Operations Guide

Contents
•  STARTUP
•  MICROSCOPE CONTROLS
•  CAMERA CONTROLS
•  SOFTWARE CONTROLS
•  EXPOSURE AND CONTRAST
•  MONOCHROME IMAGE HANDLING
Nikon Eclipse 90i Operations Guide

STARTUP
 Startup – Powering Up
Fluorescence Microscope Camera

Note: Please give microscope and camera at least 1 minute to

initialize before starting software
Please do not touch any other buttons.
 Startup – Opening Software

After logging into computer, Double left click on the icon, or right
click on and select “Open”

Note: The software will look for all microscope components when it starts
up – please make sure everything is on and initialized before starting
software.
Startup – Loading Software Defaults

Before pushing any buttons in the

software, please push the “Restore All”
button on the left hand tool bar.

The 4x lens will be rotated into place, the

focus put in range and the stage centered.
 Startup – Software Panes

Software has three main display screens:

“Docked Controls” - shows all control panels. This

is the primary display that should be used.

“Full Screen” – displays open work surface of

software without any control panels. This gives more
open space for image handling.

“ND Acquisition” – provides control box for multi-

dimension imaging – see section on ND Acquisition
Startup – Camera Imaging Mode
The DS-Ri1 is a true color camera that can also be run in a
pseudo monochrome mode.

If you are doing primarily fluorescence imaging, the monochrome

mode can make image handling easier because it creates single
pseudo colored images for each fluorescence channel which can
then be easily combined together.

The camera will always start up in the mode of its last use.

To initialize the camera for your use, push the toggle icon on
the left hand toolbar.

See Next Page>>>>>

Startup – Camera Imaging Mode
Make a selection from the dialog box that appears:

“Nikon Ri1 Camera” – Color Mode

“Nikon Ri1 Camera Emulated Mono” – Monochrome Mode

Note: it will take about 20 seconds for the change to occur.

Please wait for software screen to refresh with the change.
Startup – Toggling Between Cameras

To ensure that the software sets up properly it

is best to hit the “Restore All” button again
when changing between cameras.

During your imaging session you may use the

switch button to toggle back and forth between
the cameras.
 Startup – Optical Configurations

Optical Configuration (OC) buttons are located at top of

screen, just under the main menu option line:
•  OC’s are your main point of communication with the microscope
system.
•  Selecting an OC will put the microscope in the condition needed to
do the indicated type of imaging
•  Select an OC by left clicking on its name – its name will depress –
see “FITC” above
 Startup – Optical Configurations

Brightfield and Phase Contrast OC’s:

•  Automatically turn on the halogen light above
•  Automatically put the condenser in the correct position for each
•  Note: there is only one “Brightfield OC for all lenses, while there are
multiple “Phase” OC’s for the different lenses
 Startup – Optical Configurations

Fluorescence OC’s:
•  Automatically turn off the halogen light above
•  Automatically put the correct fluorescence filter cube in place
•  When viewing by eye, you will open/close the fluorescence shutter
as needed
•  When using the camera, the fluorescence shutter will open/close
automatically synchronized with the camera buttons, and when in
monochrome mode, the image will be pseudo colored appropriately
(eg FITC is green)
14

Startup – Objective Selection

The 90i microscope control pad is
located in the left control panel of the
software screen.

Use the objective lens buttons to select

the lens you want to use.

The objective lens in place will be

depressed in the display.

NOTE: Objective Lens Selection is the

only control that should be used in
this panel. Everything else will be
controlled the Optical Configuration
(OC) buttons.
Startup – Ocular Selection

Any time you wish to view with your eyes,

push the “eye” button on the left hand
toolbar.

Use the OC buttons to change the

microscope state as needed.

Use the “Epi” button on the top left toolbar

to open/close the fluorescence shutter as
needed.
 Startup – Camera Controls

Green Arrow – starts live video image on the screen – opens shutter
for fluorescence OC’s.

Red Square - stops live video image – closes the shutter for
fluorescence OC’s.

Camera - creates a new image in the software work space – opens/

closes shutter for fluorescence OC’s.

