Lecture 12 Applications of Confocal Microscopy

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

UPDATED March 2000

Purdue University Cytometry Laboratories

Creating Stereo pairs

Pixel shifting -ive pixel shift for left

+ive pixel shift for right z

Purdue University Cytometry Laboratories y x


3D images











Software available

• SGI - VoxelView

• MAC - NIH Image

• PC

– Optimus

– Microvoxel

– Lasersharp

– Confocal Assistant



Methods for visualization

• Hidden object removal

– Easiest methods is to reconstruct from back to front

• Local Projections

– 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



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



Additional Material

• Applications

• Live Cell studies

• Time Lapse videos

• exotic applications



Applications

• Cellular Function

– Esterase Activity

– Oxidation Reactions

– Intracellular pH

– Intracellular Calcium

– Phagocytosis & Internalization

– Apoptosis

– Membrane Potential

– Cell-cell Communication (Gap Junctions)



Applications

• Conjugated Antibodies

• DNA/RNA

• Organelle Structure

• Cytochemical Identification

• Probe Ratioing



Flow Cytometry of Apoptotic Cells

G

0

-G

1

S

G

2

-M Apoptotic cells

Normal G0/G1 cells

PI - Fluorescence Fluorescence Intensity



Flow Cytometry of Bacteria: YoYo-1 stained mixture of 70% ethanol fixed

E.coli

cells and B.subtilis

(BG) spores. mixture

BG E.coli

BG

E.coli

Simultaneous In Situ

Visualization of Seven

Distinct Bacterial Genotypes

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.

178:3496-3500.

GN-4 Cell Line

Canine Prostate Cancer

Conjugated Linoleic Acid 200 µM 24 hours

10 µM


Hoechst 33342 / PI


Visible light detector

Differential Interference Contrast

(DIC) (Nomarski)

Polarizer

1st Wollaston Prism

DIC Condenser

Specimen

Objective

Light path


2nd Wollaston Prism

Analyser


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)


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 sorting

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.



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.


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.


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



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.


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)


http://www.vaytek.com/vox.htm



Lecture 12 Applications of Confocal Microscopy

Lecture 12

Applications of Confocal Microscopy

Creating Stereo pairs

3D images

Software available

Methods for visualization

Visualization Issues

Additional Material

Applications

Applications

Flow Cytometry of Apoptotic Cells

GN-4 Cell Line

Canine Prostate Cancer

http://www.vaytek.com/vox.htm

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Lecture 12 Applications of Confocal Microscopy

Lecture 12

Applications of Confocal Microscopy

Creating Stereo pairs

3D images

Software available

Methods for visualization

Visualization Issues

Additional Material

Applications

Applications

Flow Cytometry of Apoptotic Cells

GN-4 Cell Line

Canine Prostate Cancer

http://www.vaytek.com/vox.htm

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