Olympus FLUOVIEW FV300 Guide d'installation

CONFOCAL LASER SCANNING

BIOLOGICAL MICROSCOPES

FV300

FLUOVIEW

FV300 CONFOCAL LASER SCANNING MICROSCO

PERFORMANCE FOR THE PERSONAL USER

The FV300 is the ideal choice of laser scanning microscopes for personal users.

Its optical system is fully integrated, from scanner to microscope, and not only delivers

outstanding optical sectioning, but also ensures the easy, flexible expandability required

for any future upgrade.

With its wide choice of options and configurations, including the Olympus inverted,

upright and fixed-stage upright microscope platforms, the FV300 offers excellent

versatility as well as top-class laser scanning performance.

• Highest image quality (12 bit, 2048x2048 pixel resolution)

with economical cost

• Easy operation, with user-friendly software

• Simultaneous capture of 2 fluorescence and 1 transmitted

light detector images

• Capable of the most demanding tasks, with a direct and

efficient optical system

• Optical system chromatically corrects aberrations from UV

to NIR (near infrared red) spectrum

• Fiber illumination system separates fluorescence and

transmitted light sources from the microscope body for

improved temperature stability

FV300-IX71configuration

OPE

qOptical fiber for laser introduction

wBeam collimator

ePolarizer

rDichromatic mirror

tExcitation dichromatic mirror

yXY galvanometer mirror scanners

uPupil lens

iCollector Lens

oPinhole turret

!0 Emission beam splitter slider

!1 Barrier filter slider

!2 Photo multiplier

Preset the conditions for image

acquisition and loading

Storage of Acquisition Settings enables

immediate, one-touch recall of all the

relevant experimental settings and

conditions. Adding new conditions or

altering existing ones is quick and easy.

AOTF: flexible control of the laser

intensity to meet the specific

demands (optional)

The laser exposure will be limited within

the scanning area by default, minimizing

unnecessary bleaching of the specimen.

Option includes:

•Any laser intensity for any excitation area

("Region of Excitation" mode)

•Multiple laser applications

•AOTF controller that provides easy link

with external equipment

ZoomIn scanning

Zoom scanning can be

conducted over any

designated rectangular area.

Since only the region of the

targeted, zoomed-in area is acquired,

scan time and laser exposure of the

specimen is minimized.

Point scanning

The ultimate in fast scanning,

the point scan enables

accurate quantitation of

intensity changes during rapid

physiological events.

Line scanning

A single line may be scanned,

oriented at any angle in the XY

plane. This fast scanning

option permits accurate

quantitation of physiological events such

as Calcium waves or sparks.

Free line scanning

Intensity changes may be

measured over a given period

of time along the length of a

freely drawn line, such as the

trace of an axon or along a cellular

junction.

Clip scanning

By cropping the image, selected areas

can be cut out of complex image stacks.

Flexible setting of scanning size,

zoom, movement and rotation

The observation field and scanning area

are both displayed graphically. Settings

can be confirmed while scrolling through

the zoom ratios. The “pan” button lets the

operator move the image acquisition area

at will, and rotation scanning of images is

also possible.

Simplified toolbar

A newly designed toolbar with various

dedicated buttons has greatly improved

ease of use. The user can execute a

succession of selected processes with

one-click operation for each.

Ultimate ease of operation and monitor display.

Software Graphical User Interface

Dye selection display

When a fluorescence dye is chosen, the

laser and light path settings are selected

automatically, with each of the selected

fluorescence dyes displayed graphically

on the monitor.

Innovative scanning method for

improved performance

ZoomIn

Spot

SlantLine

FreeLine

20%

50% B

Versatile display options

Exchange between condensed and full

image display modes can be performed

with a single touch. Individual panel

layouts can be changed at will, and the

panel in use can be placed in any desired

position.

Image tool bar

X-Y-Z scanning operations and time-

lapse observations both produce multiple

images, which can be displayed in

sequence simply by clicking the

sequential mode button. Channel

selection and image zooming are also

available on the same menu.

Thumbnail display

Data stored in the gallery window are

displayed as thumbnails for easy viewing.

Sequential scanning to prevent cross talk

Sequential scanning may be used to minimize the fluorescence cross talk

often seen between channels in multicolored samples. This is achieved by

exciting each fluorochrome independently, one dye at a time. With the AOTF

function, line sequential scanning is available as well.

* Once optimized: steps q-rcan be performed easily

Scanning unit set-up monitor display

Human colon crypt

Nuclei (Blue) TO-PRO-3

Actin (Green) Alexa 488

APC gene product (Red) Alexa 568

Christine Anderson,

Laboratory of Prof. Ray White,

Hunstsman Cancer Institute, Utah

q Excited by only HeNe633 w Excited by only Kr568 e Excited by only Ar488

r Composition

Easy exchange between display

methods

Independent navigation bars for each

image window enable the display method

to be changed quickly and easily.

