Olympus Fluoview-1000 User manual

Quick Start Guide

Start-Up 5

Shut-Down 5

............................................................................................................Quick Guide to Getting an Image 6

........................................................................................................Place the sample on the microscope 7

........................................................................................Finding the sample – transmitted light or DIC 7

..................................................................................................................................Brightfield vs. DIC 9

....................................................................................................Finding the sample – epi-fluorescence 9

................................................................................................................Finding the sample - confocal 10

......................................................................................................Setting Up for Confocal Microscopy 11

...................................................................................................................................Choosing the Dye 11

..............................................................................................................Changing the Dye Selection 12

...............................................................................................................Confocal acquisition controls. 12

.................................................................................................Settings for the fluorescence detectors. 13

.................................................................................................................Additional detector controls. 13

.......................................................................................................................................Laser Controls. 14

...................................................................................................................................Line Selection. 14

..............................................................................................................Line availability, Figure 7.2. 14

......................................................................................................Laser Power Selector, Figure 7.3. 15

.......................................................................................................................Scroll List, Figure 7.4. 15

....................................................................................................................Line Settings, Figure 7.5 15

.........................................................................................................................................Scan Controls 16

...........................................................................................................................Scan Mode, Figure 6.1 16

..........................................................................................................................Dwell Time, Figure 6.2 16

..................................................................................................................Scan Regions, Figure 6.3. 17

........................................................................................................Image Aspect Ratio, Figure 6.4. 17

.....................................................................................................................................Box Size, 6.5. 17

.............................................................................................................Box Size effect on resolution 18

......................................................................................................................................AutoHV, 6.6. 18

................................................................................................................................Scan Area Controls 18

..................................................................................................................................Field of View, 8.1. 18

.......................................................................................................................................Scan Area, 8.2. 18

.................................................................................................................................Scan Rotation, 8.3. 18

.....................................................................................................................................Scan Zoom, 8.4. 19

..........................................................................................................................Reset Scan Values, 8.5. 19

.....................................................................................................................................Scan Offset, 8.6. 19

..............................................................................Use of Sequential, Simultaneous and Averaging. 21

................................................................................Simultaneous Capture versus Sequential Capture. 21

....................................................................................................Two Methods for Sequential Capture 21

..................................................................................................Sequential Line Capture, Figure 9.1 21

...............................................................................................Sequential Frame Capture, Figure 9.1 21

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.....................................................................................................Scan mode and sequential capture 22

.......................................................................................................Using Channel Groups, Figure 9A. 22

....................................................................................Adjusting the channels with sequential imaging 22

...............................................................................Adjusting the channels with simultaneous imaging 22

...............................................................................................................................Collecting a Z-Series. 23

...........................................................................................Determine the depth of the optical volume. 23

..........................................................................................................Which way does the focus move? 23

..............................................................................................................Settings for z-series collection. 25

........................................................................................................................What is axial resolution? 25

...................................................................................................................................Vertical Sections. 26

...............................................................................................................Files: Saving and Transferring 26

...........................................................................................................................................Saving Files 26

.............................................................................................................The Olympus “OIB” format. 26

..............................................................................................................The Olympus “OIF” format. 26

.............................................................................................Where are Your Files on the Confocal? 27

....................................................................................................................................Export file types. 27

................................................................................................................Add a scale bar to your file 27

...............................................................................................................................Opening your Files. 27

.......................................................................................................................Olympus free file viewer 28

................................................................................................................Offline Fluoview workstation 28

.............................................................................................Importing Olympus datasets into ImageJ. 28

............................................................................................................Loci-tools Plugin for ImageJ. 28

...........................................................................................................Import Channel Order Macro. 28

...............................................................................................................................Image 5D plugin. 28

..........................................................................................File Import from the OIF.Files directory. 29

..........................................................................................................................................File Transfer. 29

.................................................................File transfer to Mac Pro ‘Fluoview’, confocal file server. 29

.....................................................................................Saving files directly to Mac Pro ‘Fluoview’. 29

..........................................................................................USB and Firewire Ports on the Mac Pro. 29

................................................................Recording to CD-ROM and DVD-ROM on the Mac Pro. 30

............................................Networked file transfer to remote servers from the Fluoview Mac Pro 30

....................................................................................................Using FTP to Windows computers 30

