Olympus FV1200 MPE User manual

Olympus FV1200 User guide June 23, 2015

Olympus FV1200 MPE Microscope User Guide

A. Microscope Start Up

1. Sign-in to the log book with the start time.

2. Turn on mercury lamp power supply by pushing the

ON/OFF button and releasing it (The light should lights

up in Blue).

3. Turn on microscope controller and XY stage

controller.

4. Turn on the touch panel controllerand touch “Start

Operation”.

5. Turn on both scanner controllers (MAIN and SIM) ;

switch ON and then turn the key ON.

6. Turn on the computer if it is not on.

7. Enter user name/password to log on to Windows7.

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Olympus FV1200 User guide June 23, 2015

B. Laser start up

1. Turn on laser power supplies

a) Multi Ar laser : Switch to ON and turn the Key to ON.

b) MCPSU (405/440/473/559/635nm): Switch ON.

c) LD559 laser: Switch on and WAIT UNTIL THE TEMPERATURE LIGHT STOPS BLINKING and THEN turn the Key

to ON.

Double click FV10-ASW program and enter User ID and password.

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Olympus FV1200 User guide June 23, 2015

C. Outline of Acquisition Setting / Image Acquisition/ Image Viewer Windows

Before taking images, let’s get familiar with buttons and sliders on the

Acquisition Setting Window (such as scan mode, scan speed, image

size etc).

Scan mode: the default is unidirectional .

Scan Speed: The higher value, the slower, the better image. Around

12.5~20 us/pixel (microsecond per pixel) is a good speed.

Image size (number of x,y pixel): 512x512 pixel is the default.

Area: the image field can be optically zoomed in and rotated.

To rotate the viewing area, click on the red dot and drag clock- or

counter-clockwise (or click ). Zoom the imaging area with the

slider (click to return to 1x). With zoomed view, you can select the

scanning area by moving the blue-lined box around.

Laser power control: Checking the box will activate the laser. The

power can be adjusted by pointing the mouse pointer to the slider

area and clicking (large change), clicking the arrow heads (small

change), or rolling the mouse wheel on the slider area.

Lambda Scan: for spectral imaging.

Microscope: Objective selection is displayed. Also selecting the

objective on the dropdown list will automatically change the objective.

Z position setting: Clicking the large arrow heads will move the

objective up or down to focus onto the sample. The extent of

movement is determined by the Step size. Clicking small arrow heads

will move objective in half the Step size.

Start Set and End Set determine the z series range.

Time Scan: set up the time lapse series condition (interval and number of image acquisition.

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Olympus FV1200 User guide June 23, 2015

D. Overview of Image Acquisition Control window

: Click Transmission light or Epifluorescent light to turn it on and off.

: Click on Focus x2, Focus x4, or XY Repeat begins scanning without

image acquisition.

XY Repeat scans at the imaging speed that you set with scan speed on Mode

panel.

Focus x2 automatically scans the sample at 2us/pixel –because of its fast

display refreshing rate, this will be good for focusing and searching for ROIs

without bleaching your sample.

Focus x4 will be faster, but the display quality may be too rough. Click Stop

scanning.

To acquire image, click XY (LZt) .

: Click Lambda to acquire lambda scan imaging (XYL).

Click Depth for z-series image acquisition (XYZ). Click Time for time-lapse

image series (XYt).

: Click on this will open the Spectral Setting window, where the range of

emission wavelength can be adjusted to prevent overlap between

fluorescences.

Olympus FV1200 User guide June 23, 2015

E. Viewing with Transmitted or Epifluorescence Light

Before scanning the sample with laser light, look at the sample and find

the region of interest first with either transmitted or fluorescent light.

MAKE SURE THE SLIDE AND COVERGLASS ARE CLEAN AND SEALED. Place the slide on

the microscope stage.

Move the stage using the joystick on the XY stage controller. Push the

speed (Fast/Med/Slow) buttonto change how fast the stage moves.

(You can also use the side wheels to control x and y separately by pushing

the XY/Z button to select XY on screen.)