Tiling display function for see-at-a-

glance comparison of multiple images

The FV300's live tiling function, which is

especially valuable in time course

experiments, allows observations of

multiple images or changes in the

specimen while the experiment is in

progress. Images in series (e.g. XYt or

XYZ) can be freely displayed.

Single monitor display is also possible.

X -t

SlantLine-t

FreeLine-t

Y -t

Y-Z-tX-Y-t

ZoomIn-t

X-Z-t

X-Y-t

X-Y-Z-t

ZoomIn-Z-t

Patch clamp

BX61WI fixed stage upright microscope+translation stage

Image acquisition ROI designation Intensity versus time

measurement

Calcium sparks in isolated cardiac myocyte

Dr. Sandor Gyorke

Texas Technical University

X -t

Time Course

Using different scanning modes to chart time-lapse changes efficiently.

High-speed (4 frames/sec) image

acquisition

For the high speed observation of the

sample, Fluoview is capable of scanning 4

frames per second in a fast scanning mode

at an image size of 512X512. By limiting the

image size, the frame rate will be even

faster. This scanning mode is suitable for

living cell observation.

Versatile line scanning modes have

many uses

The wide variety of the line scanning modes

(linear/slant/free-line) enables flexible

analysis of rapid time-lapse experiments.

Superior slice patching system

In combination with the unique fixed stage &

nosepiece focusing BX61WI microscope,

the FV300 provides a highly effective

system for slice patching. This unique set-

up has a small footprint for increased room

in a space-limited cage. The remote control

microscope options minimize the danger of

accidentally touching the delicate

experimental settings.Olympus also offers

ideal non-cover glass long working distance

water immersion objectives and an optional

XY translation stage that moves the entire

confocal microscope system while the

sample and other experimental hardware

remains in a fixed position.

Highly precise time-lapse analysis

Fluoview’s wide dynamic range of 12-bit or

4096 grey levels provides enough sensitivity

to detect even the slightest changes in

intensity. The user can designate multiple

regions of interest (ROI) by using drawing

tools. The fluorescence intensity or the ratio

can be analyzed with the intuitive GUI driven

program.

Calcium wave in Xenopus oocyte, Calcium Green staining, fluorescence

pseudo-colored fluorescence image after injection of inositol 3-trisphospate

Japan Science and Technology Corporation, Exploratory Research for Advanced Technology,

Mikoshiba cell control project, Prof. Aya Muto

Calcium wave in isolated cardiac myocyte

Dr. Sandor Gyorke

Texas Technical University

Long working distance, non-cover

glass water immersion objective

Immersion-type LUMPLFL objectives

The 40X water immersion objective in this series has a

3.3mm working distance and an extremely fine tip which

is suitable for micromanipulation using a fixed stage

upright microscope. It has a large N.A. (0.8) and is also

ideal for confocal observations. When using the BX61WI

fixed stage & nosepiece focusing upright microscope

with water immersion objectives, confocal imaging can

be used to monitor time-lapse fluorescence changes in

thick specimens such as brain slices.

The 440nm diode laser can be added

for CFP/YFP FRET imaging.

A 440nm diode laser is optionally available

for CFP/YFP imaging. The 440nm laser line

ideally excites CFP, with minimal

disturbance to YFP, and is therefore

suitable for CFP/YFP FRET experiments.

The high performance LSM objectives,

PLAPO40XWLSM and PLAPO60XWLSM,

are precisely corrected in this wavelength

range, and ensure the highest measuring

reliability.

*For simultaneous observation of CFP and YFP, 440nm

and 515nm laser lines are required.

Ratio imaging to analyze

2-wavelength images

Using time course software, the ratio image

can be continuously displayed in pseudo-

color. At the same time, the intensity of

each channel can be monitored graphically.

The analysis process is presented as an

intuitive flow chart. (optional time course

software: TIEMPO)

Hardware and software support to optimize the environment for FRET.

FRET

Input/output of external trigger signal

The optional time course software gives

control over the input/output trigger signal

by GUI. It is suitable for combined

experiments such as those involving patch

clamping.

CFP Fluorescence wavelength 485nm

Measurement

Ratio changes when cameleon is manifested on the HeLa cell and stimulated by histamine then inhibited by

cyproheptadine.

Cameleon genes provided by Dr. Miyawaki Atsushi in Brain Research Institute.

Equipment: FV300 and HeCd laser

Time period : 4 seconds.

YFP Fluorescence wavelength 530nm

CFP/YFP FRET

Calcium ion concentration in a live HeLa cell using a cameleon (split type) indicator. Energy transfer between

CFP and YFP is proportional to bound calcium. The time series shows the increase of calcium ion density

caused by stimulation of histamine and the effect of blocking by proheputajin.