........................................................................Remote access to the Mac Pro from your laboratory. 30

..................................................................................................................Optimizing Image Collection 31

..............................................................................................Qualities of an Optimal Confocal Image. 31

...............................................................................................Overview of image capture properties 31

.........................................................................................................Overview of basic image capture. 31

.............................................................................................Basic settings for confocal microscopy 31

.................................................................................................................................Labeling Controls. 32

...............................................................................................................................Negative controls 32

................................................................................................................................Positive controls 32

............................................................................................................................The confocal aperture 32

........................................................................................................................Optimal Iris Diameter 32

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.........................................................................................................................................Bleedthrough. 33

......................................................................................................................Reducing bleedthrough 33

.................................................................................................Procedure to minimize bleedthrough 33

.....................................................................................................................................................Noise. 34

................................................................................................................Signal Noise (Shot Noise). 34

..................................................................................................................................Detector Noise. 35

............................................................................................................................Kalman Averaging. 35

............................................................................................................................................Background 35

.....................................................................................Bright Images versus Scorching the Specimen 35

.....................................................................................................................Images with Weak Signals 35

...........................................................................................................................................Using Zoom 36

..........................................................................................Zoom reduces objective lens aberrations. 36

..........................................................Use Zoom to fulfill sampling requirements for the specimen. 36

.......................................................................Use Zoom to fulfill adequately sample the objective. 36

.............................................................................................................................Obtaining a Z-Series. 37

.............................................................................................................................................Resolution. 37

................................................................................Sampling frequency and the Nyquist Theorem. 38

............................................................................................................................................Box size. 38

Zoom. 38

..........................................................................................Considerations for increasing resolution. 38

.........................................................................................................................................Size of Pixels 39

..........................................Table. 5 Pixel Size (µm) versus Box Size and Magnification at Zoom = 1 39

................................................................................................................................Microscope controls. 41

.....................................................................................................................................................Focus. 41

............................................................................................................................Fine/Coarse Focus. 41

...................................................................................................................Lowering the Objectives. 41

..................................................................................................................................Manual shutter. 41

..............................................................................................Differential Interference Contrast (DIC). 41

....................................................................................................................................DIC Analyzer. 41

....................................................................................................................................DIC polarizer. 42

........................................................................................................................................DIC mirror. 42

................................................................................................................Setting Koehler Illumination. 42

...........................................................................................................................Condenser position. 43

........................................................................................................................Photoactivation Methods. 44

.....................................................................................................................Zoom versus Laser Power. 44

...........................................................................................................Recording initial recovery event. 44

..........................................................................................................Methods to photoactivate/bleach. 44

.............................................................................................................Using the Stimulus Window. 44

..................................................................................................Avoid spontaneous photoactivation. 46

...........................................................................................Controlling Computer Display Brightness. 46

..................................................................................................................Set ‘Darkroom’ color mode. 47

.............................................................................................................................Technical Information 48

.............................................................................................................................Caring for Objectives 48

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.............................................................................................................FV-1000 Optical Specifications 48

.................................................................................................................................................No Image! 50

..................................................................................................................................Start-up problems. 50

..............................................................................................................................Scans, but no image. 50

..............................................................................The confocal image appears unevenly illuminated: 50

...................................................The epifluorescent or tungsten images appear unevenly illuminated: 50

Figures

Figure 1. Main Fluoview window. 6

Figure 2. Enlarged view of Fluoview windows. 8

Figure 3. Microscope Controller Window 9

Figure 4. Image Acquisition Window – Basic controls for Finding the Image 11

Figure 5. Channel Settings for the Photomultiplier Tube (PMT) 14

Figure 6. The Scan Mode and Scan Size settings of the Acquisition Setting Window. 16

