Xenogen’s IVIS 200 Series and Living Image Software Vivo Imaging: Xenogen’s IVIS ® 200 Series and Living Image ® Software

In Vivo Imaging:

Xenogen’s IVIS® 200 Series

and Living Image® Software

2

What will be covered?

Introduction

Science of In Vivo Imaging

Xenogen IVIS® 200 Series Hardware Overview

Living Image® Software Overview

Fluorescence System

Training

Hands on Training

3

Science

Why Optical In Vivo Imaging?

Powerful labeling technique - gene expression results in

production of luciferase

Tracer Applications: Amount of light is proportional to number of cells

Functional Applications: Light is produced in response to a stimulus

Non-invasive – does not require subject to be euthanized

Relatively simple instrumentation. Users can run it

themselves – a lab instrument, not an imaging center.

4

Luciferase Emission Spectra and Tissue Transmission

Tissue is not Transparent - Light

Absorbance Depends on Wavelength

Science

5

CCD

Optics

Bioluminescent

Source

Photons Diffuse Through Tissue and the IVIS®

Views this Signal on the Surface of the Subject

Light traveling through tissue

scatters many times creating a "fuzzy" image at the surface of the animal

The IVIS® views the diffuse image on the surface of the subject

Science

6

IVIS® Imaging System 200 Series – Hardware

Customized for in vivo imaging

High sensitivity from 300-900 nm

Large dynamic range

Living Image® software

Hardware

7

IVIS® 200 Imaging System – Hardware

Chiller and

Camera controller

Lenses

CCD camera

Heated Sample

Stage

Electronics

Filter Wheels

Hardware

8 Hardware

Alignment Light Projector

Allows rapid and reproducible positioning of subjects.

Size changes with Field of View setting

9 Software

Living Image® Software

Controls all settings in the IVIS® system

Provides advanced cataloging and browsing tools

Provides analysis tools

Instrument settings are analogous to photography

Images are acquired in a two step process

10 Software – Acquisition

Standard Images are Composed of Two Images

Photographic + Luminescent = Overlay

11

Field of View

Emission Filters

Software

Camera and Lens Settings are Analogous

to Those Used in Standard Photography

Field of View (FOV) is dependent

on the distance from the lens to

the sample

Light collected is proportional

to how long the shutter is open

(exposure time)

Aperture (f/stop) controls the

amount of light collected

CCD resolution is referred

to as Binning

12

Setting Sensitivity – Luminescent Signal Level

The IVIS® CCD camera has a signal range of 0 to 65535

Analog to Digital Counts (216).

Adjust camera settings to obtain a signal level of 600 to

60,000 counts.

Settings that control signal level are:

Exposure time

Binning (CCD Resolution)

f/stop (Aperture)

Software

13

Living Image® Control Panel

Controls Sensitivity

Affects Sensitivity

Software

14

Field-of-View

FOV B: 6.5 cm FOV A: 4.0 cm FOV C: 13 cm

FOV E: 25 cm FOV D: 19.5 cm

Software

15

Exposure Time

Signal level is proportional to exposure time

Shorter exposure time improves throughput.

Longer exposure time increases signal Min exposure time = 0.5 second

Max exposure time= 5 minutes

2 sec f/1 small binning

~5000 counts peak

10 sec f/1 small binning

~25000 counts peak

Software – Acquisition

16

f/stop (lens aperture)

f/stop controls the amount of light

received by the CCD

f/1 is wide open

Max light collection,

default for luminescent

f/8 is smallest aperture

best resolution,

default for photo

f/1 f/8


17

Pixel Binning (CCD Resolution)

Large Binning Medium Binning Small Binning

10 seconds f/2 10 seconds f/2 10 seconds f/2


Binning refers to the grouping of

pixels into a larger super-pixel

Large Binning = High Sensitivity/

Lower resolution

Small Binning = High Resolution/

Lower Sensitivity

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Summary of Basic Camera Settings

Controls Sensitivity

Affects Sensitivity


19

Acquisition – Single Image

Single Image Acquisition

Overlay will automatically take Photo + Luminescent


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Acquisition – Sequential mode

Allows automatic acquisition of a series

of images separated by fixed time points.

Starts Sequential

Image Acquisition


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Series: 3 weeks, 6 weeks

Experiment: M626

Label: M626-34, M626-61

Comment: Dorsal View, Level B

Analysis Comment:

Software – Cataloging

Image Labeling

Good labeling practices are necessary

for effective data browsing

22

Standard Label Sets for Cataloging Images


23

Image Cataloging & Browsing Tools


24 Software – Analysis

Regions of Interest Tools

ROI’s available are:

Contour

Circle

Square

Grid

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2 sec f/2 Small Binning

~5000 counts peak

2.82 x 108 photons/sec

10 sec f/2 Small Binning

~25000 counts peak

2.82 x 108 photons/sec

Software - Analysis

Calibrated Physical Units

Living Image® automatically compensates for device settings: Integration

Time, f/stop, Binning, and Field of View.

