From Eye to Insight

Recent Advances in Fluorescence Microscopy

Dr. Daniel Smeets

Application Specialist Confocal Microscopy
daniel.smeets@leica-microsystems.com
Importance of Fluorescence Microscopy

Fluorescence
8000 Microscopy
Number of publications

7000

6000

5000

4000

3000

2000

1000

0
1950 1960 1970 1980 1990 2000 2010

Year

Alexandru Dan Corlan. Medline trend: automated yearly statistics of PubMed results for any query, 2004.
Web resource at URL:http://dan.corlan.net/medline-trend.html
Importance of Fluorescence Microscopy

120 Superresolution
Microscopy
Number of publications

100

80

60

40

20

0
1950 1960 1970 1980 1990 2000 2010

Year

Alexandru Dan Corlan. Medline trend: automated yearly statistics of PubMed results for any query, 2004.
Web resource at URL:http://dan.corlan.net/medline-trend.html
www.leica-microsystems.com/science-lab/
Essential Readings:
http://www.leica-microsystems.com/science-lab/step-by-step-guide-to-fluorescence-microscopy/

http://www.leica-microsystems.com/science-lab/an-introduction-to-fluorescence/

http://www.leica-microsystems.com/science-lab/deconvolution/

DMi8 S
http://www.leica-microsystems.com/products/light-microscopes/life-science-research/inverted-
microscopes/details/product/leica-dmi8/

HyVolution vs. Airyscan?

http://www.leica-microsystems.com/science-lab/super-resolution-on-a-heuristic-point-of-view-about-the-resolution-of-a-light-
microscope/

http://www.leica-microsystems.com/science-lab/hyvolution-super-resolution-imaging-with-a-confocal-microscope/

http://www.leica-microsystems.com/science-lab/white-light-laser/

MP DIVE
http://www.leica-microsystems.com/science-lab/mission-impossible-accomplished-tunable-colors-for-non-descanning-detection/

http://www.leica-microsystems.com/science-lab/laser-beam-shaping-for-multicolor-multiphoton-microscopy/
Overview

HyVolution vs. Airyscan?

DMi8 S

MP DIVE
 TCS SP8 - Ready to grow into nine directions

STED WLL

HCS-A Light Sheet

MP DIVE
SMD

HyVolution 2
CARS

CFS
Sensitivity by design
Spectral Detector

Programmable
Beam Splitter
(AOBS)

Continuous 200 nm
Excitation Spectrum
from 470 670 nm
(WLL)
Abbe Limit: Pushing or Breaking?

STED HyVolution Confocal

HyVolution
Does it colocalize?

140 nm RYR
What is HyVolution?
What does HyVolution offer?
HyVolution: Lateral resolution increase 1.5x
Axial resolution increase 2x

XY: x 1.5
Z: x 2
Contrast x 4 !
HyVolution? 140 nm resolution!
Confocal HyVolution

140 nm resolution using Origami DNA

HyVolution? True Confocal Super-resolution!

Confocal HyVolution
XY XY

XZ XZ
Mitochondrial membrane
HyVolution? Simultaneous Multicolor!

GPI-YFP
H2B-mCherry
Tubulin-SiR

In HeLa Kyoto

Sample courtesy of: Sabine Reither

H2B cell line: Steigemann et al. (2009)
Cell 136:473-84
GPI-YFP construct: Keller et al. (2001)
Nat. Cell Biol. 3:140-149
What is HyVolution?
 PMT
Possible with HyD latest generation of detectors!
50
45 HyD
40 PMT 6357
35
30
PDE(%)

25
20
15
10 GaAsP
Hybrid
5
0
400 500 600 700 800 900
wavelength (nm)

GaAsP

Cut Off ; Threshold Noise

Photon Counting Detector
Improved SN: 200x less noise compared to
(GaAsP)-PMT
Photon Counting Superior signal-to-noise ratio
HyD:
No detector dark current !
No detector noise !
HyD
Photon counting:
perfect raw data!
Only statistical noise
(Poisson, number of
photons)

PMT:
Detector dark current:
Poor signal to background
PMT ratio
Detector noise:
poor signal to noise ratio