Figure 7. Selecting laser lines and setting laser power 15

Figure 8. Scan Controls for Zoom, Pan and Rotation 19

Figure 9. Sequential Imaging Control 20

Figure 9A. Sequential Imaging Control 20

Figure 10. Z-Series Control Window 24

Figure 11. Z-Series Control Window 24

Figure 12. DIC and Focus Controls, Lower Half of the Microscope 40

Figure 13. Condenser and DIC Controls 42

Figure 14. Tilting the condenser. 43

Figure 15. The Stimulus Setting window for photoactivation 45

Figure 16. Photobleaching an ROI. 46

Figure 17. The toolbar item for control over display color 47

Figure 18. Fluoview appearance in Darkroom Color 47

Tables

Table 1. Mirror Buttons 10

Table 2. Objective Lenses Available 12

Table 3. Scan Speed and Acquisition Time 14

Table 4. Optimal Z-Step Values 37

Table 5. Pixel Size (µm) versus Box Size and Magnification at Zoom = 1 39

Table 6. Dichroic Mirrors and Barrier Filters 38

Table 7. Laser Lines on the FV-1000 38

Table 8. Index of Refraction for Common Mounting Materials 49

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Quick Start Guide

A. Start-up:

All items are lettered in the order in which they are to be turned on.

1. Enter your name and session into the Olympus FV-1000 web calendar;

2. Turn on items labeled with letters:

(A) Turn on mercury lamp, if needed - power supply is on shelf over monitor;

(B) Turn on system controllers – press black switch on power supply on right side of air table, next

to the computer.

the lasers) (405 nm, 561 nm, 633 nm);

(D) Fire up the DPSS laser (561 nm) – press the green button;

(E) Turn on Argon ion laser – turn the key to “ON” (458 nm, 488 nm, 514 nm);

(F) Turn on computer, black tower to right of confocal, press round button to right of floppy;

a. Windows user name is “Administrator”

b. password is “fluoview”

3. Double-click on the Fluoview icon, left side of computer monitor desktop, “FV-10ASW”;

4. Choose your name from the users list and enter your password;

NOTE: Fluoview user accounts open with the last settings used by the previous user.

B. Shut down:

1. Transfer files from your account (See File Transfer, Section );

2. Exit from Fluoview;

3. Ignore the warning about light coming through the microscope.

4. Turn off power strip (B);

5. Turn off power strip (C);

6. Turn off argon ion laser (E), key only;

7. Delete images from the hard drive;

8. Turn off the computer;

9. Remove immersion oil from objectives, clean the stage and platform.

10. Edit time in the web calendar, if you went longer than reserved.

Keys versus Switches.

Use the key to turn the argon laser on and off. Do not use the power switch – that will prevent the fan to

run during cooling down and potentially damage the laser.

For components controlled by power strips (B, C, D), do not fiddle with the keys or switches on the

actual components. The exception is that the 561 nm laser (D) needs its green button pushed to turn it

on, but it is turned off by the power strip (C), along with the other 2 lasers.

If the building power fails, backup batteries will provide about 10 minutes to turn off all components.

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1. Quick Guide to Getting an Image

Examples of the software control windows are on the following pages. The text contains numbers that

refer to specific numbered controls in figures.

Your overall goal with fluorescent microscopy is to image the specimen with the least amount of

photobleaching. This requires limiting specimen exposure to damaging levels of excitation light.

1. Find the specimen using the eyepieces and epi-fluorecence. This is faster than scanning.

2. Low magnification objectives allow rapidly searching over large areas, but usually dimmer.

3. Low intensity transmitted light is safest for finding and positioning the sample.

4. DIC is usually better than brightfield for low contrast samples, such as monolayers or picking out

structures in complex specimens.

5. Epi-fluorescence is usually faster than confocal to find the fluorescent sample.

6. Epi-fluorescence allows you to see a blurry glow from out of focus objects, unlike confocal.

7. Use the least amount of light necessary to find the sample to reduce photobleaching and toxicity.

8. Begin confocal imaging using low laser intensity with high PMT HV while surveying the

sample.

9. Focusing on the edge of the coverslip by transmitted light can help to find initial focus.

Figure 1. Main Fluoview window.

Drag this corner to resize the window

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1.1 Place the sample on the microscope

1. Make sure the objective lens is not extending above the level of the specimen holder;

a. If it is, rotate the focus knob to lower the objective lens (set to Coarse Focus)

2. Press ‘ESC’ button behind either focus knob to lower the objectives;

3. Move the condenser up, out of your way;

4. Install the appropriate specimen holder in the stage, if needed;

5. Place the slide on the stage with the coverslip downwards;

6. The sample should be centered in the stage opening to avoid bumping the sample holder with the

lens as you move the stage to position the desired field of interest;