Calibrated units are Photons per Second, representing the flux radiating

omni-directionally from a user defined region.

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Measurement Table

Measurement Table displays information about each

Region of Interest in the image.

Measurement Table is user configurable and can be

exported to a spreadsheet

Software - Analysis

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Fluorescence

Fluorophore

ground

state

Excitation

Wavelength

Emission

Wavelength excited

state

Fluorescence

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Twenty-two position computer

controlled Emission filter wheel

Twelve position computer

controlled Excitation filter wheel

150 Watt Tungsten/Halogen lamp

with computer controlled intensity

Low Auto Fluorescence

optics and fibers

Fluorescence

IVIS® Fluorescence System

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Fluorescence Acquisition

Lamp level

High / Low Select Fluorescent

Imaging Mode

Locks Excitation and Emission filters together

Select filters

7

Fluorescence

30 Fluorescence

Excitation and Emission Filters

400 500 600 700 800

0.1

1.0

wavelength / nm

no

rma

lize

d in

ten

sity

normalized transmission of light

through 1cm of tissue

ICG

Cy 5.5

DsRed

GFP

0.1

1

0.1

1

0.1

1

0.1

0.1

0.1

0.1

normalized

intensity

31

Instrument Background Correction

Uncorrected Image

Instrument Measurement

Corrected Image

Fluorescence

32

Fluorescent Calibrated Units: Efficiency

Emitted Light (photons/sec)

Excitation Light (photons/sec)

Efficiency =

Excitation Light Pattern

GFP Well Plate Corrected: Efficiency

GFP Well Plate Uncorrected:

Photons or Counts

Fluorescence

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Regular Rodent Food Alfalfa Free Rodent Food

Cy5.5 Cy5.5

p/sec/cm^2/sr p/sec/cm^2/sr

Fluorescence

Animal Diet and Autofluorescence

in Control Mice

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Alfalfa Free Rodent Food

GFP DsRed Cy5.5 ICG

p/sec/cm2/sr p/sec/cm2/sr p/sec/cm2/sr p/sec/cm2/sr

Fluorescence

Autofluorescence of Control Mice

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Autofluorescence Background Excitation Filters

Fluorescent

Image

Animal autofluorescence

Image using background

excitation filter

Corrected

Image

Fluorescence

36

Background Excitation Filters

400 500 600 700 800 900

0.1

1

wavelength / nm

no

rma

lize

d in

ten

sity

normalized transmission of light

through 1cm of tissue

GFPBkg, GFP

0.1

1DsRed Bkg, DsRed

0.1

1

Cy 5.5 BkG, Cy 5.5

0.1

1ICG BkG, ICG

normalized

intensity

Fluorescence

37

Click # XQA20050216121658_001Wed, Feb 16, 2005 12:18:01Em filter=560nmBin:M (8), FOV12.8, f1, 10sCamera: 23235, EEV

Series: 23235Experiment: in situ calib checkLabel: xls 58 2.89e08Comment: Analysis Comment:

8000

6000

4000

2000

ImageMin = -1550.7Max = 8722.2

counts

Color BarMin = 55

Max = 8509

bkg subflat-fieldedcosmic



8000

6000

4000

2000

ImageMin = -1563.5Max = 5442.6

counts

Color BarMin = 56

Max = 5323




8000

6000

4000

2000

ImageMin = -1568.9Max = 8222.8

counts

Color BarMin = 57

Max = 8023




8000

6000

4000

2000

ImageMin = -1571

Max = 5198.2counts

Color BarMin = 56

Max = 5047


580 nm

600 nm

620 nm

640 nm

Fluorescence

Advanced Topics: Diffuse Luminescent

Imaging Tomography (DLITTM)

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Software

For an In Depth Study – Software Manual

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What we’ve covered . . .

Science

Light is scattered and absorbed by tissue dependant on wavelength of Calibrated physical units compensate for device settings

Hardware

Custom designed for in vivo bioluminescent imaging

Settings are analogous to photography

Software

Images are acquired in a two step process

Sensitivity is controlled by Integration Time, f/stop, and Binning

Living Image® controls IVIS® and provides image analysis tools

Fluorescence

Tissue Autofluorescence and Instrument Background can be subtracted

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Xenogen’s IVIS 200 Series and Living Image Software Vivo Imaging: Xenogen’s IVIS ® 200 Series and Living Image ® Software

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