Week 3
Different types of scanning
3D construction & Various Applications
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.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 1 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 2 t:/classes/BMS602B/lecture 4 602_B.ppt
DVC Linescanner
CCD Camera
Emission Filters
Fiber Optic Link
Computer
Laser
ocular
Purdue University Cytometry Laboratories
Scanhead
© 1995-2004 J.Paul Robinson - Purdue University
Slide 3 t:/classes/BMS602B/lecture 4 602_B.ppt
DVC 250 Line Scanner
scanning mirror
Laser
Slit
Ocular
Lens
Lens
Filters
“galvanometer”
descanning mirrors
Specimen
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 4 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 5 t:/classes/BMS602B/lecture 4 602_B.ppt
Scanning
• The scanning is performed by oscillating
mirrors
• Rate of oscillation is 25-30 Hz
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 6 t:/classes/BMS602B/lecture 4 602_B.ppt
Mirrors
• DVC uses mirrors, not lenses
• Reduces chromatic aberration
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 7 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 8 t:/classes/BMS602B/lecture 4 602_B.ppt
Laser spot to line
Beam splitting lens
Laser in
Laser out
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 9 t:/classes/BMS602B/lecture 4 602_B.ppt
How the laser scans
Scan width can be adjusted
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 10 t:/classes/BMS602B/lecture 4 602_B.ppt
Light Sources - Lasers
• Argon
Ar
• Krypton-Ar Kr-Ar
• Helium-Neon He-Ne
Purdue University Cytometry Laboratories
488-514 nm
488 - 568 - 647 nm
633
© 1995-2004 J.Paul Robinson - Purdue University
Slide 11 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 12 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 13 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 14 t:/classes/BMS602B/lecture 4 602_B.ppt
Image collection
• CCD Camera (usually cooled)
• Faster - cooled and intensified camera
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 15 t:/classes/BMS602B/lecture 4 602_B.ppt
4D confocal microscopy
• Time vs 3D sections
• Used when evaluating kinetic changes in tissue
or cells
• Requires fast 3D sectioning
• Difficult to evaluate
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 16 t:/classes/BMS602B/lecture 4 602_B.ppt
4D Imaging
Time
2
3
4
5
Fluorescence
1
Time
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 17 t:/classes/BMS602B/lecture 4 602_B.ppt
4D Imaging
Time
1
2
Purdue University Cytometry Laboratories
3
4
© 1995-2004 J.Paul Robinson - Purdue University
5
Slide 18 t:/classes/BMS602B/lecture 4 602_B.ppt
4D Imaging
Time
1
2
3
4
5
This could also be achieved using an X-Z scan on a point scanner.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 19 t:/classes/BMS602B/lecture 4 602_B.ppt
Software
• Image analysis
– Universal Imaging “Metamorph”
– Image Pro-Plus
– NIH Image
• Fluorescence Ratioing “Metafluor”
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 20 t:/classes/BMS602B/lecture 4 602_B.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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 21 t:/classes/BMS602B/lecture 4 602_B.ppt
Software available
• SGI - VoxelView
• MAC - NIH Image
• PC
–
–
–
–
Optimus
Microvoxel
Lasersharp
Confocal Assistant
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 22 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Analyzer
© 1995-2004 J.Paul Robinson - Purdue University
Slide 23 t:/classes/BMS602B/lecture 4 602_B.ppt
Confocal Microscopy in the
Research Laboratory
•
•
•
•
Applications
Live Cell studies
Time Lapse videos
Exotic applications
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 24 t:/classes/BMS602B/lecture 4 602_B.ppt
Applications
Cellular Function
–
–
–
–
–
–
–
–
Esterase Activity
Oxidation Reactions
Intracellular pH
Intracellular Calcium
Phagocytosis & Internalization
Apoptosis
Membrane Potential
Cell-cell Communication (Gap Junctions)
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 25 t:/classes/BMS602B/lecture 4 602_B.ppt
Applications
Probe Ratioing
– Calcium Flux (Indo-1, Fluo-3)
– pH indicators (BCECF, SNARF)
Molecule-probe
Calcium - Indo-1
Magnesium - Mag-Indo-1
Calcium-Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Phospholipase A
- Acyl Pyrene
Purdue University Cytometry Laboratories
Excitation
351 nm
351 nm
488 nm
363 nm
488 nm
Emission
405, >460 nm
405, >460 nm
525 nm
>500 nm
515 nm
351 nm
405, >460 nm
© 1995-2004 J.Paul Robinson - Purdue University
Slide 26 t:/classes/BMS602B/lecture 4 602_B.ppt
Exotic Applications
•
•
•
•
Release of “Caged” compounds
FRAP (UV line)
Lipid Peroxidation (Paranaric Acid)
Membrane Fluidity (DPH)
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 27 t:/classes/BMS602B/lecture 4 602_B.ppt
“Caged” Photoactivatable Probes
Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester
undergoes photolysis upon exposure to UV light at 340-350 nm
Examples
•
•
•
•
•
•
•
Purdue University Cytometry Laboratories
