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to Wednesday Nite at the Lab. >> I'm Tom Zinnen and welcomeody
to the Biotechnology Center here at UW-Madison.
On behalf of our partners, the
Wisconsin Public Television, Wisconsin Alumni Association,
thanks for coming here. We do this every Wednesday
night, 50 times a year. Tonight I'm delighted to have
Steve Paddock give a talk on
Picture the World Through "A BriKaleidoscope Dyes."Lapse:
Which I think is from "Lucy in the Sky with Diamonds."
I get to date myself tonight. Sean, excuse me, Steve works in
Sean Carroll's lab here on
Howard Hughes Medical Institute. campus and is employed by the
of Bristol in the UK and then Hdid a post doc on Northwesterny
University studying cell
He came to the University of behavior.
ago to work in the microscopy Wfacility and starting about 15s
years ago, started working in the Carroll lab.
I'm looking forward to this presentation very much, and
please join me in welcoming Steven Paddock to Wednesday Nite
at the Lab. [APPLAUSE]
Steve Paddock: Thanks, Tom.
Thank you, everybody, for coming out this evening.
The title is actually a little
of two talks. I gave the first title, "A Brief
History of Time Lapse," and then
World with Kaleidoscope Dyes." I switched to "Picture the
the website I threw a few So wtime lapse images in.ed on
If you're for a Brief History of
to be incredibly brief. Time Lapse,[LAUGHTER]ry is going
So the whole talk is based
with Kaleidoscope Dyes." around "Picture the World
"Lucy in the Sky with Diamonds." As Tom said, it's from
insisted that "Lucy in the Sky And I guess Paul McCartney
daughter, Lucy, or his daughter came to him with a picture,s
a painting that she made. And he said, what's that?
And she said, "That's Lucy
So that was the story in thbehind the song.nds."
Not LSD.
But I guess maybe. [LAUGHTER]
So I'm just showing you a slide show right now up here.
I don't know if any of you
Regional Airport last year. paI had the good fortune ofnty
helping with an exhibit entitled
at UW-Madison." And it was up longer than we
expected it to be, and it got a lot of interest from people
going back and forth to the airport.
And we got many great comments, one or two negative comments
but not many. And what I thought I'd like to
do this evening would be to try and explain to you how these
images are produced, some of the
imaging that's led to these images and how we use some of
these images in our work. Most of the images in the Tiny
exhibit were actually produced
technique called laser scanning usiconfocal microscopy.rn
And we're lucky enough to have
molecular biology. an instrument in the lab in
go to my PowerPoint show. And so here's just a collectiond
of images.
These are images produced by These are not all of my images.
part of this show. people on campus here that were
the bright colors, they do have Akind of an artistic side toay
them, but they do also have scientific meaning.
And several of the labs that I
people that I have worked with have been trained in most of the
trying to make an image aesthetically appealing as well
as scientifically kind of relevant.
And I should probably stress
to view these images kind of as if they are pieces are artwork,
but they're actually the result of many long hours of
painstaking experiments, not
in the lab. aAnd these are just kind of thee
tip of the iceberg. So it's easy just to see them
and think oh, this is great, but
hard work and research. So, as Tom said, I work in Seanf
Carroll's lab, and over the years we work on developmental
biology and evolution.
the actual mechanism of how evolution works at the molecular
level.
evolution is to study Adevelopmental biology.
And it's a relatively new field,
leaders in this new field of and Sean is kind of one of the
biology or Evo Devo for short. evolutionary developmental
Stevo Devo. And my ni[LAUGHTER]actually
And it takes in from molecular
to developmental biology, bfrom comparative anatomyy
all the way out to paleontology.
that are millions of years old. So weAnd we talk about deepenes
evolutionary time of 200 million
A model organism that we use is mainly the fruit fly.
I meant to bring some, I forgot to bring them, sorry.
So but what I'd like to do is tell you a few stories about how
this imaging developed. And what I'd like to start off
with is kind of deep
So I'm going to go way back in technological time.
