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Hello All,
The ABRF Light Microscopy Research Group (LMRG) has spent considerable time designing, testing and refining the sample type, preparation and imaging protocols for our latest study. The new study will focus on metrics for spherical aberration, sensitivity and signal-to-noise for 3D imaging using a microsphere sample in a tissue like environment. The study overview is below. If you register for the study you will receive an imaging protocol and a sample - which MUST be imaged within one week of its arrival - NO ADDITIONAL SAMPLES WILL BE SENT - so we are asking you to identify an ideal time frame to receive the sample and collect the data.
Due to the data intensive nature of this current study we are only accepting 100 participants. Please fill in the survey by October 29, 2014.
https://www.surveymonkey.com/r/LMRG_Study_3
FYI aside from a pre-meeting image processing and analysis workshop, a keynote microscopy talk (John Condeelis - Albert Einstein) and a full three day microscopy track the ABRF 2015 meeting in St. Louis, MO March 28-31 will feature a session on microscope standards! For more info see conf.abrf.org
If you have any questions or concerns feel free to email me.
Sincerely,
Claire
ABRF-LMRG Study Overview
Introduction: The third study of the Association of Biomolecular Resource Facilities (ABRF) Light Microscopy Research Group (LMRG) is aimed at creating a 3D biologically relevant test slide and imaging protocol to test for 1) system resolution and distortions in 2D and 3D, 2) the dependence of Intensity quantification and image signal-to-noise of the microscope on imaging depth and 3) the dependence of the microscope sensitivity on imaging depth.
Specimen Detail: Fluorescence microspheres (Life Technologies) imbedded in a 120 µm-thick layer of CyGel (BioStatus, UK, Cat# Cy10500) with a refractive index of 1.36 closely match biological tissue. Double-sided adhesive 18 mm square spacers with a well (9 mm diameter, 120 µm deep) were used for the sample preparation (Electron Microscopy Sciences, Cat# 70327-8s).
Microspheres:
Diameter
Relative Intensity
Colour
Excitation Maxima
Emission Maxima
Catalog #
1.0 µm
100%
Red
580
605
F13083
2.5 µm
10%
Green
488
515
L14821
2.5 µm
100%
Green
488
515
L14821
6.0 µm
20%
Deep Red
633
660
L14819
6.0 µm
100%
Deep Red
633
660
L14819
15 µm
100%
Blue Core
Orange Ring
365
560
430
580
F7236
Imaging Conditions: The microsphere samples should be imaged from the coverslip up to 100 µm into the sample. The sample region should include a good selection of 5-10 of each of the different microspheres listed above. A z-stack of images should be collected as per the attached protocols with a minimal x-y and z resolution 0.35 µm. This will result in a data set with approximately 300 images having all the information required to test system resolution, quantification, the signal-to-noise ratio (S/N), and sensitivity.
Metrics: Image stacks will be sent to the LMRG for image analysis, consolidation of the data and interpretation of the results. Briefly, here are the metrics that will be measured.
1) System Resolution
a. 2D Resolution: PSF FWHM along the x-y axis for 5 microspheres at each depth of 0-25 µm, 25-50 µm, 50-75 µm, 75-100 µm.
b. 3D Resolution: PSF FWHM along the x-z or y-z axis for 5 microspheres at each depth of 0-25 µm, 25-50 µm, 50-75 µm, 75-100 µm.
c. Visual Inspection of PSF Shape: Visual inspection for aberrations and scoring of PSF shape along the x-z and y-z axes.
2) Quantification and S/N
a. Quantify Relative Microsphere Intensities: Measure 5 microsphere pair intensity ratios at each depth of 0-25 µm, 25-50 µm, 50-75 µm, 75-100 µm.
b. Measurement of S/N ratio: S/N ratio for microsphere intensity of 5 microspheres at each depth of 0-25 µm, 25-50 µm, 50-75 µm, 75-100 µm.
3) Sensitivity
a. Microsphere Intensity: Compare the intensity of 5 microspheres at depths of 0-25 *m, 25-50 *m, 50-75 *m, 75-100 *m.
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