When you push Green Arrow or Camera buttons the software will
automatically switch from eyepieces to the appropriate camera
port.
Nikon Eclipse 90i Operations Guide

MICROSCOPE CONTROLS
Microscope Controls – Stage Movement
The hanging joystick unit is used
to control XY movement of the
microscope stage.

The speed of XY movement is

automatically adjusted by the
microscope based on what
objective lens is being used.

The focus knobs on either side of

the unit can be used to focus.

No other buttons are used.

19

Microscope Controls – Transmitted Light

NCB11 Daylight - always
leave “IN”. This gives proper
white color to light.

ND 8 and ND32 – the

intensity of the light is set on
high by the software and
should not be adjusted so
that color balance is
maintained.

To adjust light level for

different lenses toggle the ND
filters IN/OUT to achieve the
desired level
Microscope Controls – Correction Collar
The 60x objective has a correction
collar, which allows the user to
adjust the lens for the thickness of
the cover slip on the slide.
The number 17 on the collar is the
setting for a #1.5 standard cover
slip
Adjust the collar relative to the
fixed reference mark to account
for different cover slip
thicknesses.
In practice you will likely turn the
collar a bit and then refocus the
microscope in repeated
successions to hunt for the ideal
setting.
Microscope Controls – Correction Collar

Correction collar setting will

produce a sharp image if
correct

And a blurry image if

incorrect. It will look out of
focus and there will be no
way to get it into focus.
22

Microscope Controls – Polarizing Filter

The Polarizing Filter allows crossed
polarized light imaging – collagen or
other tissue inclusions, etc. It mounts on
the microscope base under the stage.

Once mounted you can rotate it by hand

to vary the polarizing effect.

Use the “POL” optical configuration

which moves a motorized polarizer in the
microscope head into place.

When not in use the filter should be

stored in the drawer under the
microscope in plastic bag.
23

Microscope Controls – Fluorescence Control

“A STOP” – always leave
pushed in (right side of
microscope)

ND Filter Sliders - pushing “IN”

any of these will cut down the
intensity of fluorescence light.
The higher the number, the more
it cuts (left side of microscope)
24

Microscope Controls – Main Stand

There is no need to touch any of the buttons on the stand of the
microscope.

All of the functions of the microscope stand should be controlled by

software “Optical Configurations (OC’s)”

There is a focus knob control on the stage XY joystick. But, you

can also use the focus buttons on the microscope stand if desired.
Nikon Eclipse 90i Operations Guide

CAMERA CONTROLS
 Controls Controls – Camera Settings
Camera Settings Panel appears in the upper left corner of the software
workspace and changes as the camera is changed – the key distinction is
the additional Color features in the Color Mode Dialog (see below left)
Color Mode Monochrome Mode
Controls Controls – DS-Ri1 Settings
Mode:
Always use “Normal”

Resolution:

Fast – for live focus image

Quality – for captured image

Note: using lower resolution for live

image makes live image go faster
Controls Controls – Auto Exposure Settings
Push “Auto Exposure” button to let
software determine best exposure.

This is recommended setting.

“Analog Gain” is automatically set by

software as well when using Auto
Exposure

“Key” button will lock exposure at

current auto setting – for exposure
standardization

“AE Compensation” can be used to push software to

increase or decrease its exposure setting
Controls Controls – Manual Exposure
Settings

Unselect “Auto Exposure” and

choose exposure time from pull down
list.

“Analog Gain” can be set to fine

adjust the intensity in the image.
Controls Controls – ColorSettings
“Contrast” – different types of digital
processing are available. Standard is
“Medium”

“Auto White” will set white balance so

camera can produce proper color.

Go to empty area of slide and push

Auto White to set white balance.

