

Light sourcespecimen

Objective lens

Dichroic

mirror

Excitation

filter

Emmision

Filter

Eye piece

camera

Polarizing filter

Brightfield

Halogen

lamp

Polarizing filter

Wollastom Prism

Wollastom Prism

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s DIC and fluorescence imaging with no loss of fluorescence intensity

be where dichroic mirror acts as polariser only in red light instead of the analyser.

only for FITC now available for FITC/rhodamine/DIC and other flavours

emoved to emission filter wheel (poorer quality DIC)

tion DIC

ight field illumination (image of magnified central part of UV bulb focused directly

o Kohler).

IR filters used.

ser with oil immersion lens.

esolution (low contrast) Wollaston prism

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Quad

Dichroic

mirror

Specimen

Eye piece

Mercury

vapour

arc lamp

Excitation

Filter

(in filter wheel)

IR (heat)

filter

ExcitationShutter

Neutral density

Filters

(in filter wheel)

Objective lens

1mm

Quartz fibre

optic cable

(to scramble

the light)

Collector lens

(focusable)

Collector lens

(light into fibre)

mirror

Photosensor measures

The intensity of light

FD

Mirror

Partial mirror

CCD

lements that make up the

eltaVision widefield fluorescence

icroscope (Based on design by

ohn Sedat and David Agard)

CondenserAD

XYZ Motorized stage

Kohler or critical

illumination

Emmision filter wheelShutter

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Special features of the DeltaVision Microscope

Nanomoover XYZ stage with ramping system - the most accurate on the market

10nm error. 100nm repeatability

Quartz fiber optic light scrambling

-Achromatic optics-Efficient transmission at 340nm and approx 20mW delivered at

objective at 546nm

-Can be very carefully aligned for Kohler or critical

illumination

Intensity Correction system -adjusts for lamp fluctuations

Filter wheel accuracy to 3.5microns

-ensures no shift in image in successive optical planes

(multichannel imaging)

7 Neutral density filters

Dry Nitrogen gas supplied to all filters to slow deterioration due to

oxydation and moisture

Shutters are spring mounted - isolate vibrations

Excitation shutter mounted at an angle

-increases bulb life (reduced heating from reflection)

-prevents reflections into image light path

Camera imaging chip placed at side port with no intermediate

glass (which absorbs some light and causes chromatic aberration)

-optimal position for resolution -deconvolution on the fly on the

camera chip (DV-RT)

Software deals with 5D datasets in one file and runs on Unix platform (more

stable, better networking and deals with large files better). Probably the

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OMX - light path

Rotating ground

Glass make

non-coherent

Neutral density

Change intensityDiode lasers Rapid shutters

Choose line

Specimen

XYZ nanomover

XYZ Piezo

Objective lens

EM-CCD

EM-CCD

EM-CCD

EM-CCD

Light source

Simultaneous acquisition of

4 channels

Precisely machined

Metal block with internal sculptur

That absorbs stray light.

Tube lens

Fibre optic

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o far we have really considered image formation

y objective lenses in an over-simplified manner.

Out of focus light-Airy rings in 3D view

Different

Focal

plains

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Detectors

tographic emulsion.y high resolution, poor sensitivity but low noise.

used now and replaced by 3-CCD colour cameras, more sensitive than film, low noise if cooled.

ton Multiplier Tube (PMT). Noisy and non-linear, but very good with laser scanning confocals

dys lecture).

rge Coupled Device (CCD) -silicon. Very sensitive, linear. Excellent with widefield systems and

nning disc confocal.

Representative pixel of CCD Front illuminated vs. Backthinned

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CCDs (Charge Coupled Device)

Amplifier

Reader

A-to-D converter

Types of readout

Full frame

Frame transfer

Interline transfer

Backthinned vs. Front illuminated

Full-well capacity

Number of maximum electronsthat can be stored per well

Analog-Digital converter

Usually 12bit signal (4096 in decimal)

Some 14 bit or 16bit

e.g. Coolsnap (Roper) approx 16,000e

-

weDepth Or 4e- per 1 gray scale unit

Pixel size: 6.45m

Ixon pixel size 17m, 16bits,

well depth 100,000e-.

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most important when pixel values are low)

of number of electrons captured on each pixel).