Courtesy of Dr. Jana Doehner, Center for Microscopy and Image Analysis, University of Zurich, Switzerland.
Cos-7 cells, Oregon Green 488 (Mitochondria), TMR (Tubulin), SiR-actin.
Summary
HyVolution is

a package of
o SP8 and HyD hardware super-sensitivity

o Market-leading SVI Huygens deconvolution

o CUDA accelerated GPU processing

o Easy to use LAS X software integration with HyVolution Wizard

HyVolution offers
o Resolution of 140 nm (xy) and 400 nm (z)

o The SP8 with HyDs delivers images with a signal-to-noise ratio higher than any other
detection system (photon counting, no dark current, no detector noise).

o True confocal super-resolution

o High-speed data acquisition for fast and gentle imaging (Tandem Scanner)

o Simultaneous Multi-color imaging

Good to know

20
Good to know

Advice on Anti Airy Fast Mode

In 2016 Zeiss introduced the Fast Mode for Airyscan, because
Airyscan is too slow.

Lets recapitulate what Fast Mode Airyscan is:

Fast Mode introduces an optical element which underfills

the back aperture of the objective lens to broaden the PSF in
y-direction. This is equivalent to saying Fast Mode Airyscan
optically reduces the resolution to a level of about 1.7 AU.
This arrangement allows the LSM to span the equivalent of
four lines at once with scanning one line at a bad resolution.
Take home: Fast Airyscan is not super-resolved.

Any gain in terms of resolution with Fast Airyscan is due to

Wiener deconvolution.

21
Good to know

22
Good to know

23
Comparison 1

Airyscan Fast Airyscan HyVolution

channels 1 color 1 color 5 colors

2 colors line by line 2 colors line by line

Resolution xy 140 nm 145 nm, 180 nm < 140nm

Resolution z 450 nm 450 nm 400 nm

speed 5 fps @ 5122 19 fps @ 5122 40 fps @ 5122 (12 kHz)

28 fps @ 5122 (8 kHz)
7 fps @ 5122 (1800 Hz)

24
Comparison 2

Airyscan Fast Airyscan HyVolution

channels 2 colors line by line 2 colors line by line 5 colors

Resolution xy 140 nm 145 nm, 180 nm < 140nm

Resolution z 450 nm 450 nm 400 nm

speed 2,5 fps @ 5122 9,5 fps @ 5122 40 fps @ 5122 (12 kHz)
28 fps @ 5122 (8 kHz)
7 fps @ 5122 (1800 Hz)

25
Strategy on Anti Airy Fast Mode
Strategy on Anti Airy Fast Mode
Leica advantages

Flexible pixelsize (up to 8k x 8k)

Larger max. field size (22 mm @ FOV or 13 mm @ 8 kHz RS)
Flexible zoom
Navigator and Tile Scan w. large FOV
6 channels simultaneous detection (incl. transmission)
Transmission possible
Line average and accu possible
All technology like WLL, AOBS, SP detectors and synergies
garantee a system of best performance

27
 Paramecium Basal Bodies

Confocal HyVolution
Misrepresenting multicolor imaging

1) They call it simultaneous,

but it is line sequential 3) Lossy detection
scanning. This means for three through filters.
colors you can only use 5 fps / This is 1992 !
3 = 1.7 fps effective frame rate

2) Dual bandpass filters for

inflexible and slow LINE
SEQUENTIAL multicolor
imaging, which loses lots of
light on the way
 Still only one color

Nothing changed only one

detector, only one color at a
time. Get real-time multicolor
super-resolution with
HyVolution instead.
Airy and Airy fast mode

19.10.2017 Footer 33
What is often the perception?
Resolution XY Resolution z
At first Glance Zeiss has developed a Zeiss has developed a
dedicated Hardware which dedicated Hardware which
delivers with one shot smooth delivers with one shot
and well contrasted images in smooth and well
superresolved quality. We contrasted images in
always look at deconvolved superresolved quality.
data when talking about Airy. Deconvolved data look at
first glance of course
better than unconvolved
Looking into the details We acchieve minimum the data.
We acchieve minimum
same, often better results due same, often better results
to better deconvolution due to better
algorithms and the better deconvolution algorithms
noise behaviour of our HyDs. and the better noise
No pronounced difference. behaviour of our HyDs. No
Airy is a slow devivce, so we pronounced difference. In
should take our time and z we may be better than
collect as many photons as Airy, certainly better than
possible to make a stunning Airy fast.
image. Our resolution is
Isotropic. Resolution is not an
19.10.2017 "easy win" Footer 34
Recomendation for Resolution:

