Microscope Calibration and Alignment.

Transcription

Microscope Performance
and Calibration
Molecular Probes® Labeling and Detection
Invitrogen Corp
Imaging Tools and Accessories
 FocalCheck™ Microscope Test Slides
 Fluorescent Microspheres
– FocalCheck™ Microspheres
– Tetraspeck™ Beads
– PS Speck™ Point Spread Function Beads
– InSpeck™ Intensity Calibration Beads
 FluoCells
®
Prepared Microscope Slides
 How to Use these Tools.
Invitrogen Proprietary & Confidential
Imaging Standards…or lack thereof.
 Currently there are no established standards
used in fluorescence imaging.
 MPI’s FluoCell line of fluorescently labeled
cells and tissue sections are widely used as
standard slides by instrument vendors.
 Fluorescence Microspheres are generally
better standards as they are more uniformly
labeled and more durable.
 MPI offers a variety of microsphere products
specifically targeted at imaging applications.
F36909 FocalCheck™ Fluorescence Microscope Calibration Slide #1
15 um 3 ring
FocalCheck™
Beads
Green (ex 488)
Orange (ex 543)
Far Red (ex 633)
6 um Blue-Green
FocalCheck™ Beads
Green (ex 488) Blue
(ex 365)
4 um TetraSpeck™
Beads Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
1 um TetraSpeck™
Beads
Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
0.5 um TetraSpeck™
Beads
Blue (ex 365)
Green (ex 488) Red
(ex 543)
Far Red (ex 633)
For general alignment and calibration
6 um Green Fluorescent (ex 488) Intensity Bead Series
1
2
3
4
5
6 um
Green Beads
6 um
Orange Beads
6 um
Red Beads
6 um
Far Red Beads
A
Bead 1 503/511
Bead 2 511/524
Bead 1 541/555
Bead 2 545/565
Bead 1 578/605
Bead 2 589/613
B
Ring 503/511
Core 511/524
Ring 541/555
Core 545/565
Ring 589/613
Core 578/605
Bead 1 657/676
Bead 2 671/692
Mixture of all 8
Reference
Beads
Ring 671/692
Core 657/676
Mixture of all 4
DoubleStained Beads
Spectral Unmixing Standards
Sample
Position
Wavelengths
1
2
3
4
5
350/440
505/515
540/560
580/605
633/660
A
2.5 um Blue
Bright
100% intensity
2.5 um Green
Bright
100% intensity
2.5 um Orange
Bright
100% intensity
2.5 um Red
Bright
100% intensity
2.5 um Deep
Red
Bright
100% intensity
B
2.5 um Blue
Dim
1% intensity
2.5 um Green
Dim
1% intensity
2.5 um Orange
Dim
1% intensity
2.5 um Red
Dim
1% intensity
2.5 um Deep
Red
Dim
1% intensity
General Colors, Two Intensities
Fluorescent Microspheres
Image
registratio
n, X,Y
alignment
FocalCheck™
X
MultiSpeck™
X
TetraSpeck™
X
Point
spread
function
(PSF)
Z-step
calibratio
n
Relative
intensit
y
X
X†
InSpeck
Color
range *
B/G/O/R
/NIR‡
Mounted
on slides
(optional)
X
B/G/R
B/G/O/
NIR
X
(0.2 µm
beads)
PS-Speck™
Spectral
unmixin
g
X
B/G/O/
NIR
X
X
B/G/O/R
/NIR
FocalCheck™ fluorescent microsphere
Ring stain
* Core stain
Bead size (µm)
Cat. no.
Blue, orange
None
15
F7234
Green, dark red
None
15
F7240
Green, orange, dark
red
Green, orange, dark
red
Green
None
15
F7235
None
6
F14806
Blue
15
F7237
Green
Blue
6
F14808
Green
Dark red
15
F7238
Orange
Blue
15
F7236
Dark red
Green
15
F7239
Dark red
Green
6
F14807
* The approximate fluorescence excitation and emission maxima, in nm, of the ring and core stains are: blue, 365/430; green,
505/515; orange, 560/580; and dark red, 660/680. For current prices, please visit our website at probes.invitrogen.com.
MultiSpeck™ and TetraSpeck™ fluorescent microspheres
Microsphere
Nominal bead diameter Cat. no.
(µm)
Blue, green, red fluorescence *
MultiSpeck™
4
M7901 †
TetraSpeck™
0.1
T7279
TetraSpeck™
0.2
T7280
TetraSpeck™
0.5
T7281
TetraSpeck™
1.0
T7282
TetraSpeck™
4.0
T7283
Blue, green, orange, dark red fluorescence *
Corresponding excitation and emission peaks are 365/430 nm (blue), 505/515 nm (green),
560/580 nm (orange), 580/600 nm (red), and 660/680 nm (dark red).
† Contains a suspension of MultiSpeck™ beads and a mixed suspension of beads singly labeled
with each of the three dyes.
*
FluoCells® Prepared Slides.
Useful tools for demonstration and training
#6 NIR Nucleus
#4 For Confocal
#3 For Confocal
#2 Mitochondria
#1 Last for years
We do consider customs
FluoCells® Prepared Slides
Product #
Description
Utility
F36924
FluoCells® prepared slide #1 *BPAE cells with
MitoTracker® Red CMXRos, Alexa Fluor® 488
phalloidin, DAPI*
General 3 color RGB
fluorescence sample
F14781
FluoCells® prepared slide #2 *BPAE cells with
mouse anti-α-tubulin, BODIPY® FL goat antimouse IgG, Texas Red®-X phalloidin, DAPI*
General 3 color RGB
fluorescence sample
F24630
FluoCells® prepared slide #3 *mouse kidney
section with Alexa Fluor® 488 WGA, Alexa Fluor®
568 phalloidin, DAPI*
Tissue section. Use for
confocal and 3D imaging.
F24631
FluoCells® prepared slide #4 *mouse intestine
section with Alexa Fluor® 350 WGA, Alexa Fluor®
568 phalloidin, SYTOX® Green*
Tissue section. Use for
confocal and 3D imaging
F36925
FluoCells® prepared slide #6 *muntjac cells with
mouse anti-OxPhos Complex V inhibitor protein,
Alexa Fluor® 555 goat anti-mouse IgG, Alexa
Fluor® 488 phalloidin, TO-PRO®-3*
Use for confocal and 3D
imaging without UV
excitation capability
Applications of Fluorescent Microspheres
 Chromatic Aberration
 Color Misalignment
 Field Illumination
 Spherical Aberration
 Point Spread Function Analysis
 Z axis errors
 Field Illumination
 Intensity Calibration
Chromatic Aberration
 Chromatic aberration occurs
due to light of different
wavelengths refracting
differently through the optical
path of the microscope.
 Even expensive objective
lenses may not be fully
corrected when working
across wide spectral ranges.
Color Registration and Misalignment