- due to counting stochastic process.=10 S/N=10

ise=100, S/N=100

most important when taking short exposures 1sec)

dependent (reduced by factor of 2 for every 8-9 degrees cooling)

eltier effect (current between different metals and heat sink)

ciency

tectible electrons produced per incident photon. Varies considerably with wavelength.

ings increase UV sensitivity. (Film, QE=0.01)

s

e of pixels (generally 7-15 microns)

.e. maximum number of electrons that can be captured in a pixel)

d out of block of pixels pooled -eg 2x2 binning increases sensitivity x4. But reduced resolution).

dout 1MHz, 5MHz, 20MHz -not the only factor in how fast you can capture images.

grades of CCDs (usually scientific grades are used -only very few dud pixels)

masks, gating.

and other references for more details)

Properties of CCD cameras

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Scientific CCD cameras -Jargon Buster (Based on a document from Opto and Laser Europe http://optics.org/articles/ole/8/2/3/1)

CCD cameras may be a popular tool for scientfic imaging, but lots of technical jargon and a wide range of models can make it verydifficult to select the most suitable product. Oliver Graydon offers some useful advice for the novice.

The bewildering array of products on the market and reams of associated technical jargon can make choosing the right CCD camera a daunting task.This aims to simplify the task by indicating the key performance criteria to consider and translating the jargon.

How CCDs work

At the heart of all CCD cameras is a charge-coupled device (CCD) image chip. This is an array of light-sensitive pixels that are electrically biased sothat they generate and store electrons - electric charge - when illuminated with light. The amount of charge trapped beneath each pixel directlyrelates to the number of photons illuminating the pixel.

This charge is then "read out" by changing the electrical bias of an adjacent pixel so that the charge travels out of the sensor, is converted into avoltage and is then digitized into an intensity value. This action is performed for each pixel, to create an electronic image of the scene. Electronicsinside the camera control the read-out process.

Criteria for CCD use: The wavelength region you wish to image. The lighting level. Are you trying to detect single photons or bright events? The frame-rate required. Are you intending to perform very fast imaging?Note that the performance characteristics of a CCD camera are often interrelated, and trying to optimize one parameter will often compromiseanother.

What wavelength response do you need?

The wavelength sensitivity of a CCD camera is usually determined by the quantum efficiency (QE) of the CCD chip.

Most CCD chips with no special coatings have a QE in excess of 30% in the visible and near-infrared (400-850 nm). However, if you need to performimaging at slightly shorter or longer wavelengths, it is possible to obtain CCD chips that are coated with phosphors to increase their sensitivity in theinfrared, blue and ultraviolet.

Buying a CCD camera with a back-thinned or deep-depleted CCD chip can also enhance a camera's sensitivity in the ultraviolet, visible and near-infrared, and may be useful if you are performing low-light imaging in these regions. It is important to note that the wavelength sensitivity of a CCDcamera is temperature-dependent and will change if the camera is cooled (cooling is a popular way to reduce the dark current of a camera). Bycontrolling the temperature of the CCD chip by thermoelectric cooling (rather than cooling with liquid nitrogen) it is possible to optimize the CCD's QEin a given wavelength range.

How strong is the lighting level?

If you want to image very low light-levels, for instance in experiments involving fluorescence or bio/chemoluminescence, it is essential to use a CCDcamera with an optimized signal-to-noise ratio. In reality that means choosing between a cooled high-QE CCD camera, which has very low noise, andan intensified camera (ICCD), which makes use of an image intensifier and can image single photons. Vendors that sell both types of devices indicatethat CCD sensor technology is now so good that in many cases they recommend using cooled CCD cameras unless you need a measurement with a

nanosecond response.

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CCD Jargon Buster - page 3

CCD noise/dark current This refers to spurious electrons that are generated in the CCD chip by thermal and other effects in the absence ofany illumination. The effects of CCD noise can be dramatically reduced by cooling the image sensor.

Deep-depletion CCD A CCD chip that is designed to offer superior sensitivity in the near-infrared and high-energy X-ray region. It contains abiased area of high-resistivity silicon for capturing photons that would not normally be absorbed.

Etaloning In a back-illuminated CCD, reflections between the front and back surfaces can lead to interference effects that degrade the

performance of the camera in the near-infrared. Anti-etaloning technology in deep-depletion CCDs can overcome this effect.

Image intensifier A vacuum tube, usually 18-25 mm in diameter, that gives a significant boost in light sensitivity. It is placed in front of theCCD chip and converts incoming photons into electrons, which are multiplied before being converted back into photons. The intensifier can alsobe gated for time-resolved experiments.

Megapixel A term used to describe a CCD sensor that contains at least one million pixels.

Multipinned phase (MPP) A way of biasing the CCD chip so that the CCD's dark current is dramatically reduced. As a result, less cooling isrequired to reduce the noise of the camera, and compact thermoelectric coolers can be used instead of liquid nitrogen.

Photon/shot noise The laws of physics dictate that the number of photons striking a detector is inherently uncertain. This uncertainty isknown as photon, or shot, noise and varies with the square root of the signal level. High QE cameras improve the signal to shot noise ratio.

Quantum efficiency (QE) The probability of a CCD chip converting an incoming photon of a given wavelength into an electron. It is measuredas a percentage, and the higher the value, the more sensitive the camera.

