Quality assurance testing for modern optical imaging systems

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Transcript Quality assurance testing for modern optical imaging systems

Light Microscopy Research Group
Robert F. Stack, Richard W. Cole
Wadsworth Center / NYSDOH
Albany, N.Y.
Purpose of Phase One of the Quality Assurance study
 starting in early 2009, the LMRG formalized a list of study participants and sent out
test materials (Chroma slides and Tetraspeck bead slides) along with the proposed
procedures that had been formulated by the research group
 ascertain the current state of light microscope performance using
simple, efficient & robust tests
 three imaged-based test procedures
 LASER stability, field illumination & co-registration
 define & improve relative testing standards that will assist
core managers and users in the maintaining their microscopes
for optimal operation
 conduct a worldwide research study on instrument performance
 emphasis was on performance standards
 All microscope brands & types have their strengths &
weaknesses – the goal is Cross-platform standards that will
improve the validity of quantitative measurements in light
microscopy
History of performance standards / Light Microscopy
• current state of performance standards in light microscopy
• vendor initiated – none / acceptance specs only
• NIST developed -- none
• imaging community at large – lab specific
• Why ? – until the last 5-10 yrs, simply observing a specimen was
sufficient; recent advances in light microscopes
necessitate traceable standards & procedures
• development of performance standards (case study: mass spectrometry)
• started with minimal vendor-based tuning &
MW calibration compounds
• NIST :
•NIST Standard Reference Data Program –
mass spectra for over 15,000 compounds
• NIST Standard Reference Materials –
performance standards & mixtures available for
LC/MS, GC/MS, ICP-MS & Isotope-ratio MS
• The MS community:
• lab-specific acceptance criteria now common place
• proteomics data acceptance criteria now routine
Does anyone care ???
NIST, FDA, Congress & NIH
 Overall Goal – the creation of a range of imaging parameters traceable to standard references
 NIST – create traceable references with the goal of moving medical imaging & lab testing
from an art to a science
 FDA – device & drug approval processes ensure manufacturers systems are reliable
and drugs are safe & efficacious
 Congress – provide the financial support for standards research
What’s currently underway and or recently completed :
 NIST – development of ‘phantoms’ for CT, MRI &, standard protocols & analysis algorithms
 FDA – potential changes in drug & device approval process
 new imaging technologies will likely be subject to more rigorous quality control
standards regarding intended use
 increased imaging precision could lead to dramatically shortened clinical trials
 Congress –
 since FY2007, 4 million $$ has been provided with an additional 3.5 million $$
requested by NIST
 subcommittee hearings are ongoing regarding standards development
 Goal is to reduce Health care costs via savings in lower diagnostic imaging costs
 NIH –
 Realizes the need for and supports the “core” model – 40% of S10 grants funded
in FY2009 were for imaging in general; 13% were for confocal microscopes
 “Having a core laboratory that had not just all the instrumentation but real expertise
accelerated our research in ways that would not have happened otherwise”
(R.P.Lifton / Yale)
Quality and standards: Making bioimaging ‘measure up’
Susan M. Reiss BioOptics World, Jan/Feb 2010, Vol.3 No.1, p.14-18
Access sparks action
Lila Guterman NCRR Reporter, Winter 2010, p.4-8
LASER, stage, PMT stability:
Purpose:
Measure LASER brightness/ fluctuation and PMT sensitivity/fluctuation over time.
Protocol:
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Warm up LASERs for one hour.
Use the appropriate Chroma slide and LASER combination. Note: several different LASER lines may
work with one slide. The red slide works well for most LASERs.
With a 10x or 20x (low NA) lens focus a surface scratch, then focus down ~20um
Set up acquisition such that: Gain and offset should be set so that no PMT is saturated. The mean
value should be ~150 (out of 255 gray levels). These values as well as LASER power will vary for
each LASER used.
Collect images every 30 sec for 3 hours. Use 1 line averages per frame. Use sequential scan to
collect as many LASER lines as possible, i.e. 1 LASER line/ PMT
Collect images every 0.5 sec for 5 min., one wavelength at a time and scan faster if necessary.
