February 19th – 22nd Technologies to Enable Personalized Medicine San Antonio, TX

Point Spread Function, Spectral Calibration and beyond:Quality Assurance Testing / Phase Two

Light Microscopy Research GroupRobert F. Stack / Richard W. ColeWadsworth Center / NYSDOHAlbany, New York

History of performance standards / 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

Overall Goal – the creation of a range of imaging parameters traceable to standard references

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

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

Quality and standards: Making bioimaging ‘measure up’Susan M. Reiss BioOptics World, Jan/Feb 2010, Vol.3 No.1, p.14-18

Access sparks actionLila Guterman NCRR Reporter, Winter 2010, p.4-8

starting in early 2009, the LMRG began conducting Phase One of a worldwide research study to ascertain the current state of light microscope performance using simple, efficient & robust tests for LASER stability, field illumination & coregistration

define & improve cross-platform standards that will assist core managers and users in the maintaining their microscopes for optimal operation with the ultimate goal of improving the validity of quantitative measurements in light microscopy

the results of this study were accepted for publication in late 2010 in Microscopy and Microanalysis, one of the highest rated imaging journals (early 2011)

throughout 2010, the LMRG tested and defined additional areas of instrument performance (Phase Two) and refined the methodology for determining a system’s Spectral calibration, Spectral separation ability and finally the Point Spread Function of an imaging system

Purpose of Phase Two of the Quality Assurance studyPurpose of Phase Two of the Quality Assurance study

Phase One Study participation by country

Phase One Imaging stations by vendor

Proposed ProceduresProposed ProceduresSpectral calibration

Purpose:

Measure spectral calibration of the detection system.

MIDL lamp / mirror slide protocol:

Use 10x lens or no lens (system dependent) Set up the MIDL lamp as the illumination source or use laser(s) and mirror slide (remove blocking) Set the PMT gains to be ‘equivalent’ Perform a lambda scan and measure the signal-to-noise Compare acquired spectra with published spectra

Analysis:

1. Determine if your PMT(s) show significant spectral variation (sliders) or signs of aging

reference: http://www.lightforminc.com/MIDL/index.html

PARISS Spectral Calibration Lamp, Lightform,Inc. Asheville, NC

Overlay of 5 PMT responses and MIDL lamp calibrated output / before repair

PARISS Spectral Calibration Lamp, Lightform,Inc. Asheville, NC

Overlay of 5 PMT responses and MIDL lamp calibrated output / after repair

Proposed ProceduresProposed ProceduresQuality of Spectral un-mixing:

Purpose:

Measure the spectral un-mixing capability of an imaging system.

Protocol:

Bead slide: 6.0 µm FocalCheck Double Orange fluorescent microspheres(excitation/emission maxima: orange1 = 532/552 & orange2 = 545/565)

o use same optical settings/components (i.e. laser line/excitation, dichroic filter) to acquire reference and experimental spectrao set detection to maximize S/N without any pixel saturationo select a detection bandwidth wide enough to encompass full emission range

(e.g. DoubleOrange beads 520-575 nm) o if available, choose detection set-up (i.e. parallel vs. lambda)o split detection into smallest discreet ‘bins’ if using lambda scanning modeo select an area of the reference spectra (via ROI) with the highest S/N and store in database

Analysis:

Select the most appropriate unmixing data-processing algorithm available:

automatic mode (1st pass / not generally adequate) parallel mode (simultaneous data acquisition across multiple PMTs) lambda mode (lambda scanning utilizing one PMT)

Spectral separation

FocalCheck™ fluorescence microscope test slide

Ring 589/613 nm Core 578/605 nm

Image of a bead where the core and ring have a small spectral separation

ring and core are pseudo-colored for illustration purposes

Linear Unmixing Algorithms

The measured spectra of a ‘mixed pixel’ is broken down into a collection of component spectra (endmembers) and a set of subsequent fractions (abundances) that indicate the ratio of each endmember in the pixel

Three distinct stages of spectral unmixing :- dimension reduction (i.e. data reduction)- endmember determination (i.e. # of distinct spectra)- inversion (i.e. abundance estimation)

Employs a linear mixing model

A Survey of Spectral Unmixing AlgorithmsNirmal Keshava Lincoln Laboratory Journal, Vol.14 No.1, 2003, p.55-78

reference: http://www.lightforminc.com/ Spectral_Unmixing.html

Resolution point at which two objects are perceived as separate and distinct from one another as the Numerical Aperture (NA) of a lens increases, so does the resolution of the imaging system (up to a point) NA = measure of the acceptance angle of a lens, a.k.a. how well it bends light Resolution obtained from an imaging system is affected by:

the specific wavelength of light in usethe diffraction of light (Rayleigh, Abbe & Sparrow limits)lens aberrationssample prep (coverslip thickness, mounting media, RI matching)

Lens imperfections such as coma, astigmatism and spherical aberrations will result in a loss of resolution microscope resolution and the extent of image ‘blur’ is typically described in terms of its Point Spread Function (PSF) an ‘ideal’ PSF demonstrates symmetric balance and proportion

Limit of resolution:d = 0.612 (λ) / N.A.

