BMS 524 - 'Introduction to Confocal Microscopy and Image

Download Report

Transcript BMS 524 - 'Introduction to Confocal Microscopy and Image

Lecture 10 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 & Biomedical Engineering 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.

UPDATED March 2007

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 1 of t:/classes/BMS524/lectures2000/524lec12.ppt

Analysis of Apoptotic Cells

G 0 -G 1 S G 2 -M Apoptotic cells Normal G0/G1 cells Fluorescence Intensity PI - Fluorescence

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 2 of t:/classes/BMS524/lectures2000/524lec12.ppt

GN-4 Cell Line

Canine Prostate Cancer Conjugated Linoleic Acid 200 µM 24 hours 10 µM Hoechst 33342 / PI

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 3 of t:/classes/BMS524/lectures2000/524lec12.ppt

Differential Interference Contrast (DIC) (Nomarski)

Visible light detector Polarizer 1st Wollaston Prism DIC Condenser Specimen Objective 2nd Wollaston Prism

Light path

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Analyser Slide 4 of t:/classes/BMS524/lectures2000/524lec12.ppt

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 Slide 5 of t:/classes/BMS524/lectures2000/524lec12.ppt

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.

Amann et al.1996. J. of Bacteriology 178:3496-3500.

Slide 6 of t:/classes/BMS524/lectures2000/524lec12.ppt

Confocal Microscope Facility at the School of Biological Sciences which is 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.

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 7 of t:/classes/BMS524/lectures2000/524lec12.ppt

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.

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

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. Slide 8 of t:/classes/BMS524/lectures2000/524lec12.ppt

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.

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

This image shows a maximum brightness projection of Golgi stained neurons. Slide 9 of t:/classes/BMS524/lectures2000/524lec12.ppt

Confocal Microscope Facility at the School of Biological Sciences which located within the University of Manchester.

hair folicle sebacious gland 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

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 10 of t:/classes/BMS524/lectures2000/524lec12.ppt

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.

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 11 of t:/classes/BMS524/lectures2000/524lec12.ppt

hamster ovary cell

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

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 12 of t:/classes/BMS524/lectures2000/524lec12.ppt

3D imaging using CLSM

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 13 of t:/classes/BMS524/lectures2000/524lec12.ppt

Backscattered light and autofluorescence signals combined: collagen gel & HepG2 cells

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 14 of t:/classes/BMS524/lectures2000/524lec12.ppt

Imaging spectroscopy using CLSM

1400 1200 1000 800 600 400 200 0

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Wavelength [nm]

Slide 15 of t:/classes/BMS524/lectures2000/524lec12.ppt

Spectral imaging methodology

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 16 of t:/classes/BMS524/lectures2000/524lec12.ppt

Spectral CLSM (Zeiss, Nikon…)

Multianode PMT

Zeiss LSM 5

LIVE

A new system concept of optics and electronics

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Dispersion grating Slide 17 of t:/classes/BMS524/lectures2000/524lec12.ppt

Spectral Unmixing - General Concept

32 Channel Detector Collect Lambda Stack Raw Image

FITC Sytox-green Courtesy: Duncan McMillan, Carl Zeiss Microimaging

Derive Emission Unmixed Image

Slide 18 of t:/classes/BMS524/lectures2000/524lec12.ppt

GFP-YFP unmixing

From “Spectral imaging and its applications in live cell microscopy” by T. Zimmermann et al., FEBS Letters, 546(1) 2003, Pages 87-92 Slide 19 of t:/classes/BMS524/lectures2000/524lec12.ppt

Spectral imaging

Stains: Tetramethyl Rhodamine (TRITC; labeling actin), Rhodamine Red-X (labeling desmosomes), and To-Pro3 (labeling nuclei).

Image A was acquired using a 560-nm long-pass filter. Actin filaments, nuclei, and cell–cell junctions all appear red.

Image B was acquired using a sequential scan (multitrack), using a 560–615 band-pass emission filter for the red channel and a 650-nm long pass filter for the blue channel. It is now evident that the nuclear stain is far red, while the cytoplasmic labeling is red.

Image C was acquired using a spectral imaging device, collecting the emission as a series of 11-nm spectral bands across a total range of 552–723 nm.

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 20 of t:/classes/BMS524/lectures2000/524lec12.ppt

Unmixing autofluorescence

Bottom: Results obtained from phantom sample. (a) Image obtained at the peak of one of the quantum dots (bandpass=570+/–10 nm). (b) Unmixed image of the 570 nm quantum dot. (c) Unmixed image of the 620-nm quantum dot. (d) Combined pseudocolor image of (b) (green), (c), and autofluorescence channel (in white, not shown separately). Top left panel: RGB image of the fluorescence emission of the sample. Two species of quantum dots (570 nm, left circle; and 620 nm, right circle) were spotted onto a plastic mouse phantom. Center circle: mixture of both quantum dots. Red and green arrows indicate regions from which sample spectra were obtained.

Top right panel: Spectral data. Red and green spectra correspond to values obtained from the indicated regions. The blue spectrum is the calculated spectrum of the pure quantum dot derived from red and green spectral data.

From “Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging” by James R. Mansfield, Kirk W. Gossage, Clifford C. Hoyt, and Richard M. Levenson, J. Biomed. Opt. 10, 041207 (2005)

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 21 of t:/classes/BMS524/lectures2000/524lec12.ppt

Applications

• Cellular Function – Esterase Activity – Oxidation Reactions – Intracellular pH – Intracellular Calcium – Phagocytosis & Internalization – Apoptosis – Membrane Potential – Cell-cell Communication (Gap Junctions)

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 22 of t:/classes/BMS524/lectures2000/524lec12.ppt

Applications

• Conjugated Antibodies • DNA/RNA • Organelle Structure • Cytochemical Identification • Probe Ratioing

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 23 of t:/classes/BMS524/lectures2000/524lec12.ppt

Software available

• SGI – VoxelView (old and rarely used) • MAC - NIH Image (free) • PC

– Optimus (not now available) – Microvoxel (not now available) – Media Cybernetics software – Image Pro – Lasersharp (Biorad) – Zeiss (proprietary) – Leica (proprietary) – Confocal Assistant (free – old but good)

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 24 of t:/classes/BMS524/lectures2000/524lec12.ppt

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

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 25 of t:/classes/BMS524/lectures2000/524lec12.ppt

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

© 1993-2007 J. Paul Robinson, Purdue University Cytometry Laboratories

Slide 26 of t:/classes/BMS524/lectures2000/524lec12.ppt