Do NOT build up your sales strategy based on the assumption that we will beat the
Zeiss performance in big way. I do trust in our method that it can delivery better results
with higher dynamics as we have the better algorithms and the better detectors, but the
difference will not be mind blowing. If you raise the expectation to high we may fail

Stress other points:

black box instead of scientific proven deconvolution.
Results not trustworthy if processed with more harsh algorithms (see paper
Korobchevskaya et al.)
Data volume being 32 time higher creating lots of downstream issues. Think what it
means if a moderate datastack of 104 Mbyte (50 sections, 2ch, 1k*1k) is suddenly 3.4
Gbyte
What about non isotropic resolution for Airy fast mode (Different in x and y)?
Airy does not deliver instantaneous results. A measured example: a 2ch time lapse with
1360*1360 pixels and 50 time points takes 25sec to be processed. Yes, our solution is
competitive!

19.10.2017 Footer 35
Recomendation for Resolution:

The illumination for Airy fast mode, in fact a slit scanner, is not homogeneous. It needs
extra data tweaking to make these artefacts vanish. It actually illuminates 6 pixels at the
same time, so 2 of the 4 pixels are illuminated twice, but only once read out
Airy Scanner is not fast. It takes minimum 5 seconds to image 2k*2k and 40 seconds
for 4k*4k

19.10.2017 Footer 36
What is sensitivity?

Strictly technical speaking it is the capability to detect the highest possible portion of the
emitted photons and to turn them into a signal. Complex topic including optics,
detectors, electronics No noise should be generated on top of the inherent noise.
Difficult to really compare this, to get solid data.

Sensitivity as perceived by low bleach rate ( when rest of conditions and image quality
are similiar ) this indicates that less laser energy is needed to achieve same result.
Good indikator.

Sensitivity as perceived by a good S/N, good contrast, absence of Noise. In short: A

nice image. Probably the attribute most often being correlated with good sensitivity,
but probably the most misleading one. It is not as simple as that However, this is
what the customer sees and what makes it so dangerous.

19.10.2017 Footer 37
Perception on Sensitivity
Real Sensitivity (Transmission,
Detector Sensitivity)
At first glance In absence of measured data the
marketing message is the
decissive one. Zeiss is a master
here and has developed a
dedicated system to improve
sensitivity and resolution (claim).
Easy to grab the principle: 32
detectors see mor than one, right?
Looking into the details We do not have measured data,
but Yes, I believe that our concept
is more effective and that HyD
beats GaAsP detectors. The
beampath is simpler, less
components. But it does not
provide an advantage at first
glance, sensitivity is very difficult
to compare. Up to now no one
dared to publish trustworthy data,
all is hemi-semi-demi stated... Ir is
"US" who need to be convincec
and to convince the customer as
well.
19.10.2017
Sensitivity by low "bleach rate" Sensitivity by high S/N
Especially Airy fast is winning Zeiss Airy delivers always
points for Zeiss as it has a much deconvolved data looking of
longer pixel dwell time wich can course great. Accepted as part of
be used to reduce laser power the hardware, Airy fast adds on
(lower bleach) or to improve S/N top the much longer pixel dwell
or a combination. Pixel dwell time time improving S/N further
is for a single channel 4-6 times
longer than on our conventional
system respectively resonant
scanner.
Pixel dwell time is in itself neutralIf we pay attention to image with
but for a longer dwell time you the purpose of ecellent raw
will get a better photon statistics, images from the beginning (no
more light while you stll can time stress) plus do multicolour
afford to reduce illumination. But and always present deconvolved
of course it increases light data we can certainly draw ahead.
exposure and is hence not The difference in final quality is
beneficial for the specimen. It is probably not big enough in our
not truly less bleaching as our adavantage to compensate the
resonant is doing, And the pixel great marketing message around
dwell time advantage is gone as the Airy scanner, but every
soon as we do Multicolour customer willing to see can see
experiments: for Zeiss you MUST that we can minimum as good
do sequential plus changing for images as Zeiss.
the 3rd colour a filter wheres we
can run it simultaneous if we want
Footer 38
to do so. Good bye, Airy fast...
Recomendation Sensitivity

Sensitivity is key and and I am sure we have the lead here. So worthwhile to spend
time on this topic.