Read-out noise This is noise that is generated by the electronics that convert the charge from each pixel into a digitized light-intensity valuethat is displayed in the image. Read-out noise increases with the speed of the read-out and consequently the frame-rate. It is reduced bytechnologies such as on-chip multiplication.

Read-out rate This is often quoted in MHz and refers to the rate of read out of data per second. It should not be confused with frame-rate.

Smearing If light is still falling on the CCD chip during read-out, image distortion called smearing can result. The use of physical shutters andframe-transfer or interline CCDs minimizes the effect.

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ous types of CCD cameras

pHQ.Interline chip, fast readoutes per second). High QE, due microlenses, low noise.

ll frame readout. Even lower noise and high QE, but much slower readout.

ed - very expensive, higher QE.

x intensified CCD (I-CCD) - very expensive, very sensitive but high noise, poor resolution.

ologies:

ain (amplifier) before reading the signal and converting from analogue to digital. E.g. Ixon from A

ch pixel has its own amplifier. So far only on very cheap mass produced CCD cameras (not good in low

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Examples of CCD cameras

Now largely

Superceded

by EMCCD

technology

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Electron Multiplication CCDs (EMCCD)(same as on chip multiplication or on chip gain)

Amplifier

Reader

A-to-D converter

Back thinned or front illuminated

Dual amplifier (conventional / electron multiplicaOn chip amplification

-makes read noise irrelevant (

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Snells law: 1 sin 1 = 2 sin 2

air1=1.0coverslip glass2=1.515

1 2

Numerical ApertureNA = sin

x-y Resolution

d = 0.61 /

Brightness

I NA4 / mag2(I = intensity mag = magnification)

(d = smallest resolvable distance)

( = refractive index = cone angle )

Appendix I

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Appendix II

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ncesic focusing theory and practice. PI (www.pi.ws) http://www.physikinstrumente.com/

e - Excellent history of subject and current detailed explanations of latest equipment

alization by Fluorescence Microscopy, A practical Approach Ed. V.J. Allan OUP ISBN 0-19-963740-7

Ilan Davis: How to make and use fluorescent bead slides and how to correct spherical aberration.

avis@ed.ac.uk for a copy)

Bill Amos: Instuments for fluorescence imaging (CCD information)

, Schaefer, L. H., Swedlow, J. R. (2001) A working persons guide to deconvolution in

scopy. Biotechniques 31, 1076

R., Hu, K., Andrews, P. D., Roos, D. S., Murray, J. M. (2002) Measuring tubulin content in

gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

14

, Sedat, J., Agard, D. (1990). Biophysics Journal 57 325. Determination of three-dimentional imagin

microscope system, Partial confocal behaviur in epifluorescnece microscopy

ufacturer of CCD cameras http://www.roperscientific.com/

d application notes and information on their site. E.g. http://www.roperscientific.com/library.sht

roperscientific.com/library.shtmlhttp://www.roperscientific.com/pdfs/technotes/ccd_grading.pdf

ding manufacturer of CCDs: http://www.andor.com/

ifferent deconvolution methods used in Astronomy http://jstarck.free.fr/pasp02.pdf

and Davis, I. (2005) Deconvolution: Lifting the fog. Book chapter, in Cell Biology, a laboratory ha

.Celis. Academic Press. (Ask Richard.Parton@ed.ac.uk for a copy)

http://www.pi.ws/mailto:ilan.davis@ed.ac.ukhttp://www.roperscientific.com/http://www.roperscientific.com/library.shtmlhttp://www.roperscientific.com/library.shtmlhttp://www.roperscientific.com/pdfs/technotes/ccd_grading.pdfhttp://www.andor.com/http://jstarck.free.fr/pasp02.pdfmailto:Richard.Parton@ed.ac.ukmailto:Richard.Parton@ed.ac.ukhttp://jstarck.free.fr/pasp02.pdfhttp://www.andor.com/http://www.roperscientific.com/pdfs/technotes/ccd_grading.pdfhttp://www.roperscientific.com/library.shtmlhttp://www.roperscientific.com/library.shtmlhttp://www.roperscientific.com/http://www.roperscientific.com/http://www.roperscientific.com/mailto:ilan.davis@ed.ac.ukhttp://www.pi.ws/

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Thanks

Richard Parton

Dave Kelly

Ken Sawin

Alejandra Clark

John Sedat

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l Internal reflection fluorescence microscopy (TI

Laser light

At an angle

60X/NA1.45 oil TIRF; 60X/NA1.65 oil TIRF

Evanescent Wave

(only

penetrates

100nm below

coverslip)

Objective

lens

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Spinning disc confocal microscope

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Laser scanning confocal

microscope

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Airy rings - 2D view

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The basis of Airy ring formation

Is diffraction through a slit

(aperture of the lens)

shape of the Airy rings vary according to

elength and size of the aperture in a reproducibl

depend in details on the individual objective le

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Objective lens

Planes of

focus

(z series)

Airy rings - 3D view

Point Spread Function (psf)

Z

X

Y

X

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e lenses (single element) have spherical aberrati

ves are made of many elements to correct spherical aberration.

jectives are designed to image correctly at the surface of a cover

rticular thickness (usually number 1.5, or 0.17mm (0.15-0.19mm).