Press Esc button (on LED touch panel or on FV10 program) to move

the objective completely away from the sample and press again to bring

the objective to the original position.

Fluorescent image observation

1. Select an objective lens using the touch panel controller (TPC).

2. Click of the FV10-ASW program or [EPI] button of TPC.

3. Select the appropriate fluorescence filter by touching a mirror button on

TPC. Adjust focus using the focusing knob.

4. After finding area of interest, click or [EPI] button on the TPC to

close the fluorescence light shutter and change the light path to the laser

scanner.

Transmitted light image observation

1. Click or [DIA] button of the TPC to turn on bright field light.

2. Click on [BF], [DIC], or [Phase] button on the TPC. Adjust the light intensity with the lamp

controller or by touching Lamp arrows on [DIA] tab.

3. For DIC viewing, insert the DIC slider and adjust the DIC contrast using the prism

controller.

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Olympus FV1200 User guide June 23, 2015

4. After observation, click or [DIA] button on the TPC to close the shutter.

F. Image Acquisition (Multicolor imaging)

1. Click on Dye list button on the Image Acquisition Control Window and double click on the fluorescent

dyes you want to observe from the list. Click Apply button (if the Assign Dye Manually check box is

checked, uncheck it before pressing Apply button). It will activate the laser lines and the detection

channels according to the dyes you selected.

If the Selected dyes upon opening the program are not ones that you want to use, click the All Clear button

first before selecting your dyes.

2. Choose Image size (default- 512x512) from Size panel .

3. Press button (if not pressed) to automatically adjust HV and Offset values according to the scan

speed. . Slide the scan speed knob to select the image

scan speed (around 20 us/pixel).

4. Click button to scan the sample. It will scan fast at 2 us/pixel and show low-quality (pixelated)

image in a Live View window. While scanning, focus onto the region of interest with the remote focus

controller or by clicking arrowhead buttons ( ) in the Microscope window.

5. Click Stop button to stop scanning.

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Olympus FV1200 User guide June 23, 2015

6. Set up proper acquisition condition each fluorescence colors by adjusting laser power, PMT detector

sensitivity, and offset. The following procedure is for Alexa Fluor 405, EYFP, and Alexa 633 as an example.

a) Check the Laser 405 only (check off other lasers, i.e. 515 and 635) to set up the imaging condition for

Alexa 405.

b) Click and focus onto the brightest focal plane of your

sample.

c) Press Ctrl-H keys to change the color LUT to Hi-Lo; the live display

will change to blue/white/red color scheme, where red pixels

represent saturated intensity (beyond scale), blue ones complete darkness (0 intensity),

and white in between.

d) Adjust the laser power (at least 5%) and HV and Offset of CHS1 to obtain some

brightest pixels in red and the background in blue.

e) Click to stop the fast scanning and click to scan at the imaging speed

and to check the image quality. Adjust more if needed.

7. Repeat the above procedure for 515 nm (EYFP) and for 635 nm (Alexa fluor 633).

8. Check on all the selected lasers.

9. Set the Zoom and Rotation as required.

10. Click button to capture images. When acquisition scanning is done, a 2D view window will

appear.

11. Save the image in .oib or .oif format. (.oib format is more convenient.)

(Optional Spectral Setting for Multicolor Imaging).

Often excitation/emission profiles of fluorophores in multicolor imaging

may be close, so there could be possible bleed-through of a fluorophore

emission to neighboring channels. To minimize this artifact, the emission

detection range of each channel can be adjusted by changing Spectral

Setting.

Click on , it will bring Spectral Setting window. Change the range of

spectrum for each channel (CHS1 and CHS2) by sliding, widening, or

narrowing the tabs or arrowheads.

Olympus FV1200 User guide June 23, 2015

Sequential Scanning Mode

In many cases, the bleed-through between multicolor can’t be eliminated by a spectral setting. It can be

avoided by sequentially scanning the sample with one laser and one detector at a time.

1. Check the Sequential box and it will bring up the sequential scan information window. Choose between

Line or Frame mode.

