Download - Ilan Fluoresc3 071106
-
8/14/2019 Ilan Fluoresc3 071106
1/37
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
-
8/14/2019 Ilan Fluoresc3 071106
2/37
Last lecture
How do fluorescence microscopes work ?
Mercury arc
Light sourcespecimen
Objective lens
Dichroic
mirror
Excitation
filter
Emmision
Filter
Eye piece
camera
-
8/14/2019 Ilan Fluoresc3 071106
3/37
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
-
8/14/2019 Ilan Fluoresc3 071106
4/37
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
-
8/14/2019 Ilan Fluoresc3 071106
5/37
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
-
8/14/2019 Ilan Fluoresc3 071106
6/37
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
-
8/14/2019 Ilan Fluoresc3 071106
7/37
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
-
8/14/2019 Ilan Fluoresc3 071106
8/37
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
-
8/14/2019 Ilan Fluoresc3 071106
9/37
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
-
8/14/2019 Ilan Fluoresc3 071106
10/37
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-.
-
8/14/2019 Ilan Fluoresc3 071106
11/37
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
-
8/14/2019 Ilan Fluoresc3 071106
12/37
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.
-
8/14/2019 Ilan Fluoresc3 071106
13/37
-
8/14/2019 Ilan Fluoresc3 071106
14/37
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.
-
8/14/2019 Ilan Fluoresc3 071106
15/37
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
-
8/14/2019 Ilan Fluoresc3 071106
16/37
Examples of CCD cameras
Now largely
Superceded
by EMCCD
technology
-
8/14/2019 Ilan Fluoresc3 071106
17/37
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 (
-
8/14/2019 Ilan Fluoresc3 071106
18/37
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
-
8/14/2019 Ilan Fluoresc3 071106
19/37
Appendix II
-
8/14/2019 Ilan Fluoresc3 071106
20/37
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.
[email protected] 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 [email protected] for a copy)
http://www.pi.ws/mailto:[email protected]://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:[email protected]:[email protected]://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:[email protected]://www.pi.ws/ -
8/14/2019 Ilan Fluoresc3 071106
21/37
Thanks
Richard Parton
Dave Kelly
Ken Sawin
Alejandra Clark
John Sedat
-
8/14/2019 Ilan Fluoresc3 071106
22/37
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
-
8/14/2019 Ilan Fluoresc3 071106
23/37
Spinning disc confocal microscope
-
8/14/2019 Ilan Fluoresc3 071106
24/37
Laser scanning confocal
microscope
-
8/14/2019 Ilan Fluoresc3 071106
25/37
Airy rings - 2D view
-
8/14/2019 Ilan Fluoresc3 071106
26/37
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
-
8/14/2019 Ilan Fluoresc3 071106
27/37
Objective lens
Planes of
focus
(z series)
Airy rings - 3D view
Point Spread Function (psf)
Z
X
Y
X
-
8/14/2019 Ilan Fluoresc3 071106
28/37
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).
-
8/14/2019 Ilan Fluoresc3 071106
29/37
z
x
y
x
-
8/14/2019 Ilan Fluoresc3 071106
30/37
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
-
8/14/2019 Ilan Fluoresc3 071106
31/37
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
-
8/14/2019 Ilan Fluoresc3 071106
32/37
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)
-
8/14/2019 Ilan Fluoresc3 071106
33/37
efield Deconvolution
storing out of focus light to its point of origin
ore Deconvolution After Deconvolution
-
8/14/2019 Ilan Fluoresc3 071106
34/37
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
-
8/14/2019 Ilan Fluoresc3 071106
35/37
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
-
8/14/2019 Ilan Fluoresc3 071106
36/37
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
-
8/14/2019 Ilan Fluoresc3 071106
37/37
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