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z

x

y

x

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z microns

thick

Surface of slide

Surface of cover slip

Bead slide: 0.1micron and 0.5 micron

Tetraspeck beads: chromatic registration

DAPI/FITC/Rhodamine/Cy5

Beads (PS Spec): Single fluorochrome

Brighter -better for generatingpoint spread functions for deconvolution

Inspec Intensity beads: Measure dynamic range

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Affects of deep imaging (90m) and collarsettings on spherical aberration and psf

of 60X/NA1.2w

0.13 surf

0.13 surf0.13deep

f

0.15 surf0.15 deep

0.17 surf0.17 deep

0.19surf 0.19 deep 0.21 surf0.21 deep

x

z

y

Data from

Alejandra

Clark

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0

500

1000

1500

2000

2500

3000

3500

4000

1 8 1 5 2 2 2 9 3 6 4 3 5 0 5 7 6 4 7 1 7 8 8 5 9 2 9 9 1 0 6 11 3 1 20 1 2 7 13 4 14 1 1 48 1 5 5 16 2 16 9 1 76

z-axis(pixels)

In

tens

it

y

x-axis (pixels)

In

tens

it

y

0

500

1000

1500

2000

2500

3000

3500

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73

- a is(pie ls)

x

z

y

0.21 setting surface bead 0.21 setting deep bead

x

z

y

0

500

1000

1500

2000

2500

3000

3500

4000

4500

1 7 1 3 1 9 2 5 3 1 3 7 4 3 4 9 5 5 6 1 6 7 7 3 7 9 8 5 9 1 9 7 1 0 3 1 0 9 11 5 1 2 1 1 2 7 1 33 1 3 9 1 45 1 5 1 1 57 1 6 3 1 69 1 7 5 1 81

z-axis (pixels)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

1 3 5 7 9 1 1 1 3 1 5 17 1 9 2 1 2 3 25 2 7 2 9 31 3 3 3 5 3 7 39 4 1 4 3 4 5

In

tens

it

yI

nt

ens

it

y

x-axis (pixels)

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efield Deconvolution

storing out of focus light to its point of origin

ore Deconvolution After Deconvolution

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real image

infocuslight

out of focus light(airy rings)

observedimage

z

x

y

convolution(objectivelens)

deconvolution(+psfotf)

0.1m fluorescentbead

Planes of focus of observed image

Planes of focus of real image

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a b

c d

observed

deconvolvedfwhm

0.18

0.26

0.34

0.65

a x-y focal plane b z axis

x or y (pixels) z (pixels)

intensity

(ing

ray

scale

values)

0 510

15

20

25

30 0 5

15

20

25

10

6000

5000

4000

3000

6000 6000

5000

4000

3000

2000

1000

0

2000

1000

3000

4000

0

30

ncrease in resolution (XY and Z) after deconvoluti

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onvolution

culations done in Fourrier (frequency) space not XYZ space.

is converted to optical transfer function (only information in X and Z)

eral methods that vary in their implementation

Point Spread Function

PSF (XYZ space)

Frequency (Z)

Frequency (X or Y)Z

XYX

Optical Transfer Function

OTF (XZ frequency space)

New methods (Sedat)

Pupal functions (used to sharpen Hubble telescope) include information in otf in X,

Y and Z and phase.

Mapping psf in 3D in specimen by measuring RI using DIC to correct for specimenaberrations (dispersion, reflection, absorption, diffraction)

OMX Structural illumination- much higher resolution / Faster microscope: 3 CCD

cameras + 4 computers

Pifocal -Piezoelectric focusing on the YXZ stage (objective collar piezofocusing

changes the psf and affects DIC).

Laser illumination instead of mercury arc lamp -must make non-coherant to avoid

speckles in illumination

(spining ground glass)

Programmable Array microscope -soon to be commercialized by Cairn. Separates

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Laser Scanning Confocal

Removes out of focus lightUsing a pinhole in the light path

Less sensitive -looses light

More convenient

Image one section

PMT is more noisy than CCD

Better for diffuse signal witha lot of out of focus light

Widefield Deconvolution

Reassigns out of focus lightTo the point of origin

More sensitive and quantitative

Less convenient

-requires lots of Z sectionsand time consuming calculations

on expensive computers

Better signal to noise ratio

Better for point sources of light