Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 1 t:/classes/BMS602B/lecture 4 602_B.ppt BME 695Y / BMS

Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 1 t:/classes/BMS602B/lecture 4 602_B.ppt

BME 695Y / BMS 634 Confocal Microscopy: Techniques and Application Module

Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine

& Department of Biomedical Engineering, Schools of Engineering

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.

Week 3Different types of scanning

3D construction & Various Applications


Lecture summary

1. Line scanning confocal microscopy

2. Slit formation

3. Light sources, advantages and disadvantages

4. 4D confocal imaging

5. Applications of Confocal Microscopy


DVC Linescanner

Emission Filters

CCD Camera

Laser

Fiber Optic LinkComputer

ocular Scanhead


DVC 250 Line Scanner

Ocular

Specimen

Laser

“galvanometer”descanning mirrors

Slit

FiltersLensLens

scanning mirror


Stationary Slit Apertures

• Illuminated line must be scanned over specimen

• Emitted light must be descanned

• Light passing through slit must be rescanned to reconstruct a 2D image on the retina


Scanning

• The scanning is performed by oscillating mirrors

• Rate of oscillation is 25-30 Hz


Mirrors

• DVC uses mirrors, not lenses

• Reduces chromatic aberration


Slit

• The confocal slit is variable

• Smallest size is 20 um

• Images of excellent resolution can be collected using video cameras using small slit width


Laser spot to line

Beam splitting lens

Laser in

Laser out


How the laser scans

Scan width can be adjusted


Light Sources - Lasers

• Argon Ar 488-514 nm

• Krypton-Ar Kr-Ar 488 - 568 - 647 nm

• Helium-Neon He-Ne 633


Light Sources

• Kr-Ar lasers most common (488, 568, 647 nm)

• Ar - large (100-200 mW)

• Coupled to head with single mode optical fiber (these preserve coherence)

• Fibers usually have 60% efficiency

• Light is spread over specimen not at point so 25 mW laser produces 3-5 mW at specimen


Main Advantages• Can follow very rapid events• Up to 30 frames per second• Best when searching over large specimens for specific

features• For thick specimens provides an intermediate image

between fluorescence microscopy and point scanners• Systems are small• Can be easily changed from upright to inverted scopes• Very low level light imaging


Disadvantages• Need higher power lasers because point is spread

over line

• Can bleach specimens significantly

• Much high precision in slit manufacture (increase in $)

• Must use camera to detect signal

• Harder to use UV

• Cost is significant relative to point scanners


Image collection

• CCD Camera (usually cooled)

• Faster - cooled and intensified camera


4D confocal microscopy

• Time vs 3D sections

• Used when evaluating kinetic changes in tissue or cells

• Requires fast 3D sectioning

• Difficult to evaluate


4D Imaging

Time1 2 3 4 5

Time

Flu

ores

cenc

e


4D Imaging

Time1 2 3 4 5


4D Imaging

Time1 2 3 4 5

This could also be achieved using an X-Z scan on a point scanner.


Software

• Image analysis– Universal Imaging “Metamorph”– Image Pro-Plus– NIH Image

• Fluorescence Ratioing “Metafluor”


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


Software available• SGI - VoxelView

• MAC - NIH Image

• PC– Optimus– Microvoxel– Lasersharp – Confocal Assistant


Differential Interference Contrast(DIC) (Nomarski)

Visible lightdetector

Specimen

Objective

1st Wollaston Prism

Polarizer

DIC Condenser

2nd Wollaston Prism

AnalyzerLight path


Confocal Microscopy in the Research Laboratory

• Applications

• Live Cell studies

• Time Lapse videos

• Exotic applications


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

Applications


ApplicationsProbe Ratioing

– Calcium Flux (Indo-1, Fluo-3) – pH indicators (BCECF, SNARF)

Molecule-probe Excitation EmissionCalcium - Indo-1 351 nm 405, >460 nmMagnesium - Mag-Indo-1 351 nm 405, >460 nmCalcium-Fluo-3 488 nm 525 nmCalcium - Fura-2 363 nm >500 nmCalcium - Calcium Green 488 nm 515 nmPhospholipase A