[LAUGHTER] deep technological time to 1985.
two Englishmen sitting My first story starts with
And I'm not talking about the iSahara or the Arctic or thek.
Antarctic.
which is Cambridge in England. I'm talking about this place,
because this is the King And you can tell it's Cambridge
chapel at King's College. ColThis is the River Cam.ous
And one other clue to it being
that the people in the boat, Cambridge rather than Oxford is
Cambridge when you're punting you stand on the deck, which is
the sensible way of doing it, but in Oxford they stand
actually inside the boat on the other end.
So there's a great kind of
cricket and rugby and there's a boat race in London and they put
their milk in their tea in
So anyway, it goes back hundreds different ways.
But anyway, getting back to the two English scientists sitting
in a Bedouin tent.
microscope in the MRC labs in They were aCambridge.veloping a
And the two scientists were John
And John White just retired as White and Brad Amos.
here. tAnd John had a biologicalt
problem to solve. In growing up in England,
English science, a lot of the problems you solve you have to
start off with paper and string and try and build the equipment
yourself. And so John was a great
equipment builder.
So John was a great tinkerer and And, oops, oh, I see.
The biological problem he was trying to solve was back in 1985
many people were looking at cells and embryos using a
technique called
So they were using the power of iimmunology to label specific.
structures within cells so that people were able to raise
specific antibodies to purified proteins and then tag a
fluorescent dye to the antibody which was binding to the protein
in the cell.
back then of some cells growing This isin tissue culture.ook way
And here's my first reference to
A and B is two frames from a time lapse microscopy.
making. tSo what I was doing I wass
filming some cells in tissue
happening is I was filming the culture, and what actually is
Sometimes the nucleus will nucleus rotating.
And then I was trying to figure out what the cytoskeleton was
doing during nuclear rotation.
it, kill it and then label it Sowith a fluorescently labeledx
antibody. And then I would look at the
distribution of the cytoskeleton
figure out how the cytoskeleton wwas related to the nucleard
rotations.
culture they're kind of very So with cells growing in tissue
So you can actually resolve structures in the fluorescent
structures in cells growing in
This is a schematic of the tissue culture.
fluorescence microscope. microscope that you'd use, a
lamp, the HBO lamp house, which would supply a bright light.
And it would go through an
filter out all of the wave excitation filter which would
So a common dye that we would use would be something called
fluorescein, which is excited at
So that excitation filter would take everything else out except
for 480, and then the light would bounce off of the dichroic
beam splitter, go through the objective lens which would focus
it on the specimen. Any labeled structures within
the cell would fluoresce because
light. they would be excited by the
light of less energy would be And then aemitted.wavelength
And then that would come back up
And the dichroic beam splitter along the green path.
light through. would let that longer wavelength
would kind of clean up the And then the barrier filter
what wavelength that was coming signaoff the fluorescein. get
And then you could see it by eye
photograph of it. Okay, so it worked well for
cells growing in tissue culture,
There's a fluorescence flattened cells.
There's the light source at the back, and then you look at it by
eye on the stage and everything.
specimen is actually flooded In such a microscope, the
You can see there that the light with light.
specimen. So the specimen with significant
3-D structure, this is a HeLa cell.
I don't know if you've read,
them, but many cancer cells have many cancer cells, not all of
That's one of their properties, a they tend to round up ine.
culture.
anti-tubulin, fluorescently This is a Helabeled.labeled with
You could actually focus it up
Looking by eye, you could see microtubules but taking a
photograph was impossible.
was a nematode embryo and this And then with an embryo, this
problem. was John White's specific
divisions within a nematode He was tryinembryo.llow the cell
And the first 24 hours or so of embryogenesis there's many,
many, many cell divisions.
increase in size. They get smaller and smaller,t
but the structures get brighter and brighter because there's
more cells. And you just can't see any
structure.
the previous 14 years trying to figure out the neuro-connections
of the 302 nerve cells in the nematode using electron
microscopy. So John kind of knew about this
technique called confocal microscopy.
