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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.

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quality assurance testing for modern optical imaging systems

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

proposed procedures
LASER, stage, PMT stability:


Measure LASER brightness/ fluctuation and PMT sensitivity/fluctuation over time.


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.



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


Proposed procedures



The Good

MP = multi-photon LASER with dichroic splitter for red & green channels

Field illumination:


Measure uniformity of illumination across the entire scan field


Warm up LASERs for one hour.

Use the 488 or 543/561 LASER combination for green/orange slide from Chromaslide (cover-slipped area)

Collect scan such that the intensity is near 150 / averaging OK, zoom @ manufacturers specification, (0.7 - 1), using as many lens as possible.

Use sufficient LASER power so that the gain on the PMT is

~ ½ the maximum


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.



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


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)
    • high NA (>1.2) lens, i.e., 40X or higher
    • Collect such that the pixel size is half the resolution of the lens
    • Zoom near 10 will be needed
    • 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.



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 *

many thanks
Many Thanks

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 !