Do Not use “Scene Mode” or

“Commands” controls.
Nikon Eclipse 90i Operations Guide

SOFTWARE CONTROLS
Software Controls – White Balance
If you did not white balance ahead of acquisition, you can
activate the white balance probe – located on the upper right
of the image window - size it, and move it to a blank area in
the existing field to perform white balance.
 Software Controls – Live Mouse XY
At the top of the “Live” image window is a button to use
the Mouse XY. Push this button in and you can use the
mouse to move the stage in XY by left clicking with the
mouse and dragging the image in the direction you want
to move it. This stays active until you push it off again.
 Software Controls – Live Focus
The mouse wheel can be used to focus the microscope in
live mode. It will stay selected until someone turns it off.
When active, left click the mouse in the live image window
once, and then turn the mouse wheel to focus up/down

Note: Mouse Joystick Setup allows you to set the Z

step size of each mouse wheel click
 Software Controls – Auto Contrast
Auto Adjust Contrast – sets contrast to best viewing setting – more on
contrast settings in following pages

Red “X”
resets
contrast to
null

Keep Auto Adjust - It is common in the live image window

to use the “keep auto adjust” which will continuously
adjust the contrast for changing conditions in the live
image window, which can be helpful when scanning
around and changing between phase contrast and
different filter cubes.
Software Controls – Other Window Controls
Pixel Saturation Indicator – when pushed it will show
overexposed pixels in color you select from a pull down menu
found with the black arrow

ROI controls activates a sub-region box – pull down arrow

let’s you start this feature and position box. Pushing ROI
button turns sub-region imaging on/off
 Software Controls – Scale Bar

Every Image has a Once active on the screen,

scale bar button on the the scale bar can be
upper right hand side. grabbed with the mouse
and moved around the
Push it to activate scale
screen to location you
bar in the image.
desire.
Software Controls – Scale Bar Burn
Right click the mouse on the scale bar and you get two options:
“Burn” – which permanently stamps the bar into the image.
If the image is not already an 8-bit RGB it will now be converted to
that format.
Always save out your original image before burning the scale bar,
which should then be saved out as a copy of the image.
You may want to go back to the original without a scale bar. Also,
initially it may take some trial to get the scale bar properties to
your liking.
“Change scale properties” – set the size, color, font, etc.
Note that you can let the software auto size the length of the scale
bar or uncheck and select from a pull down of different sizes.
 Software Controls
Scale Bar

Every Image has a Once active on the screen,

scale bar button on the the scale bar can be
upper right hand side. grabbed with the mouse
and moved around the
Push it to activate scale screen to location you
bar in the image. desire.
 Software Controls
Scale Bar Burn and Properties

Right click the mouse on the scale bar and you get two
options:

“Burn” – which permanently stamps the bar into the image.

If the image is not already an 8-bit RGB it will now be converted to
that format.
Always save out your original image before burning the scale bar,
which should then be saved out as a copy of the image. You may
want to go back to the original without a scale bar. Also, initially it
may take some trial to get the scale bar properties to your liking.

“Change scale properties” – set the size, color, font, etc.

Note that you can let the software auto size the length of the scale
bar or uncheck and select from a pull down of different sizes.
41

Software Controls – Merge Channels

Merge Channels – overlays multiple color images to create a

multi-color fluorescence image.

Use the pull downs for each color component to select the
desired image for that component.
Software Controls – Autofocus
If the focus is in close range, and when
changing objective lenses, you can use the
software autofocus feature to critically
focus:

AF1 – Primary Coarse Autofocus

AF2 – Secondary Fine Autofocus

Software Controls – Other Controls
Arrow – return to normal mouse arrow

Magnifying Glass – Click on this control to get magnifier

icon on mouse. Left click activates zoom window within
any image window.

Close All – closes all open images, with a dialog prompt to

be sure you want to discard all open images.

Convert Image to 8-bit RGB – converts any image to 8-bit

RGB color image - discussed in detail in “Exposure and
Contrast” section.
Nikon Eclipse 90i Operations Guide

EXPOSURE AND CONTRAST

 Exposure and Contrast – LUT’s
The best possible image is one that fills the dynamic range of the
camera without overloading any pixels and causing saturation.

The “LUTs” window in the bottom left corner of the software window
shows a graphic representation of the dynamic of the camera and
the intensity of the image.
Monochrome Image Color Image
Exposure and Contrast – Methods for Use
1)  Use “Auto Exposure” button in camera control box – software
will determine the longest possible exposure before saturation
occurs.