While chromatic
aberration can be a
serious issue, Color
Registration is a more
common problem in
fluorescence imaging.

Misregistration is caused
most commonly by
alignment differences
between the optical
filters.

Misalignment in a
confocal system caused
by using different light
path configurations
between scans.
Color Registration/Misalignment
 While chromatic aberration can be a
serious issue, Color Registration is a more
common problem in fluorescence
imaging.
 Misregistration is caused most particularly
by alignment differences between the
optical filters.
 Any of the multicolor beads we sell can be
used to diagnose and correct this problem
 Misregistration shown using TetraSpeck
beads 1 um.
Field Illumination
 Uneven field illumination is a serious matter
particularly for quantitative imaging applications.
 If the images are not corrected for unevenness in
illumination then segmenting the image may be
very challenging. In addition the distribution of
intensities in the image will be much greater than
expected making statistical analysis difficult.
 The best method for performing this correction is
to use a uniform fluorescent sample.
 However a crude assessment can be made by
comparing intensity values of beads distributed
throughout the field of view.
Field Illumination: Lamp Alignment
10x objective
100x objective
Flat Field Correcting Images
Uncorrected Image
of microscpheres.
Shading image,
pseudocolored to
show intensity
range.
Image
Shading
Uncorrected
after
image,
applying
Image
shading
pseudocolored
of
microscpheres.
correction.
to
show intensity
range.
Spherical Aberration using 200 nm beads
RI 1.5 mounting media
RI 1.38 mounting media
Coverslip Position