7. Press ‘ESC’ button to return the lens to focus;

8. Lower the condenser back into position;

9. Do not use the keypad on the front of the microscope, it has been disabled;

10. All microscope control must be through the Fluoview software windows, Figures 2, 3;

11. Open the Microscope Controller window, if it is not open: Fluoview

menu>Device>Microscope Controller…, Figure 2.1;

12. Select an objective: Microscope Controller Window>Objective Lens>click on desired lens,

Figures 2.4 and 3.2, see Table 2;

13. Condenser setting: Microscope Controller Window>Condenser sets condenser automatically –

don’t touch, Fig. 3.1;

14. Select mirrors (fluorescence filter cubes): Microscope Controller Window>Mirror>click on

desired mirror, Fig. 3.3, see Table 1;

15. Fine focus/coarse focus: press the ‘F/C’ button below each focus knob, Figure 12.2, to toggle

between focus modes;

16. Lower/raise lens to apply immersion oil: press the ‘ESC’ button behind each focus knob, Figure

12.3;

17. Center the region of interest in the field of view with the joystick;

1.2 Finding the sample – transmitted light or DIC

1. Center the sample over the stage opening to avoid bumping the stage with the lens;

2. Refer to Microscope Controls, Sections 7.2 and 7.3, for details on using DIC and setting Koehler

illumination;

3. Open the Microscope Controller window, Fluoview menu>Device>Microscope Controller…,

Figure 2.1;

4. All microscope control must be through the Fluoview software windows, Figures 2.1, 2.4;

a. Select lens: Microscope Controller Window>Objective Lens, Figure 3.2, Table 2;

b. Condenser controls are set automatically: Microscope Controller Window>Condenser –

don’t touch, Fig. 3.1;

c. DIC condenser settings are automatically selected when transmitted lamp is on:

Microscope Controller Window>Mirror>Select DIC (DICT), Fig. 3.3;

5. Turn on the transmitted light;

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a. Image Acquisition Control Window>Click on transmitted light, Figure 4.1;

b. Image Acquisition Control Window>adjust brightness (TR Lamp) (click or scroll),

Figure 4.4;

6. Bring sample into focus;

7. Set Koehler illumination [optional, Section 7.3];

8. Center the region of interest in the field of view;

9. Turn off the transmitted light with the Image Acquisition Control Window, Figure 4.1;

10. Switch to fluorescent imaging.

Remove the DIC analyzer from the light path for optimal confocal images!

Figure 2. Enlarged view of Fluoview windows.

2.1 Menus

2.2 Acquisition settings

2.4 Microscope controller

2.3 Image acquisition

2.5 Dye list window

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1.3 Brightfield vs. DIC

The default setting for plain brightfield imaging is with the condenser DIC prism in place, “DICT”.

True brightfield illumination is obtained by removing the condenser polarizer and analyzer from the

light path then selecting a “--“ setting for the condenser in the Microscope Controller window.

Refer to section 7 for more information.

1.4 Finding the sample – epi-fluorescence

1. Sample should be brought into focus and centered, as described above;

2. Open the fluorescent shutter

a. Image Acquisition Control Window>Click on the fluorescent shutter button, Fig. 4.2;

b. The Microscope Controller will switch to the last filter used;

3. Select fluorescence filter, if desired

a. Microscope Controller Window>Mirror>click on ‘DAPI’, ‘FITC’ or ‘TRITC’ (blue,

green or red fluorescence), Fig. 3.3, Table 1;

4. Adjust focus and stage to position sample;

5. Adjust fluorescent intensity with the aperture lever on the mercury lamp;

6. Close the mercury lamp shutter

a. Image Acquisition Control Window, Fig. 4.2;

b. System will switch to ‘LSM’;

Figure 3. Microscope Controller Window

3.2 Click on buttons to

select objectives

3.1 Condenser buttons

[automatic, don’t touch]