- Acyl Pyrene 351 nm 405, >460 nm


Exotic Applications

• Release of “Caged” compounds

• FRAP (UV line)

• Lipid Peroxidation (Paranaric Acid)

• Membrane Fluidity (DPH)


“Caged” Photoactivatable Probes

• Ca++: Nitr-5

• Ca++ - buffering: Diazo-2

• IP3

• cAMP

• cGMP

• ATP

• ATP--S

Examples

Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm


Applications

Organelle Structure & Function– Mitochondria (Rhodamine 123)– Golgi (C6-NBD-Ceramide)– Actin (NBD-Phaloidin)– Lipid (DPH)– Endoplasmic Reticulum


Applications

• Conjugated Antibodies

• DNA/RNA

• Organelle Structure

• Cytochemical Identification

• Probe Ratioing


Flow Cytometry of Apoptotic Cells

G0-G1

SG2-M

Fluorescence Intensity

# of

Eve

nts

PI - Fluorescence

# E

vent

s Normal G0/G1 cells

Apoptotic cells


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

Sca

tter

Sca

tter

Fluorescence

Simultaneous In Situ Visualization of Seven Distinct Bacterial GenotypesConfocal 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.


GN-4 Cell LineCanine Prostate Cancer

Conjugated Linoleic Acid 200 µM 24 hours

10 µM

Hoechst 33342 / PI Hoechst 33342 / PI


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


Step 1: Cell Culture

Step 2: Cell Wash

Lab-Tek

1 2

3 4

5 6

7 8

top view

side view

170 M coverslip

Step 3: Transfer to Lab-Tek plates

confocal microscopeoil immersionobjective

37o heated stage

stimulant/inhibitor added

Step 4: Addition of DCFH-DA, Indo-1, or HE

Below: the culture dishes for live cell imaging using a confocal microscope and high NA objectives.

Live cell studies


Confocal System

Culture SystemPhotos taken in Purdue University Cytometry Labs

Photo taken from Nikon promotion material


Example of DIC and Fluorescnece

Human cheek epithelial cells (from JPR!) stained with Hoechst 33342 - wet prep, 20 x objective, 3 x zoom (Bio-Rad 1024 MRC)

Giardia (DIC image) (no fluorescence) (photo taken from a 35 mm slide and scanned - cells were live when photographed)


Fluorescence Microscope image of Hoechst stained cells (plus DIC)Image collected with a 470T Optronics cooled camera


• Use for DNA content and cell viability– 33342 for viability

• Less needed to stain for DNA content than for viability– decrease nonspecific fluorescence

• Low laser power decreases CVs

Measurement of DNA

G0-G1

SG2-M

Fluorescence Intensity

# of

Eve

nts


PI - Cell ViabilityHow the assay works:• PI cannot normally cross the cell membrane

• If the PI penetrates the cell membrane, it is assumed to be damaged

• Cells that are brightly fluorescent with the PI are damaged or dead

PIPI

PIPI

PIPI

PIPI

PIPI

PIPI

PIPI

PIPIPIPI

PIPI

PI

PIPI

PIPI

PIPI

Viable Cell Damaged Cell


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 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 VonWillebrands factor (A) and Collagen IV (B). Capturing was carried out using a x16 lens under oil immersion. This study was part of aninvestigation 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 andcaptured 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 muchstructural information as possible. ReferencesForeman D, Bagley S, Moore J, Ireland G, Mcleod D, Boulton M3D analysis of retinal vasculature using immunofluorescent staining and confocal laser scanning microscopy, Br.J.Opthalmol.80:246-52


SINTEF Unimed NIS Norway

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.

http://www.oslo.sintef.no/ecy/7210/confocal/micro_gallery.html


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)


Summary

•Linescanning allows faster imaging•Usually requires a CCD camera•4D imaging•Application of fixed cell imaging•Introduction to live cell imaging

Documents

Purdue University Cytometry Laboratories © 1995-2004 J.Paul Robinson - Purdue University Slide 1 t:/classes/BMS602B/lecture 4 602_B.ppt BME 695Y / BMS