Ca++: Nitr-5
Ca++ - buffering: Diazo-2
IP3
cAMP
cGMP
ATP
ATP--S
© 1995-2004 J.Paul Robinson - Purdue University
Slide 28 t:/classes/BMS602B/lecture 4 602_B.ppt
Applications
Organelle Structure & Function
–
–
–
–
–
Mitochondria (Rhodamine 123)
Golgi (C6-NBD-Ceramide)
Actin (NBD-Phaloidin)
Lipid (DPH)
Endoplasmic Reticulum
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 29 t:/classes/BMS602B/lecture 4 602_B.ppt
Applications
•
•
•
•
•
Conjugated Antibodies
DNA/RNA
Organelle Structure
Cytochemical Identification
Probe Ratioing
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 30 t:/classes/BMS602B/lecture 4 602_B.ppt
G0-G1
S
G2-M
Apoptotic cells
# Events
# of Events
Flow Cytometry of Apoptotic Cells
Fluorescence Intensity
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Normal G0/G1 cells
PI - Fluorescence
Slide 31 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Fluorescence
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
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 32 t:/classes/BMS602B/lecture 4 602_B.ppt
GN-4 Cell Line
Canine Prostate Cancer
Conjugated Linoleic Acid 200 µM 24 hours
10 µM
Hoechst 33342 / PI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 33 t:/classes/BMS602B/lecture 4 602_B.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)
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 sorting
© 1995-2004 J.Paul Robinson - Purdue University
Slide 34 t:/classes/BMS602B/lecture 4 602_B.ppt
Live cell studies
Step 1: Cell
top view
Culture
Step 2: Cell
Wash
Step 3: Transfer to LabTek plates
side
view
Step 4: Addition of DCFHDA, Indo-1, or HE
1
2
3
4
5
6
7
8
170 M coverslip
Below: the
culture dishes for
live cell imaging
using a confocal
microscope and
high NA
objectives.
stimulant/inhibitor
added 37o heated
stage
oil
immersion
objective
Purdue University Cytometry Laboratories
confocal microscope
© 1995-2004 J.Paul Robinson - Purdue University
Slide 36 t:/classes/BMS602B/lecture 4 602_B.ppt
Confocal System
Culture System
Photos taken in Purdue University Cytometry Labs
Photo taken from Nikon promotion material
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 37 t:/classes/BMS602B/lecture 4 602_B.ppt
Example of DIC and Fluorescnece
Giardia (DIC image)
(no fluorescence)
Human cheek epithelial cells (from JPR!) stained with
Hoechst 33342 - wet prep, 20 x objective, 3 x zoom
(Bio-Rad 1024 MRC)
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
(photo taken from a 35 mm
slide and scanned - cells were
live when photographed)
Slide 38 t:/classes/BMS602B/lecture 4 602_B.ppt
Fluorescence Microscope image of Hoechst stained cells (plus DIC)
Image collected with a 470T Optronics cooled camera
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 39 t:/classes/BMS602B/lecture 4 602_B.ppt
Measurement of DNA
G0-G1
S
# of Events
• Use for DNA content and cell viability
G2-M
Fluorescence Intensity
– 33342 for viability
• Less needed to stain for DNA content than for
viability
– decrease nonspecific fluorescence
• Low laser power decreases CVs
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 40 t:/classes/BMS602B/lecture 4 602_B.ppt
PI - Cell Viability
How 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
Viable Cell
Damaged Cell
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
PI
Slide 41 t:/classes/BMS602B/lecture 4 602_B.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)
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 sorting
© 1995-2004 J.Paul Robinson - Purdue University
Slide 42 t:/classes/BMS602B/lecture 4 602_B.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.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 43 t:/classes/BMS602B/lecture 4 602_B.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.
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.
© 1995-2004 J.Paul Robinson - Purdue University
Slide 44 t:/classes/BMS602B/lecture 4 602_B.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.
This image shows a maximum
brightness projection of Golgi
stained neurons.
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 45 t:/classes/BMS602B/lecture 4 602_B.ppt
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
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 46 t:/classes/BMS602B/lecture 4 602_B.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.
Purdue University Cytometry Laboratories
The above image shows a x-y section in the same
metallic lacquer as the image on the left.
© 1995-2004 J.Paul Robinson - Purdue University
Slide 47 t:/classes/BMS602B/lecture 4 602_B.ppt
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)
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 48 t:/classes/BMS602B/lecture 4 602_B.ppt
Summary
•Linescanning allows faster imaging
•Usually requires a CCD camera
•4D imaging
•Application of fixed cell imaging
•Introduction to live cell imaging
Purdue University Cytometry Laboratories
© 1995-2004 J.Paul Robinson - Purdue University
Slide 49 t:/classes/BMS602B/lecture 4 602_B.ppt