DeltaVision Imaging System

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

1/
GE Healthcare
4/28/2014

What we need from a Microscope

1) Magnification

Intensity

2) Resolution
Ability to distinguish between objects in 3D space

Distance (D)
3) Contrast
Ability to see objects of interest against background or surroundings

4) Good Cell Viability Characteristics for live cell applications

Sensitivity
Speed
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

2/
GE Healthcare
4/28/2014

Contrast
Signal-to-Background

Background is any systematic

addition to the intensity of the
signal. It represents intensities in
the signal that do not rightfully
belong there.

Signal-to-Noise

Noise is any random variation

that adds to the uncertainty of
the signal.

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

3/
GE Healthcare
4/28/2014

Introduction to DeltaVision
Widefield Restoration Deconvolution Imaging Systems

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

4/
GE Healthcare
4/28/2014

Image Large Structures

10 m

10 m

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

5/
GE Healthcare
4/28/2014

Image Small Organisms

Small structures can
be imaged in detail.
Extracellular parasite
Trypanosoma bruceii
Labeled with calflagin
(green) and cytosolic
proteins (red).
5 m

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

6/
GE Healthcare
4/28/2014

Image VERY Small Particles

HIV particles are
approximately 120
nm in diameter and
can be visualized
within the cell.
HIV (green) and
microtubules (red).

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

7/
GE Healthcare
4/28/2014

Image Thick Specimens

Chick Embryo Neural
Tube.
Fixed tissues up to 70
- 100 microns thick.
Labeled for
microtubules (green)
and DNA (blue).

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

8/
GE Healthcare
4/28/2014

Image Live Cells Over Time

Collect time-lapse
images to study
processes in real time.
Drug-resistant
Mycobacterium
tuberculosis grown in
culture for over four
weeks.

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

9/
GE Healthcare
4/28/2014

Capture Dynamic Events

Follow small, fast
moving objects in live
cells.
Microtubule End
Binding Protein (EB3)
fused to mEmerald.

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

10 /
GE Healthcare
4/28/2014

Sample Types
Mammalian cells
Yeast
Bacteria
Viruses
Samples less than 30 m thick
Fixed AND live cells

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

11 /
GE Healthcare
4/28/2014

Restoration Deconvolution

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

12 /
GE Healthcare
4/28/2014

Widefield microscope
Excitation
Emission
Bright field

coverglass

light source
objective

Light
source

CCD

Inverted microscope
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

13 /
GE Healthcare
4/28/2014

Point Spread Function (PSF)

100 nm (0.1 mm) Fluorescent Bead in a Light Microscope

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

14 /
GE Healthcare
4/28/2014

PSF in a Sample

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

15 /
GE Healthcare
4/28/2014

Principals of Deconvolution
Widefield fluorescence microscopy collects
light from other focal planes and blurs the
image.
Deconvolution mathematically restores the
light to the plane of origin to:
Reduces blur
Improve contrast

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

16 /
GE Healthcare
4/28/2014

Restoration Deconvolution

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

17 /
GE Healthcare
4/28/2014

How Restorative Deconvolution Works

Convolution

Deconvolution

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

18 /
GE Healthcare
4/28/2014

Gaussian Fit of 100nm Beads

~300
~250nm
nm

Deconvolved
Conventional

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

19 /
GE Healthcare
4/28/2014

Diffraction limited volume

250-300 nm
250-300 nm

500-600 nm
Voxel = Volume Pixel

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

20 /
GE Healthcare
4/28/2014

Restoration Deconvolution
Uses PSF to return scattered light to plane of
origin
Constrained Iterative algorithm
Quantitative data
Improves contrast
Maintains data integrity

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

21 /
GE Healthcare
4/28/2014

The Benefit of Deconvolution

original data
deblurred image data
restored image data

As is apparent in the data, deblurring causes a significant loss of pixel intensity

over the entire image, whereas restoration results in a gain of intensity in areas
of specimen detail. A similar loss of image intensity as that seen with the
deblurring method occurs with the application of any two-dimensional filter.
Courtesy: BioTechniques 31: 1076-1097 (2001)

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

22 /
GE Healthcare
4/28/2014

Image Restoration and Contrast

Microtubules stained with anti-tubulin antibody,

Actin stained with Rhodamine-Phalloidin

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

23 /
GE Healthcare
4/28/2014

Deconvolution in Practice
Raw Image (x, y)

Deconvolved Image (x, y)

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

24 /
GE Healthcare
4/28/2014

Deconvolution in Practice

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

25 /
GE Healthcare
4/28/2014

Confocal Microscopy

Wide field v.s confocal

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

26 /
GE Healthcare
4/28/2014

Confocal Microscopy

pinhole
Pinhole
eliminates the
interference
signal

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

27 /
GE Healthcare
4/28/2014

The TruLight Illumination System

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

28 /
GE Healthcare
4/28/2014

The TruLight Illumination System

Faster wavelength switching with solid state
illumination
Optimized light path increases power to the
sample 5 fold
Automated switching between Viewing and
Imaging modes
Supports key applications for multiple imaging
needs

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

29 /
GE Healthcare
4/28/2014

Delivers 5x More Light to the Sample

Standard Light Path

TruLight Illumination

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

30 /
GE Healthcare
4/28/2014

TruLight Illumination Systems

Viewing Mode

Imaging Mode

Same field of view, same exposure conditions

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

31 /
GE Healthcare
4/28/2014

Khler vs. Critical Illumination

Khler Illumination
Light is focused at the
back aperture of the
objective and is defocused
at sample plane.