Focus on beautiful images right from the start. They will be compared with deconvolved
data, which are considered as raw. Beautiful is sensitive
Explain clearly the great advantages of HyD, underline Photoncounting as the ultimate
proof of sensitivity (well, it is not, but it delivers hard facts and Zeiss can not do it)
Stress the filter less concept, absence of fibercoupling , simply all our great
beampath
We do not collect the noise of 32 detector elements just to remove it afterwards with a
lot of effort
The Airy fast throws away the light of 16 of the 32 detectors. Does it mean that they are
useless in the normal Airy? Does it really contribute to the sensitivity of Airy fast to
throw away light? Either it proves our version that Airy is a sham or they are stupid
enough to throw light away.
Neither Airy nor Airy fast is truly quick, at least not for larger Pixel formats. We can
afford to collect photons, lets take the time for a good image.

19.10.2017 Footer 39
What is speed?

A very high frame rate.

A maximum number of channels (multicolour) and frames per second

The ability to image a large area at high frame rate in order to generate an overview at
as little time as possible or to study more than a compartment of a cell.

19.10.2017 Footer 40
 Perception Speed
Speed in fps
First Glance Based on paper Specs Zeiss equals
or wins against our SP8. Excellent
numbers for conventional and Airy
fast. Airy fast convinces with best
S/N at high speed. Multispot
illumination and detection is an
intuitive and highly promising
approach. The best of two worlds,
spinning disk and single point
scanner...
Looking into the Details But real high speed goes along on
the Zeiss System with small FOV
and for the Airy scanner with the
loss of simultaneous multichannel
recording. The FOV at highest
speed is really small
19.10.2017
fast Multicolour imaging
Paper specs are better on the
Zeiss machine for conventional
Scanner. Airy Fast vs. Resonant:
Leica wins as it is capable to do
more than one ch at a time. But in
the end Airy fast is at first glance a
full match. Zeiss might be
perceived as better in this
dicipline, but no marked
perceived difference.
Neither Airy nor Airy fast have the
slightest chance against SP8 as
everything needs to be done
frame by frame sequential. No
transmitted light in parallel.
data plus is not truly fast at high
Footer
Fast Overview (Tile Scan)
It you define the goal as taking
and stitching as many as possible
tiles in one colour Airy fast gives
the impression that it is optimal
for tile scan, providing at the same
time better reolution.
Thanks to our much larger FOV it is
a clear advantage for Leica. Zeiss
Airy is unsuitable due to the
extreme amount of data and Airy
fast still is producing 4 times the
pixel formats(large FOV as they
have a bottleneck in terms of read
out events (separate explanation)
41
Recomendation Speed
Albeit the Airy fast is at first glance such a convincing solution the disadvantages of the
concept as it is realized are outweighting the advantages. Worthwhile to spend some
time here, many points to collect.

The Zeiss system is in all scan modes (Airy, Airy fast, normal detectors) based on a
conventional scanner. If you want to be fast you sacrifice FOV big time.
Maximum FOV for both Airies is 12 mm (larger FOV not recommended), shrinking
further for high speed. Our resonant is capable of 13mm.
Airy can not be combined with transmitted light.
Airy is always just one detector. Multicolour? One after the other, like good old SPE. For
3 colours you MUST change even a filter
You have double bandpasss filters in front of the detector; so even if you DO sequential
excitation (well: you must) you will not be able to eliminate the cross excitation.
Drive the demo to more than 2 colours plus TLD, if possible simultaneous aquisition.
You will blow Zeiss out of the water. We have crosstalk? So what, Zeiss has cross
excitation to take care of
Zeiss will avoid to record large FOV (well, anyway only 12mm possible), so no need for
us to take large images when we want to show off with HyVo. Take the spare time to do
better S/N in a smaller region.
 To use Airy fast for tilescans is nonsense: probably multicolour Small FOV makes
more stitches necessary. Creates artefacts, loss of the 10% overlap (in each direction,
so in total you scan 19% of the area double)
If you aim for high pixel formats as you need it for a well resolved Tile Scan you will
loose drastically in terms of aquisition speed. Due to the detector the data amount is
tremendous and they have internal limits for the number of pixels which can be taken
per second. Similiar as we see it for our resonant scanner especially with the old
electronics See separate Excel sheet.
BiDir mode on Zeiss seems to show often (always?) inhomogeneous phasing artefacts
(different in the center than at the border) which you can not correct for. Show what we
can Draw the customers attention to it. All the speed claims of Zeis are done for bidir.
Maybe start the demo in resonant mode Certain advantages.