3.3 Mirror buttons control

imaging mode and filters

3.4 Indicate & toggle

coarse/fine focus

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Mirror Button

Function

Mode

Visualization

LSM

Confocal Imaging

Confocal

Confocal only, automatic

DAPI

UV excitation

Epi-fluorescence

Eyepieces only

FITC

Blue excitation

Epi-fluorescence

Eyepieces only

TRITC

Green excitation

Epi-fluorescence

Eyepieces only

Cy5

Red excitation

Epi-fluorescence

Eyepieces or Camera

DICT

DIC/ Brightfield

Transmitted light

Confocal or Eyepieces

---

Brightfield

Transmitted light

Confocal or Eyepieces

Table 1. Mirror Buttons

1.5 Finding the sample - confocal

1. Select the dyes from the Dye List Window (Section 2.1);

2. Set collection parameters for minimal photobleaching – See “Optimizing Image Collection”;

a. Minimal laser intensity, Acquisition Setting Window>Lasers, Fig. 7, start with 1%;

b. set detector HV fairly high ~1000 V, Image Acquisition Control Window (See section

2.7.1 regarding Auto HV)

3. Set the Live View to tile view, each channel is displayed in a separate window;

4. Set to gray scale (control-h);

5. Set scanning to simultaneous (Sequential checkbox, Fig. 4.9, is cleared);

6. Initiate scanning: Image Acquisition Control Window>Focus X2 or Focus X4, Fig. 4.5;

7. Adjust focus and position of sample;

8. Roughly adjust laser power and HV to reduce saturation;

9. Stop scanning, Image Acquisition Control Window>Stop Fig. 4.8.

10. Refer to Section 2 for more information on settings.

11. Select Sequential, Frame, Fig. 4.9;

12. Select each channel and adjust to minimal laser power and HV less than 750 to avoid saturation

(red masked pixels);

13. Set the Offset to see only a few or no pixels of intensity = 0 (blue masked pixels)

14. Set the dwell time, 2 or 4 µs are good starting points

a. Acquisition Setting Window>Fast-Slow slider, Fig. 6.2;

15. Scan XY Repeat, Image Acquisition Control Window, Fig. 4.6 to preview full resolution;

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Figure 4. Image Acquisition Window – Basic controls for Finding the Image

2. Setting Up for Confocal Microscopy

This section covers basic settings for obtaining a confocal image.

2.1 Choosing the Dye

Each combination of fluorescent labels requires specific laser lines, dichroic mirrors and filters. These

are selected automatically when you choose the fluorophores from the list in the Dye List Window.

1. Open the Dye List Window, if it is not currently visible, Fig. 2.5;

a. click on Image Acquisition Control/Dye List button, Fig. 4.3, to open;

2. Selected Dyes – upper window displays the currently selected dyes

a. click on “All Clear” to remove all current selections

b. double-click on any selected dye to remove it

3. Scroll through the list of dyes in the lower window;

4. Select each desired fluorophore by double-clicking on the name;

a. if your particular dye does not appear in the list, contact facility manager, or choose one

with similar characteristics

b. dyes with conflicting optical requirements will generate an error message

5. Activate the settings by clicking on the “Apply” button, lower window;

4.1 Tungsten Lamp

4.2 Fluorescence

shutter

4.3 Dye List

4.5 Fast scan

4.6 XY Repeat

4.8 Stop scan

4.4 Tungsten Voltage

4.7 Acquisition scan

4.10 Confocal Aperture

4.9 Sequential

4.11 Kalman

Averaging

4.12 Information

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Dye names are written into the image file metadata. However, fluorophores with broadly similar

excitation and emission spectra will use the same laser line and filters. For example, FITC, EGFP,

Alexa488 and Mitotracker Green will all use the 488 nm laser and a bandpass filter of 500 nm to 550 nm

2.1.1 Changing the Dye Selection

1. Remove a selected fluorophore by double-clicking on the dye name

2. To add a dye

a. double-click on the new label from the dye list

b. set the detectors for the new dye by clicking on “Apply”.

Q. My fluorescent stain is not on the list – what do I do? Refer to “Optimizing the Image”.

Table 2. Objective Lenses Available

*Special purpose, not usually kept on microscope.

*Correction collar allows correction for coverslip thickness and refractive index mis-match.

20X LWD is intended for use with plastic culture dishes.

60X/W lens is used for live tissue or fixed tissue mounted in low RI media such as PBS

2.2 Confocal acquisition controls.

1. Focus X2, 4.5 - Scans only the odd lines and replicates them, at 2 µs pixel dwell time.

a. rapidly scanning is very helpful when focusing and moving the stage.

b. may be used for recording rapid fluorescence changes, such as ion measurements.