This is a pattern from 1957 from
And so rather than kind of Marvin Minsky.
light, the idea of confocal flomicroscopy is to scan thewith
specimen with a discrete beam of
And in Minsky's microscopy light.
at cells in living brains, apparatus, his aim was to look
But the idea, all of the living nerve cells in brains.
are based on this basic design. But his idea was way before its
time. The technology wasn't really
available to him. So the technique is the light
path, this would be a transmitted light microscope.
The light source, a very bright light source on this side, the
light passes through a pin hole,
lens into the specimen, and then number 14, it's focused by a
specimen is again focused by any light going through the
hole, number 24, and then that another lens on a second pin
detects the point of light. isSo in that kind of setup weat
would just get a point of light.
actual specimen moved. So in Minsky's apparatus the
mechanism. It was called a stage scanning
fork device. And he used a primitive tuning
was in a telescope so the images It worked but his visual display
impressive, especially to biologists who were using
microscopes with stained
good images. specimens and could get really
epifluorescent, a reflected In hilight version which iso an
incredibly similar to the first diagram I showed you of the
fluorescence microscope.
you only use one lens, number A11, which got rid of a lot of,
alignment problems which you had
So John knew about the stage with the transmitted system.
kind of not good for biology scanning systems but they were
tend to be wet and wobbly. because biological specimens
lose your resolution. If you move your camera taking a
picture, then goodbye clarity.
is they were trying to figure out how they could actually scan
the beam itself.
time in the Bedouin tent and the And they, after a lot of kind of
making kind of modifications to that apparatus, they hit upon a
two-galvanometer scanning scanner.
I'm not going to go into the
really understand them, but dbasically they were able tot
figure out a way of scanning the
And it was about this time I beam in a pretty efficient way.
microscopy facility, and we actually took delivery of one of
the first four prototype
They hooked up with Bio-Rad. microscopes.
instrument. You can see the fluorescence
microscope on the bottom here,
microscope. And then this was their scan
head which produced a scanned beamed of the specimen.
The detectors were
the output was sent into a photomultiplier detectors and
that time computer frame stores compwere kind of new too.ch at
So not only did they have to
the beam, but they also had to solve the problem of scanning
write them the program and get tame a computer programmer to
scanning beam. the computer hooked up with a
mirrors have to, the computer has to keep track of where the
mirrors are in order to build the image on the screen.
So as the beam is scanning
computer kind of puts the image acrossonto the screen.en the
And this system would produce an
in about one second. image of a fluorescent specimen
microscopy when this thing came Abut everybody really wanted to
use it. It was really amazing and fun
technique to use. So John and Brad and Mike
Fordham, who I guess was a
paper in the Journal of Cell computer programmer, published a
of confocal versus conventional imaging of biological structures
by fluorescence microscopy. And this paper literally took
the cell biological community by
they just went around and collected specimens and took a
picture in the regular
took a picture in the confocal fluorescence microscope and then
So here's or HeLa cell which is how it looked in the regular
microscope. You could actually see
structures by eye because your
than anything else. But looking in the confocal mode
you start to see spindle
And if you move the fine focus microtubules.
different view. jusSo the actual out-of-focus a
fluorescence was just gone. So these images were actually
called optical sections because you were actually sectioned the
structure by light rather than a
So the paper really was just physical means.
a real proof of the art. amazing because people just saw
the regular epifluorescence microscope, and suddenly you'ren
seeing structures.
Again, what people used to see, and then what they say with the
confocal microscope.
to worry about was that the Omicrotubules looked kind ofed
wiggly and wavy in the confocal.
specimen preparation was not up And it tto scratch.hat their
Once they kind of figured that
more normal. out, the microtubules looked
in tissue cultures the images And so ewere improved. growing
You could start to see
the nucleus. microtubules kind of going about
from that with my nuclear rotation studies, my time lapse.
So you could optically section, because as long as the specimen
stayed still, it didn't move, you could take an image, the
computer would move the stage by
take another image, and so you a certain set amount and then
through something like a mitotic could take a Z series section
into another program to make a spindle and then send the images
And a lot of these programs already existed for MRI and
other data. So it's kind of like an MRI at
the microscopic level.