19.10.2017 Footer 43
Overview

HyVolution vs. Airyscan?

DMi8 S

MP DIVE
 Add triggered components Send and receive trigger signals
LED and laser light sources Simple interface
Infinity TIRF Triggering Robotics Triggering independent from acquisition
Microfluidic devices Record analog data with experiment
Integrated TIRF and alignment sensors Sensors
Azimuth shifting TIRF Microcontrollers etc.
Flexibility, reproducibility and ease of use
Largest TIRF FOV (19mm) on the Market
63x,100x &160x PL APO TIRF objectives Infinity Scanner
TIRF via the Infinity port (easy upgrade)
Same lasers as Infinity TIRF
TIRF High power option for Super Vector Scanning for speed and accuracy
Resolution Any shape, any wavelength, any power
WSU supports 1-5 lasers lines High power for FRAP
405 50mW Low power for Optogenetics
445 80 mW Scanning can be mixed freely with any imaging sequence
488 50mW & 150 mW Easy wizard without restrictions
515 60 mW ROI Dependent Settings
561 50mW & 120 mW Fill
Outline
638 150mW
Laser wavelength
Simultaneous multi-color excitation Global Scanner settings
TIRF with Photo manipulation Scan speed
High power TIRF for Localisation Aperture
microscopy and STORM ND Filter
Free space port for additional laser
Pulsed laser Unit for ablation etc. in October 2017

Infinity TIRF
Module
Infinity TIRF
Module HP
DMi8 S
ALL motorized components Improved camera control
real-time controlled sCMOS Cameras
Z drive Rolling Shutter
Stages Global shutter
DMi8S Filter cubes Streaming mode
Synapse TL/IL mixed modes CCD Cameras
Vertical shift
compensation
Infinity Scanner Modules
anti-smear mode
Streaming Mode
 FRAP (Fluorescence Recovery After
Photobleaching)
Area of a cell is photobleached and
then the recovery of the fluorescence
in the ROI is followed over time.
iFRAP, whole cell is photobleached
except a small region and loss in
fluorescence from the ROI is followed
over time
Applications : studying protein
mobility in cells
FLIP (Fluorescence Loss In
Photobleaching)
Area of a cell is photobleached
repeatedly, time lapse is used to observe
loss or not of fluorescence in a close by
ROI. If ROI becomes bleached then
structures are continuous, if not, ROI is
in a different compartment
Applications : Signalling events,
organelle continuity e.g. endoplasmic
reticulum, receptor motility
FLAP (Fluorescence Localisation After
Photobleaching)
Molecule of interest is labelled with
two fluorescent labels. Only one label
is bleached. Absolute FLAP image is
from subtracting bleached signal from
the unbleached. A ratio can be
obtained by comparing the bleached
area to a second non bleached area
Applications : molecule mobility in
cells
Photoactivation Protein Dynamics
A fluorescent label, e.g. a protein, is
activated from a dark state to a bright
fluorescent state by illuminating the
Protein Interaction
sample with a specific wavelength.
Fluorescent intensity changes are
monitored in the activated region and
non activated regions to follow the Pulsed Laser Unit
protein movements in the cell.
Applications : protein tracking, similar
to iFRAP but less damaging Sample Stimulation

Optogenetics Photoconversion
The combination of genetics and optics to The fluorescence colour changes, e.g.
control well-defined events within specific Kaede, a protein, is irreversibly
cells of living tissue. Light responsive converted from green fluorescence to
proteins allow scientists to turn neurons red by a pulse of UV light.
on or of selectively. Channelrhodopsin is Applications : Protein tracking