Line: by line-by-line, it scans one channel with only a specific laser line and detector on and then sequentially

scans the next channel with only a another laser line and detector on.

Frame: it scans sequentially frame-by-frame; it finishes scanning one channel first and then scans the next

channel.

If there is no bleed-through

between the emissions of some

fluorophores you are using, you

can group them together to scan

them simultaneously as a group. Simply click on the fluorophore name on the list and drag to the other group.

(Note: it will be good to set up the acquisition condition for each channel before using the Sequential mode. To

do so, first in simultaneous mode (unchecked Sequential box), set the acquisition condition by turning on only

the specific laser for the specific channel. Once you set the imaging condition for each colors, check the

Sequential box.)

2. Scan with Focus x2 or XY Repeat button to see to check if there is any bleed-through. If

not, stop scanning, and set the Scan speed at a slower rate and click button to acquire the image.

3. Save the image.

Olympus FV1200 User guide June 23, 2015

G. Z-Series (xyz) Image Acquisition

Use this mode to obtain optical section through the depth (z dimension) of your sample that can be used for

3D visualization. Before starting the following procedure, make adjustment for xy multicolor imaging

condition as described above.

1. Click button to scan.

2. Use the arrowheads buttons or the focus knob on the

remote controller to focus into different Z-axial planes (large

arrowhead buttons shift a full step size and small ones a half

step size that you define).

3. When you find an upper limit of your sample by moving the

objective up, click Start Set button. Bring the objective down

until you find lower limit and click End Set button.

4. Determine the Step Size and the number of Slices, which correlate with each other. It is recommended to

set the step size similar to the optical section thickness of the objective you are using, so that there is no

gap between the optical sections upon projection into 3D. The step size can be fixed by checking the box.

5. Click Stop button. Adjust the Scan Speed if needed, click Depth button (“Z”

will be appear on the XY button to become XYZ), and then click XYZ button.

6. When acquisition is done, Append

Next/SeriesDone button will appear over the

Stop button. If you need add additional sections,

click Append Next button (enter the number of

sections you want to add into the Append box

) or click SeriesDone to finish the

acquisition.

7. Save the image.

H. Time-Series (xyt) Image Acquisition

Use this mode for imaging time-lapse of live specimens. Before

starting time series imaging, make adjustment for xy imaging as usual.

1. Click Time button . It becomes XY_t.

2. In the TimeScan option, enter the interval time (in second)

between one acquisition start and the next acquisition start (for

example; to set up a 1 min interval, type 60 and press Enter key. If

you put 0 and enter (FreeRun), the interval will be the acquisition

time that is required for each frame).

Olympus FV1200 User guide June 23, 2015

3. Enter the number of acquisition frame in Num box.

4. Click button to start the timelapse.

5. Click SeriesDone button to finish the sequence and save the image.

I. Saving and Exporting Images

1. Click on the image window to be saved.

2. Click on icon or select File/Save or Save as from

menu.

3. ASave as window will appear. In case the image should

be saved in the Image folder of a Log-in user, click on

button and it will direct to your image folder.

Select Olympus Image Binary Format *.oibfile type,

type file name, and click on Save button. (Log-in user

and its associate folder will be created as you become

self-user).

4. To set up your image folder where you can jump directly by

clicking My Image Folder button, select Tools/Option menu.

Click Generaltab and specify the path of your image folder

with Browse..button. [Upon creating a user account, each

user will have its own folder in Ddrive (Do not create any

folder in Cdrive.). Click OK button.

5. Oib file type contains all the metadata including all the

acquisiotn parameters and it can be opened in FV10-ASW

program. The light version of this program is available for

installing in user’s own computer. Otherwise, the image

file can be exported as other file format so that it can be opened and processed in other imaging software.

To export images:

1. Select File/Export or File/Export Multi-Tiff menu. Click My

Image Folder button if the current folder is not yours.

2. With Export Multi-Tiff option, it saves the image as a

single tiff file that can contain multiple frames, like z- or

timelapse- series. This file type can be opened in Image J

software.

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