I'm just telling the story as if One of the advantages, I mean
the only people doing this, but Jothere were other confocalre
microscopes coming out. There was another, Brakenhoff in
the Netherlands had one.
these microscopes were so popular then Zeiss and Olympus
and all the different companies got on board.
But the great advantage of the
because of the way they were White and Amos microscope,
could adjust the pin hole in scanning the beam, was that you
could increase or decrease the front of the detector so you
that you were taking. oYou could also, so this is a
tissue taken at 4X where you can
And this is taken at 40X where take kind of a map view of it.
structures. But then you can decrease the
area of specimen that you scan
not changing the actual lens. anSo you can impart differentut
magnifications to the same lens.
that I kind of drew. And this is a little schematic
micrometers. A human egg is at 130
you can get the whole egg. Using a 4X with a pin hole open
down you get a smaller optical With the pin hole kind of closed
So these are the kinds of structures that you can kind of
visualize using the confocal
The other thing you could do was system.
different immunofluorescent you coulddyes.l with two
So the green one would be fluorescein and the red one
would be rhodamine. And as long as the specimen
stayed steady and your dichroic mirror angle was consistent and
you used a lens which was corrected for chromatic
aberration, then you could look at two different proteins in the
same cell and then color them green and red.
Another thing you could do would
this is a nuclei in an be to take an optical section,
another layer of nuclei at a epithelium and then there's
them. So that's kind of the basic kind
of story behind the development of the MRC microscope.
And when I moved up to Sean Carroll's lab, I was hit with
some more kind of interesting kind of problems.
At that time we were working
development. exclusively on fruit fly
And he were interested in the Agenes that control the setting
up of the different segments of the body early on in
embryogenesis.
graduate student then, Jim And I was working with a
the gene Harry which is Lexpressed in seven stripes inon
the early embryo. And he was wanting to look at
different genes that related to Harry at the same time.
And at the time we could do double labeling so we could look
at Harry and Kruppel, or Giant and Harry.
And Jim said to me, well, Steve,
at the same time? And we had a krypton/argon laser
on our microscope at the time which had three lines in it,
568, 488, remember that was the fluorescein line, and 647.
And there was a knew dye that had just come out called
cyanine 5, and Jim figured out
protocol of staining three the immunological staining
labeled proteins in one cell. And at that time it was really
hard to get an image from a PC
So we had to get another to a Mac.
wrote this program called RGB Merge where we took the threee
images and then it clunked
it turned it into a TIF file. Most of the microscope companies
have their own propriety formats
to give out the code for that. too which then they didn't used
in Photoshop. And at that time it came up with
this triple-labeled embryo. And we presented this at a fruit
fly meeting, I don't know,
kind of quite well received. 15 years ago, and it was really
can immediately see is the And one of the things that you
actually nuclei and where the same gene is being expressed in
a nucleus then you get an additive color.
So where the green and the red
you get yellow. So you get these kind of rainbow
effects.
meaning. And it also has biological
Bio-Rad came out with this And then a couple of years later
on a light path whereby you sycould actually excite threers
fluorochromes at the same time
And then on the screen you'd get simultaneously.
image and the merged image at tthe touch of a button within ae
second. Whereas RGB Merge took about an
hour to figure out one image but
So the idea is that we can look at development in a series of
fixed and stained cells and figure out which genes are being
turned on.
you kind of get a few domains Tcoming up and then the seven
stripes of Harry and then gastrulation occurs and then
engrailed comes on and you get
starting to know where they're 14 stripes and these cells are
they're getting their fates kind going to be in the embryo and
because they're not in the right of worked out and some are dying
So people work on this kind of place.
cell develops into a multi, and we're using microscopy to figure
these things out. So with fruit flies, the
appendages develop in the, for
larva, in the maggot, from these want of a better word, in the
discs, which are really great stfor confocal imaging.nal
They're relatively flat sheets
And this one here is going to be of cells.
