Transcript BMS 524 - 'Introduction to Confocal Microscopy and Image

Lecture 12
Applications of Confocal Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of
Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students
encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce
the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson
unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops.
It may not be used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number
of the ideas and figures in these lecture notes are taken from this text.
Slide 1 of t:/classes/BMS524/lectures2000/524lec12.ppt
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UPDATED March 2000
Creating Stereo pairs
Pixel shifting -ive pixel shift for left
+ive pixel shift for right
z
x
Purdue University Cytometry Laboratories
y
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3D images
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Software available
• SGI - VoxelView
• MAC - NIH Image
• PC
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Optimus
Microvoxel
Lasersharp
Confocal Assistant
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Methods for visualization
• Hidden object removal
– Easiest methods is to reconstruct from back to front
• Local Projections
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Reference height above threshold
Local maximum intensity
Height at maximum intensity + Local Kalman Av.
Height at first intensity + Offset Local Ht. Intensity
• Artificial lighting
• Artificial lighting reflection
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Purdue University Cytometry Laboratories
Visualization Issues
Volume rendering is a computer graphics technique whereby the object or phenomenon of interest is
sampled or subdivided into many cubic building blocks, called voxels (or volume elements.) A voxel
is the 3-D counterpart of the 2-D pixel and is a measure of unit volume. Each voxel carries one or
more values for some measured or calculated property of the volume (such as intensity values in the
case of LSCM data) and is typically represented by a unit cube. The 3-D voxel sets are assembled
from multiple 2-D images (such as the LSCM image stack), and are displayed by projecting these
images into 2-D pixel space where they are stored in a frame buffer. Volumes rendered in this manner
have been likened to a translucent suspension of particles in 3-D space.
In surface rendering, the volumetric data must first be converted into geometric primitives, by a
process such as isosurfacing, isocontouring, surface extraction or border following. These primitives
(such as polygon meshes or contours) are then rendered for display using conventional geometric
rendering techniques.
http://www.cs.ubc.ca/spider/ladic/volviz.html
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Additional Material
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Applications
Live Cell studies
Time Lapse videos
exotic applications
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Applications
• Cellular Function
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Esterase Activity
Oxidation Reactions
Intracellular pH
Intracellular Calcium
Phagocytosis & Internalization
Apoptosis
Membrane Potential
Cell-cell Communication (Gap Junctions)
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Applications
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Conjugated Antibodies
DNA/RNA
Organelle Structure
Cytochemical Identification
Probe Ratioing
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G0-G1
S
G2-M
Apoptotic cells
# Events
# of Events
Flow Cytometry of Apoptotic Cells
Fluorescence Intensity
Normal G0/G1 cells
PI - Fluorescence
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Scatter
Flow Cytometry of Bacteria: YoYo-1 stained
mixture of 70% ethanol fixed
E.coli cells and B.subtilis (BG) spores.
mixture
Simultaneous In Situ
Visualization of Seven
Distinct Bacterial Genotypes
Scatter
BG
BG
E.coli
E.coli
Confocal laser scanning image of an
activated sludge sample after in situ
hybridization with 3 labeled probes.
Seven distinct, viable populations can be
visualized without cultivation.
Amann
et al.1996. J. of Bacteriology
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Fluorescence
Purdue University Cytometry Laboratories
178:3496-3500.
GN-4 Cell Line
Canine Prostate Cancer
Conjugated Linoleic Acid 200 µM 24 hours
10 µM
Hoechst 33342 / PI
Purdue University Cytometry Laboratories
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Differential Interference Contrast
(DIC) (Nomarski)
Visible light
detector
Polarizer
1st Wollaston Prism
DIC Condenser
Specimen
Objective
2nd Wollaston Prism
Light path
Purdue University Cytometry Laboratories
Analyser
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Flow-karyotyping of DNA integral fluorescence
(FPA) of DAPI-stained pea chromosomes. Inside
pictures show sorted chromosomes from regions
R1 (I+II) and R2 (VI+III and I), DAPI-stained;
from regions R3 (III+IV) and R4 (V+VII) after
PRINS labeling for rDNA (chromosomes IV and
VII with secondary constriction are labeled)
Purdue University Cytometry Laboratories
A-B): metaphases of Feulgen-stained pea (Pisum sativum
L.) root tip chromosomes (green ex), Standard and
reconstructed karyotype L-84, respectively. C) and D):
flow-karyotyping histograms of DAPI-stained chromosome
suspensions for the Standard and L-84, respectively.
Capital letters indicates chromosome specific peaks, as
assigned after sortingSlide 18 of t:/classes/BMS524/lectures2000/524lec12.ppt
Confocal Microscope Facility at the
School of Biological Sciences which located within the
University of Manchester.
These image shows twenty optical sections projected onto one plane after collection. The images are of the human retina stained with Von
Willebrands factor (A) and Collagen IV (B). Capturing was carried out using a x16 lens under oil immersion. This study was part of an
investigation into the diabetic retina funded by The Guide Dogs for the Blind.
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Examples from Bio-Rad web site
Paramecium labeled with an
anti-tubulin-antibody showing
thousands of cilia and internal
microtubular structures. Image
Courtesy of Ann Fleury, Michel
Laurent & Andre Adoutte,
Laboratoire de Biologie
Cellulaire, Université, Paris-Sud,
Cedex France.
Whole mount of Zebra
Fish larva stained with
Acridine Orange, Evans
Blue and Eosin. Image
Courtesy of Dr. W.B.
Amos, Laboratory of
Molecular Biology,
MRC Cambridge U.K.
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Examples from Bio-Rad Web site
Projection of 25 optical sections of a
triple-labeled rat lslet of Langerhans,
acquired with a krypton/argon laser.
Image courtesy of T. Clark Brelje, Martin
W. Wessendorf and Robert L. Sorenseon,
Dept. of Cell Biology and Neuroanatomy,
University of Minnesota Medical School.
This image shows a maximum
brightness projection of Golgi
stained neurons.
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Confocal Microscope Facility at the
School of Biological Sciences which located within the
University of Manchester.
The above images show a hair folicle (C) and a sebacious gland (D) located on the human scalp. The samples were stained with eosin and
captured using the slow scan setting of the confocal. Eosin acts as an embossing stain and so the slow scan function is used to collect as much
structural information as possible.
References
Foreman D, Bagley S, Moore J, Ireland G, Mcleod D, Boulton M
3D analysis of retinal vasculature using immunofluorescent staining and confocal laser scanning microscopy, Br.J.Opthalmol.
80:246-52
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SINTEF Unimed NIS
Norway
http://www.oslo.sintef.no/ecy/7210/confocal/micro_gallery.html
The above image shows a x-z section
through a metallic lacquer. From this
image we see the metallic particles
lying about 30 microns below the
lacquer surface.
The above image shows a x-y section in the same
metallic lacquer as the image on the left.
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Purdue University Cytometry Laboratories
http://www.vaytek.com/
Material from Vaytek Web site
The image on the left shows an axial (top)
and a lateral view of a single hamster ovary
cell. The image was reconstructed from
optical sections of actin-stained specimen
(confocal fluorescence), using VayTek's
VoxBlast software.
Image courtesy of Doctors Ian S. Harper,
Yuping Yuan, and Shaun Jackson of Monash
University, Australia. (see Journal of
Biological Chemistry 274:36241-36251,
1999)
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http://www.vaytek.com/vox.htm
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