2. Focus X4, 4.5 - Scans alternate odd lines, replicating the scanned lines to generate an image of

4X reduced resolution in the Y-axis, at 2 µs pixel dwell time for extremely fast scanning.

3. XY Repeat, 4.6 – Scans at full resolution, at the user’s chosen dwell time.

4. XY, 4.7 – Acquires one image, or defined set of images, using the current settings.

5. Stop, 4.8 – Stops any scan, retaining the current image in the Live Image Window.

6. Sequential Acquisition, 4.9 – collect each channel separately, scanning with one laser line at a

time.

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Objective

Working

Distance

Immersion

Resolution

Objective

Working

Distance

Immersion

Lateral

Axial

4X UplanSApo

0.16

13 mm

Air

1.906 µm

54.3

10X UplanSApo

0.40

3.1 mm

Air

0.601 µm

8.56

20X LWD*

0.45

7.8 mm

Air

0.677 µm

3.56

20X UPlanSApo

0.75

600 µm

Air

0.319 µm

2.12

40X UPlanFL N

1.30

200 µm

Oil immersion

0.181 µm

0.948

60X PlanSApo

1.35

150 µm

Oil immersion

0.226 µm

0.782

60X/W collar*

1.20

280 µm

Water immersion

0.254 µm

0.800

100X UPlanSApo

1.40

139 µm

Oil immersion

0.168 µm

0.778

7. Confocal Aperture, 4.10 – Controls the diameter of the pinhole.

8. Kalman Averaging, 4.11 – Allows averaging signals from multiple scans to reduce noise.

9. Information, 4.12 – Opens a window displaying the current pixel size.

Focus X2 and Focus X4 settings switch to 2 µs dwell time. These scan settings reduce resolution in

Y axis by 2-fold and 4-fold, respectively. Use these settings with the AutoHV switch selected.

2.3 Settings for the fluorescence detectors.

Each fluorophore selected from the Dye List will activate one of the photomultiplier tube (PMT)

channels and set its appropriate filters. Three settings control the sensitivity and signal to noise ratio for

each detector. Refer to “Optimizing the Image” for details on these settings.

1. HV, 5.1 – The primary adjustment for the PMT. HV controls the detector sensitivity and

amplification.

a. try to stay below 750, higher settings will introduce detector noise in the image

b. higher HV may used with averaged images to allow reduced laser intensity

2. Gain, 5.2 – This multiplies the analog electrical signal created by the PMT by a constant, before

it is digitized. While this can provide ‘brighter’ images, the noise and background intensities are

increased as well.

a. leave set to “1” unless working with very low HV settings.

3. Offset, 5.3 – The Offset will raise or lower all pixel intensities to control the “Black Level” of

the image. It should be set so that the darkest values in the image are above zero.

a. leave set to default values of approximately 5 to 7

b. set the display to gray scale, pixels with intensity = 0 will be pseudocolored blue

c. set the Offset so that no blue pixels ( intensity = 0) are present

2.4 Additional detector controls.

1. Laser power level, 5.4 - changing from this window may be more convenient than scrolling over

to the laser control window.

2. Active detector check mark, 5.7 – removing this check mark will turn off the channel and its

associated laser line.

3. Group number, 5.5 - allows channels to be grouped for sequential channel acquisition.

a. channels that do not share bleedthrough, e.g. Alexa488 and Cy5, they can be combined

into the same group.

b. channels in the same group are scanned simultaneously to reduce the amount of time

required for sequential acquisition

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Figure 5. Channel Settings for the Photomultiplier Tube (PMT)

2.5 Laser Controls.

Each fluorophore selected in the Dye List will select an appropriate laser line and some default intensity

setting. The Line Setting values are given as a percentage of full rated output of the laser (mW) entering

the scanhead. You can change the power setting for each line or turn lines off to assess bleedthrough

(details of bleedthrough are in section 6).

2.5.1 Line Selection.

Active lines are indicated by a check mark, Figure 7.1. Clicking in these boxes will toggle lines

between active and blocked. This control blanks the lines in the scanhead, it does not turn the laser itself

on or off.

2.5.2 Line availability, Figure 7.2.

Laser lines associated with each set of controls are listed in this column. This list is not affected by a

whether or not a laser has been powered up.

5.6 Channel

5.2 Amplifier Gain

5.1 PMT Voltage

5.5 Channel group

5.7 Active if

checked

5.3 Offset (black level)

5.4 Laser Settings

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2.5.3 Laser Power Selector, Figure 7.3.

