Quality assurance testing for modern optical imaging systems

1. Quality assurance testing for modern optical imaging systems Light Microscopy Research Group Robert F. Stack, Richard W. Cole Wadsworth Center / NYSDOH Albany, N.Y.

2. 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

3. 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

4. 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

5. LASER, stage, PMT stability: Purpose: Measure LASER brightness/ fluctuation and PMT sensitivity/fluctuation over time. Protocol: 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: 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. Proposed procedures http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm

7. The Good MP = multi-photon LASER with dichroic splitter for red & green channels

8. The Good

9. The Bad

10. The Bad

11. The Ugly

12. The Ugly

13. Field illumination: Purpose: Measure uniformity of illumination across the entire scan field Protocol: 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 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

14. Zoom = 1 Zoom = 1.25 Images of fluorescent test slide (20X) & results of line scans

15. The Good Modern light microscopy is a Quantitative technique; it is critical to know whether measurement variations are due to uneven photon strength

16. The Bad 20x objective zoom=1 uneven illumination at corners and edges

17. The Ugly

18. 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. http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm

19. “most in focus” slices from each PMT & center of mass values (µM) for top most bead

20. The Good -- No lateral shift a/o width differences (good lateral & axial co-registration )

21. The Bad -- lateral co-registration good -- axial (Z) co-registration bad

22. The Ugly -- lateral co-registration bad (center of mass = *) -- axial (Z) co-registration bad (note size difference)

23. 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

24. Acknowledgements 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 *

25. 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 ! CHEERS !!