Nikon Eclipse Ti2

Contents

Eclipse Ti2 specifications

User guidelines
Nikon Eclipse Ti2 Microscope: general information and care (objectives, lens care, immersion oil)
Startup and sample setup
System startup
Accessory equipment
Sample setup and observation with the microscope
Köhler Illumination
NIS Elements acquisition setup
Progam interface
Basic image acquisition
Microscope settings
Acquisition
Advanced image acquisition
Multi-channel
Z-Stack
Large image
Multi-point imaging
Time lapse
Combined ND acquisition
Perfect Focus System (PFS)
Viewing options
(adding a scale bar, switch view between channels, LUTs, ND views, volume viewer)
Image processing
Clarify.ai and Denoise.ai
ND2 files
Examples (thresholding, create ROIs, create an EDF image, deconvolution, colocaliza-
tion, measurements)
Macros (record, save, run)
Data management
(file format, image layers, saving images, save as .avi or .mov, export analyis data,
create reports)
Shutdown

March 2022
Nikon Eclipse Ti2 2

Nikon Eclipse Ti2

The inverted Nikon Eclipse Ti2 microscope, equipped with a high precision encoded stage and
Perfect Focus System is specifically designed for automated acquisition of large image
files (time lapse, stitched images, high-content screening), and fast ratiometric imaging.
Furthermore, a laser engine makes the system capable of TIRF and FRET imaging.

Camera: Hamamatsu Orca Flash4.0 LT camera

LED excitation lines for fluorescence: 395 nm, 440 nm, 470 nm, 508 nm, 555 nm, 640 nm

Fluorescence filters for DAPI, CFP, GFP/FITC, YFP, RFP/TRITC, CY5

LED-based Fura-2 illuminator for ratiometric Ca2+ imaging: 340 nm and 380 nm

Laser engine with 488 nm and 561 nm lasers

Objectives: 4x, 10x (Air); 20x, 40x (Air; ELWD, long working distance); 60x, 100x (Oil), 100x
TIRF (Oil)

Software: NIS Elements - with modules AR (Advanced Research) and HC (High Content), includ-
ing high content analysis (HCA)

Contact:
Dr. Michaela Strüder-Kypke
Office: Summerlee Science Complex,
room 1253
Tel: (519) 824-4120 ext. 52737
E-mail: [email protected]
Nikon Eclipse Ti2 3

User Guidelines

1. When you begin to use these facilities please provide a Billing Authorization Sheet including the supervisor’s
signature and the Trust Fund Account number.

2. Before any unsupervised access is granted, users must enroll in supervised training sessions during which
they will review with the confocal manager how to operate the equipment properly and safely. The time
required for the training sessions will vary depending upon the user’s demonstrated competency with the
equipment. Billing will be at the “Training” fee rate.

3. When using these facilities you must clearly write in the sign-up book the date, your name, department, log
on and log off time, and total number of hours you used the equipment.

4. For any planned after hours use of the system, please make arrangements with the confocal manager for
access to the hallways.

5. Users are expected to bring all their own supplies including pipets, slides, coverslips, computer disks, etc.
However, the facility will provide immersion oil and lens paper.

6. Files saved to computer hard drives must be removed as soon as possible. All computer hard drives will be
cleared on a regular basis - it is the users’ responsibility to manage their own image files. USB keys are not
allowed on any of the instrument computers! The facility offers a variety of other options for data transfer.

7. Please notify the confocal manager immediately of any problems that you encounter with the equipment - it
is essential that we work together in taking care of the facility. Improper care of the equipment will result in
rejection of access to the facility.
Nikon Eclipse Ti2 4

Nikon Eclipse Ti2 Microscope

General Information & Care

Objectives

Refractive Numerical Coverslip Recommended Step Size

Lenses Immersion
Index Aperture (NA) µm (#) for Image Stacks (µm)

4x Dry 1 0.13 < 1.2 29.6

10 x Dry 1 0.3 < 1.2 5.6

20 x Dry, ELWD 1 0.45 0-2 2.5

40 x Dry, ELWD 1 0.6 0-2 1.4

60 x Oil 1.52 1.4 0.17 (1.5) 0.3

100 x Oil 1.52 1.3 0.17 (1.5) 0.3

100 x TIRF Oil 1.52 1.49 0.17 (1.5) 0.3

Immersion oil
Use only the immersion oil (Type F) provided by the facility. Do not mix the immersion oil with other substances
including immersion oil from another source. If you used any other immersion oil during previous observations
with a different microscope, remove all traces with Windex.
Carefully apply the immersion oil to the lens.

Cover slip
Sealing the coverslip completely with nail polish or other sealing materials is highly recommended. This prevents
mixing of immersion oil and embedding materials, movement of the coverslip when moving the stage, contam-
ination of the objectives, and moving embedded materials during z-sectioning. Be sure that any nail polish is
completely dried before you place the slide onto the stage.