What is a Point Spread Function & why is it so important ?

a measure of the degree of blurring of an object being imaged and any potential optical path aberrations speaks directly to the quality/resolution of an imaging system a point of light (object plane) spreads out in the image plane ‘Image’ = convolution of an object and the point spread function In 1835, G.B. Airy described a diffraction-limited spot of light focused by a ‘perfect’ lens with a circular aperture an object plane light wave refocused by a lens produces a blurred focal plane point commonly referred to as an ‘airy disc / airy pattern’sub-resolutional beads are typically usedthe PSF of an imaging system directly impacts image refinement using deconvolution algorithms

Proposed ProceduresProposed ProceduresPoint Spread Function:

Purpose:

Measure the point spread function of an imaging system.

Protocol:

Bead slide: 175 nm PS-Speck beads (mixture of blue, green, orange & deep red single-color beads)

o test multiple lens: i.e. 20x, 40x , 63x & 100x (all objectives routinely used for imaging in your lab)o collect a Z series or scan in XZY modeo if needed, suitably rotate image to obtain a ‘side view’o if your system is filter based (non-AOBS), check various dichroic filters

Analysis:

use the MetroloJ plug-in (Fiji / ImageJ) to determine the FWHM lateral & axial resolution compare the experimental vs. theoretical resolution values check the curve fits for all three

Leica SP5 / PSF set-up

MetroloJ plug-in

special thanks to Fabrice Cordelieres / Microscopie Photonique de Fluorescence Multidimensionnelle

MetroloJ PSF report

http://pacific.mpi-cbg.de/wiki/index.php/Fijispecial thanks to Fabrice Cordelieres

MetroloJ PSF report

http://pacific.mpi-cbg.de/wiki/index.php/Fijispecial thanks to Fabrice Cordelieres

‘Idealized’ PSF images

courtesy of Zeiss

Theoretical PSF images / Confocal vs. Widefield

courtesy of Media Cybernetics

Widefield PSF of thick specimencoverslip

decreasingdepth &

worsening PSFs||V

‘3D’ Widefield PSF

20x oil immersion lens NA 0.7

20x / Refractive Index mismatch

collar incorrectly set to water // RI(water)=1.33, RI(Leica imm.oil)=1.518

PSF / 40x oil NA 1.25 / pinhole = 1 airy unit

40x oil NA 1.25 / pinhole = 0.5 & 2 airy units

40x oil NA 1.25 / pinhole = 3 & 5 airy units

40x oil NA 1.25 / pinhole = 7 & 8 airy units

63x N.A. 1.4 oil immersion lens / Brownian motion

Corrective ActionsSpectral Calibration –a.Service call

Spectral Unmixing –a.Try a different unmixing algorithm

- avoid using ‘automatic’- try various linear algorithms- try non-linear (e.g. SWCCA) algorithms

b.Try a different detector set-up- use (5) PMTs with simultaneous scanning OR- use (1) PMT with lambda scanning

c. Improve the signal-to-noise

Point Spread Function –a.Clean the lens and optics / remove all air bubblesb.Check for any possible refractive index mismatchesc.Try a different lensd.Open pinhole aperture to mimic widefield conditionse.Check for optical misalignment

** It is important to note that the above suggestions DO NOT encompass all possible solutions to these issues **

The test specimens proposed for both phases of this study were decided upon byThe test specimens proposed for both phases of this study were decided upon bythe members of the LMRG for their applicability, robustness, ease-of-use and relativethe members of the LMRG for their applicability, robustness, ease-of-use and relative

cost. While the phase I & II tests utilize materials from specific vendors who offercost. While the phase I & II tests utilize materials from specific vendors who offerexcellent products for these purposes, neither the members of the LMRG nor theexcellent products for these purposes, neither the members of the LMRG nor theABRF ABRF endorseendorse the use of these specific the use of these specific vendorsvendors, and fully acknowledge the use of, and fully acknowledge the use of

legitimate alternatives for the purposes of instrument performance testing.legitimate alternatives for the purposes of instrument performance testing.

AcknowledgementsLight Microscopy Research Group

Richard Cole(Chair) Wadsworth Center / NYSDOHCarol Bayles Cornell UniversityClaire Brown McGill UniversityKaren Jonscher(EB Liaison) University of ColoradoKaren Martin West Virginia UniversityCynthia Opansky Blood Center of WisconsinKatherine Schulz Blood Center of WisconsinRobert Stack Wadsworth Center / NYSDOHAnne-Marie Girard Oregon State University

Ad hoc members:Jay Jerome Vanderbilt UniversityGeorge McNamara University of MiamiRobert Zucker US EPA

* We would also like to thank the ABRF for their financial support and commitment to this project *