PAPP for FRAP Application

PAPP: Programmable Acquisition Protocol Processor

Easy, reliable flow of experiments for fluorescence recovery after photobleaching.

AOTF and PAPP function for effective

FRAP (Fluorescence Recovery After

Photobleaching)

Fluorescence recovery after photobleaching

can be analyzed on any designated area by

means of the AOTF-equipped laser

combiner. During the processes of

photobleaching and recovery, the PAPP

function enables time scales to be freely

and easily programmed to suit different

experiment purposes.

New PAPP (Programmable Acquisition

Protocol Processor) makes it easy to

program a wide range of experiments

Using the new PAPP function, which is

included in standard software, the

experiment protocol is created by

describing the individual steps or phases

within the experiment. Users can specify

detailed conditions and parameters for each

step. This function enables users to

construct complex experimental protocols

with minimal effort. PAPP is suitable, for

example, for FRAP experiments that require

more flexibility.

Mouse; hippocampal neurons;

fluorescence of GFP

Living neurons expressing GFP were maintained in

culture and fluorescent images were obtained.

Subsequently, FRAP analysis was performed on

the same cell to determine the diffusion rate of

GFP proteins into the dendritic spines. Rapid

fluorescence recovery ( within seconds ) was

observed.

Shigeo Okabe

Department of Anatomy and Cell Biology

Tokyo Medical and Dental University

Fluorescence quantitation

Line graph depicting average

fluorescence intensity versus time.

qFluorescence Baseline

w- t

Photobleaching at 0.22 sec interval

y- o

Fluorescence recovery at 0.32 sec interval

we t

yu oi

Multi-point time lapse system

High-magnification multi-point time lapse observation of living cells.

Wide-ranging specimen observations

for improved experiment throughput

Use of a motorized XY stage allows the

analysis of time lapse changes in many

points scattered over a wide area. The

system is therefore effective for work with

thick specimens, such as observing

changes in the states and movements of

stem cells using a brain slice, or analyzing

expression mechanisms at the individual

level in an embryo. In wide-ranging tasks

such as analyzing cell functions using GFP,

the system provides many different kinds of

data at the same time, enabling a higher

overall level of experiment efficiency even in

long-lasting observations. In addition, using

separate chambers at the same time makes

it possible to perform different experiments

at the same time. These are just some of

the ways in which this system dramatically

improves the throughput of experiments

requiring long-duration observations.

Features

1. Measure up to 254 points

A variety of scan conditions can be set for each point,

such as XYZ coordinates, the Z-axis acquisition range

and the detector sensitivity.

2. Up to 5 X 5 adjacent fields of view registered

automatically

Since adjacent fields of view are registered

automatically, it is possible to broaden any given field

while maintaining a high magnification level.

3. High-precision XY stage scanning

A wide area can be observed with highly precise position

reproduction. Errors from repetition are not

accumulated. (High-precision XY stage complies

exclusively with the "PROSCAN" model from PRIOR

Scientific)

4. Flexible scanning conditions

The system combines scan flexibility with time lapse

imaging for monitoring changes in the specimen over

time.

No1 No2

No3

No4

No5

No6

Introduction of DsRed2 expression vector into brain of 14th day

mouse embryo through womb electroporation. Taking a specimen

slice from the 15th day embryo and observing the living nerve stem

cell and nerve cells subsequently born from it.

Image courtesy of:

Dr. Kazunori Nakajima

Dr. Hidenori Tabata

School of Medicine, Keio University

Using multi-point software*, it is possible to acquire an XYt, an XYZ or and XYZt image series

at multiple positions through automated software control of the motorized XY stage.

* Multi-point software and motorized XY stage are optional

Tile display

Topographic projection

Height of 3D structure indicated by color.

X-Z

Y-Z

X-Y-Z

X -Z

Y -Z

Using multiple 3D images to obtain accurate 3D structure analysis.

3D Imaging

Easy Z axis operation and setting

The upper and lower limit of Z scanning can

be specified interactively by actually

scanning the sample or by direct input of

the numerical value.

Acquire X-Y-Z images and display X-Y

cross-sectional images quickly and

continuously in increments of 0.01*µm

Thanks to the precision driving mechanism

that enables 0.01µm step control within the

BX61, BX61WI and IX81 motorized

microscopes, high-quality continuous

cross-sectional images can be acquired.

The 3D function also provides extended

focus projections, red/green stereo views,

topographic projections and 3D animations

for exploring the structure of the sample.

Multi-plane images can be created from an

XYZ image series, enabling easy

measurement and observation of horizontal

and vertical cross sections. Other useful

procedures include 3D image cropping,

series animation and simple volume

measurement.

* 0.025µm is the smallest increment for other

microscope combinations.

X-Y-Z

ZoomIn-Z

Y -ZX -Z

SlantLine-Z

FreeLine-Z

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