Axial Stretching and Signal Degradation due to Spherical aberration.
100x Oil Immersion objective , 1.4 NA.
25 micron depth, imaged at 0.25 micron steps, Zeiss 510 META.
Focus Motor Calibration
 Slippage of the Z axis
(focus) motor can lead
to some very puzzling
problems in confocal
systems.
 Z axis calibration using
FocalCheck Beads can
be performed with a
single XZ line scan
through the bead then
measuring for the
correct diameter.
 These beads can also
be used to establish the
correct XYZ ratio for the
image display.
Pinhole Misalignment
 Incorrect alignment of the
pinholes in a confocal
system not only degrades
the signal it can also lead
to some strange image
artifacts.
 Pinhole alignment can be
performed using a small
target object such as our
TetraSpeck beads or PSSpeck beads.
Intensity Calibration



Calibrating intensities between microscopes is difficult due to
variation between the system components.
We offer a series of intensity beads that provide a relative scale that
can be established for each instrument.
Sample intensities can then be made against this scale
independently of the actual instrument intensity values.
100%
Relative
Intensity
33%
Relative
Intensity
10%
Relative
Intensity
3%
Relative
Intensity
0.667%
Relative
Intensity
Intensity Bead Comparison Between Systems.
Measured Intensity Plots of Intensity Series Beads from 3 different Microscope Systems.
9000
8000
Measured Nominal Intensity
7000
6000
5000
4000
3000
2000
1000
0
0
20
40
60
80
100
Nominal Relative Intensity from Flow Cytometry.
Intensity Bead Comparison Between Systems.
Normalized Plots of Intensity Series Beads from 3 different Microscope Systems.
100.00%
90.00%
Measured Nominal Intensity
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
0
10
20
30
40
50
60
70
Nominal Relative Intensity from Flow Cytometry.
80
90
100
Intensity Variations
 The recommended working life for a mercury
arc lamp is about 200 hrs. Beyond this the
performance of the lamp can degrade both in
terms of output and stability.
 Using any of our standard beads as a target
one can follow the performance of the light
source over time to help spot problems.
 Similarly variations in intensity due to an
unstable arc can be detected by time lapse
imaging a field of beads and plotting the
intensities over time.
Fluorescence
00:00.0
07:12.0
14:24.0
21:36.0
28:48.0
Elapsed time (minutes)
1
0.995
0.99
0.985
0.98
0.975
0.97
0.965
0.96
00
09
17
26
35
43
52
00
Time (seconds)
Dimensional Calibration

Stage Micrometer, scale imaged
with 4 x lens
Summary
 Minimize spherical aberration by using correct lens
and mounting media.
 Identify and correct chromatic aberration and color
registration problems.
 Correct images for uneven field illumination.
 Periodically check stability of lamp.
 Use intensity beads to establish instrument
independent standard curves.
 Perform dimensional calibrate the optical system.
Resources for the Inquisitive





Spectraviewer
– http://www.invitrogen.com/spectraviewer
CCD Camera Technologies:
– http://www.microscopyu.com/articles/digitalimaging/ccdintr
o.html
Digital Imaging:
– http://micro.magnet.fsu.edu/primer/digitalimaging/digitali
magingdetectors.html
Nikon’s Microscopy U:
– http://www.microscopyu.com
Optical Filters:
– http://www.omegafilters.com/front/curvomatic/spectra.p
hp
Imaging Tools and Accessories
 FocalCheck™ Microscope Test Slides
 Fluorescent Microspheres
– FocalCheck™ Microspheres
– Tetraspeck™ Beads
– PS Speck™ Point Spread Function Beads
– InSpeck™ Intensity Calibration Beads
 FluoCells
®
Prepared Microscope Slides
 How to Use these Tools.
THANK YOU!
Questions?
techsupport@invitrogen.com
800.955.6288

Microscope Calibration and Alignment.

Transcription

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