At the end of test, shift the slide ~1/2 of the field of view and collect another image. Measure the
intensity across the field to check for photobleaching.
Analysis:
Calculate:
1.
2.
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4.
mean brightness should be ~150
standard deviation
the range in brightness (highest value-lowest value)
longest time the LASER stayed within 10% & 3% of the mean value for 3hr & 5 min test
respectively.
http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm
Laser power Vs. Time
160
Gray scale values
155
150
145
140
135
130
125
120
0
20
40
60
80
100
120
Time (mins)
488
543
594
633
140
160
180
The Good
MP = multi-photon LASER with dichroic splitter for red & green channels
The Good
The Bad
The Bad
The Ugly
The Ugly
Field illumination:
Purpose:
Measure uniformity of illumination across the entire scan field
Protocol:
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Warm up LASERs for one hour.
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Use the 488 or 543/561 LASER combination for green/orange
slide from Chroma slide (cover-slipped area)
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Collect scan such that the intensity is near 150 / averaging OK,
zoom @ manufacturers specification, (0.7 - 1), using as many
lens as possible.
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Use sufficient LASER power so that the gain on the PMT is
~ ½ the maximum
Analysis:
Using entire image: perform a line scan profile diagonally and
horizontally across the image to check for drop off near the
edges. The typical 1X zoom variations are 10% in horizontal
and 20% in diagonal.
http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm
Zoom = 1
Zoom = 1.25
Images of fluorescent test slide (20X) & results of line scans
The Good
Modern light microscopy is a Quantitative technique; it is critical to know whether measurement variations are due
to uneven photon strength
The Bad
20x objective zoom=1 uneven illumination at corners and edges
The Ugly
PMT co-registration:
Purpose:
Determine to what extent images of an object (bead) collected with different
PMTs will co-register/superimpose to each other
Protocol:
Bead slide: (will be provided) 4.0 µm Tetraspeck beads (B , G , O & dark R)
o high NA (>1.2) lens, i.e., 40X or higher
o Collect such that the pixel size is half the resolution of the lens
o Zoom near 10 will be needed
o use a standard three or four color protocol.
Collect a Z series using sequential scans of three or more PMTs
Do not forget to use the NDD for MP scopes.
Analysis:
 Using a line scan function, plot the intensities across the bead for each
slice in the stack. The brightest slice is the “most in focus” This should be
the same Z position for all PMTs.
 Using the ImageJ measurement function, determine the center of mass
for the “most in focus” slice for all the PMTs. Determine the displacement
among the PMT’s.
 Performing this on >1 bead will help to separate aberrant beads
Single beads should be “cropped out” for the measurements.
http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm
“most in focus” slices from each PMT & center of mass values (µM) for top most bead
The Good
-- No lateral shift a/o width differences (good lateral & axial co-registration )
The Bad
-- lateral co-registration good
-- axial (Z) co-registration bad
The Ugly
-- lateral co-registration bad (center of mass = *)
-- axial (Z) co-registration bad (note size difference)
Phase Two / Future directions
detailed data analysis in conjunction with a statistician
publication detailing study findings & recommendations
develop standards (procedures & samples) that provide
broad applicability and are widely accepted by the greater
imaging community
 identify specimens with structure that are excitable over
multiple wavelengths
 interface with NIST
traceable reference standards are their area of expertise
Light Microscopy Research Group
Richard Cole (Chair) - Wadsworth Center / NYSDOH
Carol J Bayles - Cornell University
Karen Martin - West Virginia University
Cynthia Opansky – Blood Center of Wisconsin
Katherine Schulz - Blood Center of Wisconsin
Robert F. Stack - Wadsworth Center / NYSDOH
Pamela Scott Adams - (Ad hoc) (EB Liaison) - Trudeau Institute
Anne-Marie Girard – Oregon State University
* We would also like to thank the ABRF for their financial support and commitment to this
project *
To all of the dedicated researchers who took time out of their busy schedules to
participate in this study – we received data from 23 PIs across 7 countries !
CHEERS !!