These controls regulate the percentage of laser power allowed to pass through the acoustico-optical

tunable filter (AOTF) located in the scanhead. Each individual laser has a unique output power. Thus, if

all laser lines are set at any given percentage, each will be providing a different amount of energy to the

sample.

2.5.4 Scroll List, Figure 7.4.

If there are more lines than can be displayed in the window, scroll through the list by clicking on the

black triangles or by dragging the slider.

2.5.5 Line Settings, Figure 7.5

As with other controls, the laser power may be adjusted by clicking anywhere in the control area,

scrolling the thumbwheel (1% increments) or by clicking on the black triangles (0.1% increments).

Using the thumbwheel to go below 1% will set the laser power to 0%. Use the triangles to increment

laser intensities to values between 0% and 1%.

Figure 7. Selecting laser lines and setting laser power

7.1 Laser Line

Selection

7.3 Line Power Selector

7.2 Laser Line

7.5 Line Settings

7.4 Scroll List of Lines

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2.6 Scan Controls

2.6.1 Scan Mode, Figure 6.1

1. Unidirectional - Standard scan mode for routine imaging. Each line is scanned from left to right,

the laser beam is blanked while the scanner jumps back to the left side to scan the next line.

2. Bi-Directional – the specimen is scanned in both directions, odd lines are recorded from left to

right, even lines from right to left. This allows for extremely fast scanning of brief, rapid events.

There is a 30 sec. limit for bi-directional scanning.

2.6.2 Dwell Time, Figure 6.2

The Dwell Time is the length of time allotted for the focused laser spot to scan across each sample point

in the scan box. A longer dwell time increases the length of time spent moving over each point to

deliver more photons exciting the fluorescent label to generate signal. However, a longer dwell time

will increase photobleaching. The time required to scan a field is determined by the product of the

number of times each line is scanned, number of lines in the field, number of sample points per line and

the dwell time. Zoom does not affect scan time.

The numbers below the dwell time slider provide the effect of dwell time on image collection, for

example - P:4.0 µs L:3.144 ms F:1.644 s S:4.932 s

Figure 6. The Scan Mode and Scan Size settings of the Acquisition Setting Window.

6.5 Box Size

6.4 Image Aspect Ratio

6.6 Auto HV

6.3 Scan Regions

6.2 Dwell Time

6.1 Scan Modes

Olympus Fluoview-1000 User’s Guide

V.M. Bloedel Hearing Research Center, Core for Communication Research

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Dwell Time

per Pixel (P:)

Box Size

Scan Time

Per Line (L:)

Seconds

per Frame (F:)

Time for Set

(S:)

2.0 µs

512X512

1.572 ms

0.822 s

2.446 s (Sequential)

2.0 µs

512X512

1.572 ms

0.822 s

0.822 (Simultaneous)

8.0 µs

512X512

6.288 ms

3.288 s

9.862 s (Sequential)

8.0 µs

512X512

6.288 ms

3.288 s

3.288 s (Simultaneous)

4.0 µs

1024X1024

6.288 ms

3.288 s

9.862 s (Sequential)

4.0 µs

1024X1024

6.288 ms

3.288 s

3.288 s (Simultaneous)

Table 3. Effect of Scan Speed and Box Size on Acquisition Time - single plane, 3-channel image

Scan Time per Line (L) is determined by Dwell Time (P) X length (e.g. 512, 1024, etc.) plus the

“flyback” time required to return the laser spot to the start of the next line. The time to record a Frame

(one image, F) is L X number of lines in the box size. The Set (S) is F X the number of scans required

to record each 2D image plane. S = F for simultaneous collection, but S = F X # channels for sequential

acquisition. Kalman averaging will increase scan time by the number of averages, and a z-series will

increase S by the number of focus steps (F X z-steps).

2.6.3 Scan Regions, Figure 6.3.

These buttons select the type of region to scan.

The leftmost “Normal” rectangle defines the scanned region as the selected box size. The Polygon

options allows user-defined regions within the box. These may be rectangular, circular or irregular

polygons. The laser beam is blanked outside of the selected scan region.