Area captured by camera

Illuminates full field of

view through oculars.
Illuminates area greater
than CCD chip.
Back Aperture of
Objective
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

Area of Khler
Illumination 32 /
GE Healthcare
4/28/2014

Khler vs. Critical Illumination

Critical Illumination
Light is focused at the
sample plane.
Illuminates area
surrounding CCD chip.
Increases intensity of light
over Khler illumination.
May decrease viewing
area of the oculars.

Area captured by camera

Back Aperture
of Objective
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

Area of Critical
33 /
Illumination GE Healthcare
4/28/2014

The TruLight Illumination System

Use lower exposure times to collect signalimproves viability
Collect images more frequently to capture faster
events
Better signal to noise ratio
Restrict illumination to the region of the camera
chip-- protects cells in neighboring areas

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

34 /
GE Healthcare
4/28/2014

InsightSSI Illumination Units

High performance light technology
Increased power across ALL wavelengths
Uniform and consistent illumination across channels
Discrete channels of illumination eliminate UV leakage

Faster performance
Instant on/off
Microsecond switching between wavelengths
Electronic shuttering eliminates moving parts for maximum speed

Flexible options
4 color Standard Set: DAPI, FITC, TRITC, Cy5
4 Color Fluorescent Protein Set: CFP, GFP, YFP, mCherry
7 Color Combined Set for maximum flexibility

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

35 /
GE Healthcare
4/28/2014

Stages
25 mm x 50 mm Flexure Stage
Precision control over stage motion minimizes axial and
lateral drift
Minimal drift that occurs is localized to within optical
resolution of the camera

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

36 /
GE Healthcare
4/28/2014

DeltaVision Flexure Stages

Precision and accuracy are critical in motion control
Enabled by patented NanoMotion motors and controls
Absolute Accuracy

Repeatability

Step Resolution

< 10 m per 25 mm (x,y)

< 0.2 m (x,y)

20 nm (x,y)

< 0.6 m per 13 m (z)

< 0.1 m (z)

5 nm (z)

DeltaVision stages confine drift to levels undetectable by the

camera for superior time lapse-imaging
Pixel= 64 nm in x, y
Optical Resolution= 200 nm in x, y

x
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

37 /
GE Healthcare
4/28/2014

Environmental Control
Control over the sample
Cell viability improves when the
system mimics the incubator
Humidified CO2
Dark environment
Control over the system
Mechanical drift is decreased with
thermal control
Eliminate drafts in the room
Enclose as much of the system as
possible

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

38 /
GE Healthcare
4/28/2014

Hand-selected Objectives
Objectives are hand selected in order to obtain the best
PSF possible for every DeltaVision.
Astigmatism &
Coma

Spherical
Aberration

Axial Skew

Accept

Reject

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

39 /
GE Healthcare
4/28/2014

Oil kit
To get perfect PSF
Oil match calculator
Temperature
Refractive index of medium
Distance to sample

Perfect PSF

Coverslip thickness

Spherical aberration

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

40 /
GE Healthcare
4/28/2014

Camera
Evolve

HQ2

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

41 /
GE Healthcare
4/28/2014

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

42 /
GE Healthcare
4/28/2014

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

43 /
GE Healthcare
4/28/2014

Advanced Imaging

X4 Laser Module and

Multi-line TIRF Module

InsightSSI
Module
UltimateFocus
Module

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

44 /
GE Healthcare
4/28/2014

UltimateFocus Module
Even with Precision Motion
Control positions the stage for
the customer.
BUT even small changes in
room temperature cause shifts
that are detectable when
measuring in microns.
UltimateFocus provides realtime corrections.

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

45 /
GE Healthcare
4/28/2014

Ultimate Focus
UltimateFocus corrects when the stage moves outside the
user-defined Move Threshold
Automatically maintains focus with 25 nm precision during
experiment
UltimateFocus Correction
150

Stage Movement

100
50
0
0

Stage Position

-50
-100
-150
Time (min)

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

46 /
GE Healthcare
4/28/2014

Find the Sample: Focus Assist

Focus Assist guides the user to bring the

sample to the plane of focus without using the
eyepieces.
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

47 /
GE Healthcare
4/28/2014

Long Term Time Lapse

Imaging
Long Term Live Imaging

Cell Tracking

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

48 /
GE Healthcare
4/28/2014

Cell Dynamics
Turn-over rate of clathrin
coated pits in the cell. WF
and TIRF.

Microtubule End Binding

Protein (EB3) fused to
mEmerald.

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

49 /
GE Healthcare
4/28/2014

Advanced Imaging
X4 Laser Module
-

Supports up to 4 lasers:
405 nm, 445nm, 488 nm, 515 nm, 561 nm

Multi-line TIRF, Protein Kinetics (FRAP FRET)

- Automated to multiplex up to 4 laser lines
- Full chromatic correction (Patent Pending)

Localization microscopy (STORM, PALM)

- Single molecule localization 10-15 nm (depending on number of

photons collected)
- Proprietary Mixed Model Algorithm for fluorophore localization
- Much denser sampling (10-100x)
- Fewer acquisition scans needed
- Can work with photoswitchable or photoactivatable localization
systems
GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

50 /
GE Healthcare
4/28/2014

Nanometer Precision XYZ stage For PointVisiting and Stitching

Example of stitching

Cell tracking

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

51 /
GE Healthcare
4/28/2014

Software interface

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

52 /
GE Healthcare
4/28/2014

Find the Sample: Spiral Mosaic

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

53 /
GE Healthcare
4/28/2014

Thanks and any question?

GE COMPANY CONFIDENTIAL PRIVILEGED & CONFIDENTIAL - INTERNAL USE ONLY

54 /
GE Healthcare
4/28/2014