Light Microscopy Research Group Robert F. Stack, Richard W. Cole Wadsworth Center / NYSDOH Albany, N.Y.

 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 Purpose of Phase One of the Quality Assurance study

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

Quality and standards: Making bioimaging ‘measure up’ Susan M. Reiss BioOptics World, Jan/Feb 2010, Vol.3 No.1, p 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) 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:  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. mean brightness should be ~ standard deviation 3. the range in brightness (highest value-lowest value) 4. longest time the LASER stayed within 10% & 3% of the mean value for 3hr & 5 min test respectively.

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

The Good

The Bad

The Ugly

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 Chroma slide (cover-slipped area)  Collect scan such that the intensity is near 150 / averaging OK, manufacturers specification, ( ), 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.

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.

“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 ColeRichard Cole (Chair) - Wadsworth Center / NYSDOH Carol J BaylesCarol J Bayles - Cornell University Karen MartinKaren Martin - West Virginia University Cynthia OpanskyCynthia Opansky – Blood Center of Wisconsin Katherine SchulzKatherine Schulz - Blood Center of Wisconsin Robert F. StackRobert F. Stack - Wadsworth Center / NYSDOH Pamela Scott AdamsPamela 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 !!