This is a wing imaginal disc. You can dissect them out and
then stain them, fix and stain
the microscope. them, This is the wing.them in
This is the haltere.
of wings and then they have Frthese reduced wings calledir
halteres which are balance
the wings. organs that flap along behind
antenna imaginal disc. And then this is the antenna,
pattern formation where you So tcould look at the genelled
expression patterns and figure
development. out what's going on in
turned on at the same time. ThAnd these are wing halteres
imaginal discs. And Scott Weatherbee in the lab
many years ago came up with these beautiful images of
different genes being expressed all at the same time.
And these are all fixed and stained different discs.
So with the multiple labeling you can get an idea of how these
fields of cells are being set up.
So some of the biology here.
this whole area. This is called the wing pouch,
vestigial, and that labels all And the red gene is called
form the wing. of the cells that are going to
kind of looks purple here, And then the blue gene, which
apterous, and those are going to overlapping the red, is called
the wing. And later on in development this
whole structure is going to fold
And then the green gene is along this boundary here.
actually not a nuclear, one sothat's outside of the cell.'s
You can see it's around all the little circles.
All the circles nucleus.
what are called transcription Manfactors, but CI is on theare
outside.
imaginal disc. And again here's another wing
then these yellow dots here are Tgoing to form the sensorynd
bristles. These cells are being told by
a gene that they're going to be the sensory bristles.
So then you can play around with
presentation purposes too, which the colors for kind of
Photoshop. is a lot of fun within
generally recommended because a Alot of people with red/greens
color blindness have trouble
So if you're giving a presentation you want everybody
to see it.
the best ones to use. AOkay, so Sean gives a lot of
talks, he gives a lot more talk now than he did, but he was at
the University of North Carolina
given his talk and he was being gwalked across campus by thist
really great biologist, Fred
world's expert in butterfly Nijhout, wbiology.nd of the
And he said, gee, Sean do you think all this fruit fly stuff,
what do you think happens with
And to cut a long story short we butterflies?
with butterfly development. sButterflies have a lot morect
colorful patterns on their wings
And this is a butterfly embryo. And just to pick a gene, this
gene is called distalis in red. And it stains the distal
portions, it tells the cells in the distal portions of the
developing limbs that they're going to be the ends of limbs.
And distalis is expressed in
appendages too. Distalis is also expressed in
centipede limbs and something very basic to the insects
called -- limbs.
flavor of how we study evolution So this kind of gives you a
We kind of look at the at the molecular level.
patterns and find them. Evolution doesn't use different
genes for different animals, it uses the same genes in different
ways. So it's not the gene itself,
it's what regulates the gene
limb development. thaAnd then one of the greatfor
surprises is when we looked at
These butterfly imaginal discs, you dissect them out of a
caterpillar.
This is the fifth instar and you They have five instars.
the wing starting to develop can actually see the shape of
The peripheral tissue actually within this beautiful wing disc.
So it's kind of like a cookie dies ocutter.he end.
The shape kind of comes out with
part just dies away. a cookie cutter, and the cutter
developing here, and then the engrailed, the genes that set up
the territories in the fruit fly
the same as the ones that set up wing imaginal disc are exactly
imaginal discs. One of the great surprises was
that when we looked at distalis in imaginal discs, it was
actually setting up the positions of where the eye spot
field is. So another theme of this Evo
Devo is that genes actually recycle during development.
They're used for different
different things. Distalis was used back in the
embryo for setting up the limb
the eye spot fields. fields and now it's setting up
clearly here. This is a 4X confocal image that
I showed you before and.
actually see the cells within a Thesingle eye spot field.can
And it's probably kind of a gradient of gene expression
that's set up.
looked at different wing discs And when Julie Gates in the lab
ones with eye spots had the hot spots of distalis where the eyee
spot fields were. And if we looked at mutant cells
this kind of finding held up. And then Craig Brunetti in the
lab looked at some other genes, and then the actual color seemed
to be related to other gene expression.
So engrailed is in purple with
being recycled. diSo that kind of gives you ais
flavor of this is all fixed and stained tissues.