Infinity Scanner -
a protein that can be used to label cells
and then turn them on when exposed to Also, Photoswitching
blue light. Halorhodopsin can be used in Some proteins can be switched on and
the same way and will switch cells off off by the user by using pulses of light of
when yellow light is used. Scientists can, different wavelengths

thus, turn individual neurons on or off as
required.
Applications : Frequently carried out on
live brain slices - studying the connection
and signalling of neurons in neural circuits
is one application for optogenetics, the
responses are monitored with electrodes,
patch-clamping or fluorescence.
Uncaging
Caged compounds are synthetically
designed, chemically coated compounds
which are biologically inert but when they
are uncaged or broken open they release
the active compound. Uncaging is
achieved by exposure to UV light
(photolysis).
Applications : Neurobiology
Introduction of neurotransmitters as
caged compounds into synapse junctions,
UV is then used to uncage the
neurotransmitters
Applications
Ablation
Cutting
The pulsed laser can be used to
The pulsed laser can be used to
completely ablate cells, parts of
cut connections between cells,
organisms etc.
cellular structures etc.
Applications : Ablation is used for
Applications : Cell repair, wound
wounding studies, users cause wounds
healing
in tissues and research the repair
mechanisms and cellular processes
resulting from the wounding. In
developmental biology users may
ablate parts of an embryo to see how
development proceeds.
DNA Damage
The UV laser can be used to induce DNA
damage in the cell and then time lapse
imaging can be used to observe the
subsequent effects, this could be done on
many cells so is very efficient.
Applications : observe the movement of
green fluorescence when a nucleus is
damaged and the DNA repair enzyme has
ben tagged with GFP, the area targeted
becomes brighter while the repair is
running and disperses when it is complete
FRET (Forster/Fluorescence Resonance
Energy Transfer)
Energy is transferred from an excited
donor to an acceptor (e.g. CFP/YFP
when CFP is excited, emission is from
YFP when the right filters are used).
Donor and Acceptor need to be very
close together (less than 10nm) thus
giving greater resolution than a standard
light microscope
Applications : protein-protein
interactions eg enzyme activation

TIRF
Infinity
TIRF
Fluorescence illumination from evanescent
(electromagnetic) field
Evanescent field generated by reflection of laser
TIRF
at the glass water interface of the sample as
above
Evanescent field reaches to a depth of upto
200nm
Evanescent Field is the same wavelength as that
of the laser used
Sample must be on a glass coverslip, in aqueous
medium and a TIRF oil objective has to be used,
we have 63x, 100x and 160x.
TIRF imaging gives a high signal to noise ratio as
only a very small volume (upto 200nm) of the
sample is illuminated
TIRF is suitable for membrane studies such as Cell
Membrane imaging, trafficking, Single molecule
tracking, Receptor dynamics/clustering, Receptor
Super resolution, Purified protein preps,
Cytoskeletal organization, Exocytosis Endocytosis,
Cell adhesion, Cell motility
PALM (PhotoActivated Localisation Microscopy)
Nanometer scale resolution, uses properties of photoswitchable fluorophores
Fluorophores are exposed to certain wavelengths and emission spectra
change
Photoactivatable and reversible photoswitchable fluorophores become
fluorescent
Reversible photoswitchable fluorophores change from one fluorescent state
to another
Low power laser will randomly (stochastically) convert the fluorophores so
only a few will be switched on
Image is acquired using high power illuminating laser which switches them off
again
The last two steps are repeated thousands of times to capture all of the
molecules.
As the fluorophores are excited under controlled conditions and
stochastically, can ensure that each dot only comes from one fluorophore,
there are no clumps of fluorescence in the image
XY position of each dot can be determined by fitting the PSF intensity to a
Gaussian distribution
XY position of the molecule can be located to within tens of nanometers
Whole image set of tens of thousands is processed and compiled to form a
map of the location of each molecule, a super resolution image
Resolutions of down to 10nm can be achieved
dSTORM (direct Stochastic Optical Reconstruction Microscopy) (GSD)
Uses standard organic fluorophores like Alexa and ATTO dyes which can
be used as reversible photoswitches
Uses specific buffers
Fluorescence is recovered spontaneously or is light-induced through
oxidation
Using a high power laser with high intensity will send molecules into the
dark state
Photon bursts from individual molecules can be captured, only single
events will eventually be recorded for the highest resolution
Sample depending, there may only be a few events per image, this can
be increased by using the UV laser in parallel which reduce the length of
the Off state and thus increases the number of events per frame
10,000 images or more are acquired to capture single blinking
molecules as they spontaneously come out of the dark state as there
are only a few single molecules in each image
A data set may take between 5 and 10 minutes to acquire
Each single molecule or event is mapped by the software
All of the images are compiled to give a single super resolution image of
up to 20nm
Overview

HyVolution vs. Airyscan?