The ‘Line’ and ‘Point’ options are specialty scans that may be used for creating a “vertical” section by

scanning through a focus range or to record rapid dynamic processes, such as calcium spikes. All of the

scan regions may be used for techniques such as photoablation, photoactivation or photobleaching.

2.6.4 Image Aspect Ratio, Figure 6.4.

The default scan region is 1:1 (width:height), or square. The “4:3” option provides rectangular boxes,

e.g. 768 X 512. The “arbitrary” option allows the user to define any needed box, e.g 129 X 17.

2.6.5 Box Size, 6.5.

Box Size controls the “sampling density” of the image, the number of sampling intervals in the X-axis

and Y-axis of the scanned field. The scanned region will be divided into the stated number of lines with

each line divided into the selected number of sample points. In turn, the acquired image will possess

corresponding dimensions, in pixels.

Olympus Fluoview-1000 User’s Guide

V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

May 11, 2011 17

2.6.6 Box Size effect on resolution

The resolution of an image is determined by dividing the scanned field by the box size to set the area

represented by each pixel in the final image. Dividing the field into fewer sample points requires that

each sample point will encompass a larger area. Conversely, doubling the box size (e.g. from 512X 512

to 1024X1024) reduces the area of each sample point by 2-fold. However, a 2-fold increase in the box

size will also give a 4-fold increase of the file size because this is an area (width X height).

The box size and zoom setting (section 2.10) are used together to control the size and resolution of the

scanned region for any selected lens.

Refer to the later section on Optimizing the Image.

2.6.7 AutoHV, 6.6.

This only needs to be set once after a user account is established. If you don’t activate the Auto HV

setting, then every time laser and HV are set with a selected dwell time (such as with Scan X2 or Scan

X4), switching to a different dwell time may cause your image to become saturated.

1. Begin scanning with Focus X2 or Focus X4 Focus mode and find your sample;

2. Adjust the laser and detector settings;

3. Click on ‘Auto HV’ to set the reference histogram;

4. Switch to Repeat XY or XY to scan with a longer dwell time, e.g. 4 µs;

5. Notice that the HV is reduced to maintain image intensity and prevent saturation.

2.7 Scan Area Controls

This panel controls the scanned area.

2.7.1 Field of View, 8.1.

The circle represents the area imaged by the objective lens. The scanner will record the selected sub-

region.

2.7.2 Scan Area, 8.2.

This is the portion of the objective field of view that will be recorded. A square region at Zoom = 1

represents the maximum field, as displayed in figure 8. A Zoom = 2 would scan only the central 1/2 of

the area.

2.7.3 Scan Rotation, 8.3.

The scan region may be freely rotated by clicking on the red dot and dragging it around the circle.

Clicking on the curved arrows will increment rotation in 0.1° increments.

Olympus Fluoview-1000 User’s Guide

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2.7.4 Scan Zoom, 8.4.

This controls the size of the scanned region and the resolution. Click in the slider area to increment scan

zoom by 1X increments or click on the arrows to increment by 0.1X increments. Increasing the zoom

will reduce the pixel size and the field of view. The rectangular outline in the Scan Area, 8.2, updates to

display the scanned region.

Figure 8. Scan Controls for Zoom, Pan and Rotation

2.7.5 Reset Scan Values, 8.5.

Return the field Offset and Rotation to “0”, and zoom to “1”. Changing the Zoom is most quickly

accomplished by clicking on the Zoom Reset then changing the zoom to the new value.

2.7.6 Scan Offset, 8.6.

Move the Scan Area within the Field of View by either clicking on the scan area and dragging it, or by

clicking on the sliders and arrows. The only works with a zoom > 1, of course. Be aware that moving

the scanned field to an extreme edge of the objective field of view will result in slightly degraded

resolution and distortion due to parallax.

8.3 Scan Rotation

8.4 Scan Zoom

8.2 Scan Area

8.1 Field of View

8.5 Reset Scan Values

8.6 Scan Offset

Olympus Fluoview-1000 User’s Guide

V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

May 11, 2011 19

Figure 9 – Sequential Acquisition and Averaging Controls

Figure 9A – Sequential Acquisition and Averaging Controls

9.1 Sequential

Line vs. Frame

9.3 Channels

9.3 Kalman Averaging

9.2 Groups

9.2A

9.3A Grouped Channels

Olympus Fluoview-1000 User’s Guide

V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

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