There's another engrailed.
started to figure out that the So around 1990 or so people
heard of green fluorescent green fluorescent, has anybody
So many jellyfish have this protein that fluoresces.
It happens to fluoresce, excited
fluorescein at 488. And it's turned out, to cut a
long story short and one Nobel
the gene sequence to green Prize later, that figuring out
actually genetically engineer animals so that they, you can
tag the gene for GFP to a gene
So where the gene of interest is of interest.
the GFP will also be expressed. So if you shine a 488 nanometer
light on a transgenic animal
that's green will be where that protein is being expressed.
So here's John White's nematode embryo circa 2009.
This is taken by Kevin
And it's a transgenic nematode. This is GFP, a nuclear GFP,
I think, or maybe it's a
Kevin is able to do is follow the cell divisions in exquisitet
detail. His microscope is fast enough to
take Z series over time of the
mitotic spindles. This is called multi-dimensional
imaging.
expression in living cells using So you can actually look at gene
Here's a fruit fly labeled with engrailed GFP.
If you shine a 488 nanometer
engrailed gene is being expressed fluoresces green.
And there's been much work in kind of engineering GFP, genetic
mutants of GFP. So now there are many, many
color variants available of these called fluorescent
reporter proteins.
what they did was streak a In the Tsien lab in California
GFPs, different colored GFPs and bacterial plate with different
California scene. And Roger Tsien just recently
won a Nobel Prize for his work
Chalfie and a Japanese worker whose name I don't remember.
So the modern confocal
White and Amos, technology has microscopes from the days of
incorporated, lasers have become moved on and the companies have
So rather than one laser, we a lot cheaper to put in.
kind of diode lasers. But one of the great advances is
what's called an AOTF which is
allows you to kind of, rather athan using a glass filter, youh
use an electronic filter that filters the light really, really
fast, kind of really high rates of speed.
So you can kind of collect
You can filter the light a lot images a lot more quickly.
And this is kind of the light path within the system that we
have in the lab.
of a neat new technique that I What I'm leading up to is kind
can genetically engineer three think is so cool in that now you
proteins into a single mouse. And so the cells in the mouse
will randomly incorporate the three different reporters.
So if one cell has three copies of CFP, it will fluoresce blue,
in two CFPs one YFP will be fluoresced light blue and so on
and so on.
reporters are going to be You can'texpressed.here the
But using the new kind of microscopes with the AOTFs and
filter switching and kind of
scientists in Jeff Lichtman's lab have been able to delineate
up to 99 different colors.
in such a mouse, which is called So this is how the brain looks
So rather than two or three you can look at 99 different.
So this is really kind of incredibly artistically
beautiful.
mapping, it's much easier to map But it's also used for kind of
colored cables than all gray a whole bunch of different
So people are using it to map out kind of neuro-networks.
One thing Jeff Lichtman is
hard to do this, but also that finding out is it's incredibly
complicated and the nerve becomes simpler as you get older
rather than the other way around.
But even using this beautiful brain-bow technique, they're
calling these things
And they're actually having to connectomes.
to actually figure out the go back to electron microscopy
gray one in. whole, oh yeah, I did put the
about how the confocal So I've told you a little bit
How we're using it to study the mmechanisms of evolution.
And some of the new kind of techniques of imaging living
cells.
moving and exciting kind of Microscofield right now.bly fast
Not to short change you on the
I want to just show you just how time lapse aspect of the thing,
is. coAnd there's a relatively newly
microscope that's come out
where the field sweeps this way called a and that way.icroscope
And Ernst Stelzer in Germany has
of zebrafish development. These are GFP labeled zebrafishs
embryos.
interested in where the cells actually go, and so he's able to
add color to the actual direction of where the cells are
going.
So with thawith this.t leave you
So it's a whole jumble of cell
some order. m>> Is that a single embryos
developing? >> This is a single zebrafish
embryo developing.
there. That's time going by at the top
kind to cells. So this technique is incredibly
One of the problems with the They don't die.
that they were very hard on early coliving cells.copes was
And that's it.
Well thank you very much
[APPLAUSE]