DMi8 S

MP DIVE
Imagine.
you have a full spectral choice and freedom in MP!

From DEEP blue to DEEP red , Up to 3 Multiphoton lines simultaneously

Imagine.
You can have Deeper insights in complex biological processes
Imagine.
You can visualize a 4Tune of existing and new transgenic
markers!
Flexibility in Emission with in 1 Photon, 2 Photon, 3 Photon, SHG, THG
Imagine.
Imagine.
you have a infinitive FOV with your SP8/Resonant/Camera!
SP8 DIVE
The worlds first tunable deep imaging solution
SP8 DIVE
Deep In Vivo Explorer

DEEP Insights
Spectral FREEDOM
FUTURE Proof
SP8 DIVE with 4Tune detection

The worlds first tunable deep imaging solution.

Adapt to different sample requirements

From Nature Methods, 2005 (Helmchen et

al.)

Allowing to focus into the depth, if required

Mouse brain cortex., Thy1-eYFP, Tx Red
IRAPO 25x1.0 W motCorr
Sample courtesy of Dr. Fuhrmann, DZNE, Bonn
Best Depth Best Resolution

Mouse brain cortex, Thy1-eYFP

IRAPO 25x1.0 W motCorrSample courtesy
of Dr. Fuhrmann, DZNE, Bonn
 Spectral Freedom with 4Tune

Worlds only true spectral NDD

Spectral Freedom with 4Tune

Variable Dichroic Splitter

Variable Bandpass Filter

NON descanned area.

Worlds only true spectral NDD

Spectral Freedom with 4Tune

Enables YOU to adapt to existing and new transgenic markers

2-4 PMTs or HyDs, free positioning

Fully tunable from 380 800 nm
10 nm min bandwith
1 nm step size

Up to four colors simultaneously. Infinite colors sequentially.

DIVE provides Spectral Freedom

Confetti Mouse, intestine tumor

gray SHG Collagen1 Sample courtesy of Jacco van Rheenen.
magenta, blue, green, yellow, red lineage traced University of Utrecht, NL
tumor cells
DIVE provides Spectral Freedom

Second Harmonic Generation

= Frequency Doubling

/2

Sample courtesy of Dr. Eva Rog-Zielinska,

Human heart, valve Institute for Exp. Cardiovascular Med.,
SHG Collagen1 University of Freiburg, DE
DIVE provides Spectral Freedom:
Three photon excitation and third harmonic generation

Mouse brain cortex., Thy1-eYFP cortical neurons, THG

blood cells, 1300 nm
IRAPO 25x1.0 W motCorr
70 fps, 256x256
Sample courtesy of Dr. Fuhrmann, DZNE, Bonn
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Vibrational Fluorescence
Imaging Lifetime Imaging

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DIVE expands your insights towards functional imaging

Funding is heavy beyond Neuroscience

Sources:
NIH Website, Scios360, Nature, IBO, Economist, internal estimations

Great opportunities to address

1 No 1: Core Imaging Facility Manager

Urs Ziegler
Head of Core Imaging Facility, University of
Zrich

Offers equipment and support to a wide range of

scientists/institutions (> 100 user)
High performance highly configurable equipment serving a
wide variety of applications

Need:
Minimum time to result, reliable systems
Breakthrough technology

Pain Point:
Flexible solution to trace multiple varying markers within
deeper layers of model organisms or tissues

need future proof systems with full flexiblity for a variety of applications
2 No 2: Principle Investigator
(Cancer, Immunology)

Jacco van Rheenen

Prof. Intravital Microscopy, University Medical Center
Utrecht

Utilizes a defined range of scientific tools to drive research in a

well funded scientific area
Explicit need for a certain technology

Need:
Imaging machine that allows focus on biology not technology

Pain Point:
Solution to trace an established set of transgenic marker
(> 2) within deeper layers of model organisms or tissues

have an explicit need for a certain technology

We have clear targets

No.1 in
MP!!!
From Eye to Insight

Thank you for your attention! Questions?