Point Spread Function, Spectral Calibration & Spectral Separation: Quality Assurance Testing Light Microscopy Research Group Richard W. Cole Wadsworth Center / NYSDOH Albany, New York
Point Spread Function, Spectral Calibration & Spectral Separation: Quality Assurance Testing Light Microscopy Research Group Richard W. Cole Wadsworth
Point Spread Function, Spectral Calibration & Spectral
Separation: Quality Assurance Testing Light Microscopy Research
Group Richard W. Cole Wadsworth Center / NYSDOH Albany, New
York
Slide 2
Why ? until the last 5-10 yrs, simply observing a specimen was
sufficient; advances in light microscopes necessitates 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
were imaging in general; 13% confocal NIST goal of moving medical
imaging & lab testing from an art to a science FDA ensure
manufacturers systems are reliable, guaranteeing that the drugs
will be safe & efficacious Congress provide the financial
support for comparable standards of 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 action Lila
Guterman NCRR Reporter, Winter 2010, p.4-8
Slide 3
Phase One 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--assist core managers & users maintaining 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 throughout 2011, the LMRG tested and defined additional
areas of instrument performance (Phase Two) and refined the
methodology for determining a systems Spectral calibration,
Spectral separation ability and finally the Point Spread Function
of an imaging system What we have done Stack, R., Bayles, C.,
Girard, A., Martin, K., Opansky, C., Schulz, K., and Cole, R.
(2011) Quality Assurance Testing for Modern Optical Imaging
Systems. Microscopy & Microanalysis 17(4):598-606. Cole, R.W.,
Jinadasa, T., and Brown, C.M. (2011) Resolution and Quality Control
of Confocal Microscopy Optics. Nature Prot. 6 (12): 19291941.
Slide 4
Spectral 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
Slide 5
Slide 6
PARISS Spectral Calibration Lamp, Lightform,Inc. Asheville, NC
Overlay of 5 PMT responses and MIDL lamp calibrated output / before
repair
Slide 7
PARISS Spectral Calibration Lamp, Lightform,Inc. Asheville, NC
Overlay of 5 PMT responses and MIDL lamp calibrated output / after
repair
Slide 8
Slide 9
Quality of Spectral un-mixing: Purpose: Measure the spectral
un-mixing capability of an imaging system. Protocol: Bead slide:
6.0 m FocalCheck Double Orange fluorescent microspheres
(excitation/emission maxima: core = 532/552 & shell = 545/565)
o use same optical settings/components (i.e. laser line/excitation,
dichroic filter) to acquire reference and experimental spectra o
set detection to maximize S/N without any pixel saturation o 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 mode o 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 (1 st pass /
not generally adequate) parallel mode (simultaneous data
acquisition across multiple PMTs) lambda mode (lambda scanning
utilizing one PMT)
Slide 10
Spectral separation FocalCheck fluorescence microscope test
slide core = 532/552 & shell = 545/565 ) Image of a bead where
the core and ring have a small spectral separation Ring and core
are pseudo-colored for illustration purposes
Slide 11
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 Algorithms Nirmal Keshava Lincoln
Laboratory Journal, Vol.14 No.1, 2003, p.55-78
Slide 12
Brain tissue (5 different labels)
Slide 13
blue = cell nuclei, green = Nissl-specific for neurons, yellow
= reactive astrocytes, red = microglia, purple = endothelial cells
representing blood vessels.
Slide 14
Slide 15
Resolution point at which two objects are perceived as separate
and distinct from one another Resolution obtained from an imaging
system is affected by: the specific wavelength of light in use the
diffraction of light (Rayleigh, Abbe & Sparrow limits) lens
aberrations sample 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 () / N.A.
Slide 16
What is a Point Spread Function & why is it so important ?
a measure of the degree of blurring of an object & any
potential aberrations speaks directly to the quality/resolution of
an imaging system Image = convolution of an object and the point
spread function 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
used
Slide 17
Point 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 mode o 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
Theoretical PSF images / Confocal vs. Widefield courtesy of
Media Cybernetics
Slide 21
Widefield PSF of thick specimen coverslip increasing depth
& worsening PSFs | V
Slide 22
3D Widefield PSF
Slide 23
20x / Refractive Index mismatch collar incorrectly set to water
// RI(water)=1.33, RI(Leica imm.oil)=1.518
Slide 24
40x oil NA 1.25 / pinhole = 0.5 & 5 airy units
Slide 25
63x N.A. 1.4 oil immersion lens / Brownian motion
Slide 26
Corrective Actions Spectral 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 bubbles b.Check for any possible
refractive index mismatches c.Try a different lens d.Open pinhole
aperture to mimic widefield conditions e.Check for optical
misalignment ** It is important to note that the above suggestions
DO NOT encompass all possible solutions to these issues **
Slide 27
The test specimens proposed for both phases of this study were
decided upon by the 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 offer
excellent products for these purposes, neither the members of the
LMRG nor the ABRF endorse the use of these specific vendors, and
fully acknowledge the use of legitimate alternatives for the
purposes of instrument performance testing.
Slide 28
Acknowledgements Light Microscopy Research Group Carol
BaylesCornell University Claire Brown (chair)McGill University
Richard ColeWadsworth Center / NYSDOH Brady Eason McGill University
Anne-Marie GirardOregon State University Jay JeromeVanderbilt
University Tushare Jinadasa McGill University Karen Jonscher(EB
Liaison)University of Colorado Cynthia OpanskyBlood Center of
Wisconsin George McNamaraUniversity of Miami Katherine SchulzBlood
Center of Wisconsin Marc Thibault Ecole Polytechnique * We would
also like to thank the ABRF for their financial support and
commitment to this project *