2)  Adjust exposure manually and view histogram in LUT’s window

to fill dynamic range of camera to your liking.

Manual exposure adjustment is usually preferable for fluorescence

imaging where auto exposure will may you an unnecessarily long
exposure.
Exposure and Contrast – Saturation

The toolbar at the top of the live image window has a

saturation indicator button. Pushing this will show any
overexposed areas in the image.

The black arrow next to it will show a list of options to change

the color of the indicator, etc.
Exposure and Contrast – Adjusting Contrast
It is not necessary to only use exposure to get the best and brightest
possible image. It is often desirable to use a shorter exposure and
then use the software contrast controls to adjust the image brightness
to your liking.

The black bar The white bar is

is the contrast the brightness
setting. setting.
The best The best setting is
setting is generally just at
generally just the very right edge
at the left of the image
edge of the histogram.
image
histogram.
Exposure and Contrast – Adjusting Contrast
The auto adjust button will set the brightness bar at the right edge of
the image histogram.
Red “X” button will reset the
contrast to non-adjusted

Use the mouse

to drag the
You can use
the mouse to brightness bar
if you want to
drag the
contrast bar up manually adjust
brightness
over from the
left to adjust
the background
darkness.
Exposure and Contrast – Adjusting Contrast
Adjusting image contrast in Elements does not change the image data,
just the way it looks on the screen.
If you want to permanently fix the contrast for viewing in other
programs as well, push the “Apply LUTs” button.
A warning message will indicate you are about to permanently change
the image.
Note: It is best to
save a raw original
before applying LUT
changes
Exposure and Contrast – Saving Image
Contrast
Adjusting image contrast in Elements does not change the image data,
just the way it looks on the screen.
If you want to permanently fix the contrast for viewing in other
programs as well, push the “Apply LUTs” button.
A warning message will indicate you are about to permanently change
the image.

You can save an LUT setting and then later reapply it to another image
using the “Save LUTs” button.
Exposure and Contrast – RGB Contrast
Real color images from the Fi1 color camera will have 3 look up tables
– Red, Green, and Blue
Sliding the contrast bars will move all colors equally to maintain color
balance. Holding shift key while sliding a color bar will adjust only that
color, and color balance will change.
 Exposure and Contrast – RGB Contrast

AWB – for post capture Change orientation of LUT boxes

white balance adjustments

Pixel Saturation Indicator – will

color any pixels in the image that
are overexposed
Nikon Eclipse 90i Operations Guide

MONOCHROME IMAGE
HANDLING
Monochrome Image – Pseudo Color
By default Elements will display a monochrome image in the
pseudo color dictated by the Optical Configuration. To turn
off this aid and view images in true monochrome, right click
in the image window and unselect “View Channel in Color”
Monochrome Image – Pseudo Color
The pseudo color you see in Elements software will not follow
the image if you try to just save it and open in another
program. See the section that follows later on converting
images to 8-bit RGB.

Note: Any single pseudo color image must have “View

Channel in Color” active before it can be made into a
color 8-bit RGB image.
 Monochrome Image – Overlay
To make a multi-color image from single pseudo color images
you can overlay them with each other – including overlaying
phase contrast with fluorescence.
To overlay, use the mouse to left click on
the name tab of the image you are
moving, and then holding down the
mouse drag and drop it into the image
space of the other image.

The result image will now have multiple

name tabs – “All” which will show all
colors, and then a tab for each color
that will just show that individual color in
the image window.
Monochrome Image – Pseudo Color Multi
Channel Image Contrast
When you overlay multiple monochrome images, you will get multiple
contrast windows for each color of your overlay. Colors can then be
adjusted separately to balance the look of the image.
Single Channel View Multi-Channel View
Monochrome Image – Pseudo Color to RGB
To take images out of Elements and display them in other programs,
you can convert to an 8-bit RGB image.
Press the quick button on the left hand side of the screen to make the
conversion.
It is prudent to save the original image before making the 8-bit RGB
copy so you have a raw file.

Note: The image name tab will change to RGB tabs.