Lens care
After use, clean the lenses with lens paper only. Fold the paper and hold both sides of the paper, keeping the
folded edge straight. Draw the folded edge back and forth over the lens surface. Repeat with another piece of
lens paper and continue until there are no more traces of immersion oil on the paper. Use Kim Wipes to remove
excess oil on the objective (around the lens).
Nikon Eclipse Ti2 5

Nikon Eclipse Ti2

Startup and Sample Setup

System Startup

1) Hamamatsu camera (Fig. 1, arrow)

2) Microscope (Fig. 2, arrow)
3) Computer, no password required
4) Start NIS Elements and log into your lab account, select ‘Widefield’

Accessory equipment

Lumencor: LED light source (Fig. 1B), currently with 4 excitation wavelengths: 395 nm, 470 nm, 550 nm, and 640
nm. The LEDs are constantly on standby. Please don’t switch off the power supply.

Specialized LED-based Fura-2 Illuminator: The Fura-2 illuminator excites at 334 nm and 380 nm with a fast exci-
tation wavelength switcher. It is used for ratiometric Ca2+ imaging.

Microscope Controller: Core Power Supply (Fig. 1C) – this is also permanently on standby, there’s no need to
switch it on or off.

Camera: The system has a monochrome Hamamatsu Orca Flash 4.0 for fluorescence or black/white transmission
imaging (Fig. 1A).

Stage: The stage is fully motorized and the software gets the coordinates automatically. It is encoded for high
precision but still fast. Two inserts are available: one for regular slides and petri dishes and one for well plates.
Nikon Eclipse Ti2 6

Sample Setup and Observation With the Microscope

The microscope is fully automated and all functions can be controlled through the software. Therefore, you will
likely not need to use the buttons on the microscope with the exception of setting the Köhler Illumination.
Nevertheless, a quick overview of the controls available is given here (Fig. 3).
Please also refer to Fig. 9 on page 9 for setup using the NIS software controls (recommended!).

5 6 4
10 7 10
8
1
2 3
1 9

3A B C

1 - Focus Wheel 6 - Change Objectives

2 - Light Intensity 7 - Ocular Observation
3 - Transmitted Light On/Off 8 - Camera Observation
4 - Escape Button 9 - Fluorescence Filter Wheel
5 - Limit (= upper threshold) Button 10 - PSF Button

Tilt the microscope pillar carefully back, place your slide, petri dish, or well plate on the stage and - if you use
any of the oil immersion objectives - add the immersion oil (carefully place a drop of oil on the lens). Bring the
pillar carefully back down. Select ‘Eyepieces’ as observation mode (Fig. 3B - 7; Fig. 9B).

If you use the 20x or the 40x ELWD (Long Working Distance) objectives
(Fig. 4), you can not only image through a glass coverslip but also through E
relatively thin plastic (e.g., specific well plates). To achieve this, both ob-
jectives have a correction collar (Fig. 4, arrows) that needs to be adjusted F B F
for your sample (glass - setting closer to ‘0’; C D C
plastic - setting closer to ‘2’). A

The X/Y of the stage drive is the joystick on

the controller box (Fig. 5A). Switching be-
tween fast and precise movement is done by a
click on the ‘XY’ button (Fig. 5B) - the speed
is indicated by the number of arrows on the
controller display ( ^ ). 4 5
^ ^
Using the focus wheels on the side of the Nikon controller box (Fig. 5C), move the objective nosepiece up until
your sample is in focus (towards you = up, towards the wall = down). Switch to ‘Fine Focus’ by clicking on the ‘Z’
button on the controller box (Fig 5D). Fine and coarse focus are also indicated on the display ( ^ ).
^ ^
Nikon Eclipse Ti2 7

Centre your sample. Switch the joystick to slow movement.

Observe through the oculars, find your ROI (region of interest) and change to the objective you will use for imag-
ing (Fig. 3B - 6; Fig. 9A).

Adjust the focal plane again in your sample with the fine focus. Once your sample is in focus, press the ‘Limit’
button (Fig. 3B - 5) to set it as elevation limit. Pressing and holding the ‘Limit’ button will release the elevation
limit setting. Press the ‘Reset Z’ button (Fig. 5E) on the on the controller box to reset the reative Z coordinates
to Zero.

You now have saved your focal plane as ‘upper threshold’ and can move the nosepiece down quickly by pressing
the ‘ESC’ (= Escape) button (Fig. 3B - 4; Fig. 9G), e.g., to change your sample or add immersion oil. To bring the
objectives back up to your focal plane, press the ‘ESC’ button again.

Köhler Illumination

Köhler Illumination provides homogeneous illumination of your specimen without stray light. You get images with
optimum contrast and resolution. Please do this in the BF setting!

These are the steps (Figs. 6, 7):

6
Focus the image
A Close the field diaphragm (Fig. 7, FD), adjust the aperture dia-
phragm (Fig. 7, AP) if necessary.
B Adjust condenser height with condenser focus wheel (Fig. 7, ar-
FD
rows) until the edge of the field diaphragm is in focus.
C Centre the field diaphragm with the screws on the condenser (Fig.
7, *).
D Open the field diaphragm (Fig. 7, FD) until the edge just disap-
pears from the field of view.
E Adjust the aperture diapragm (Fig. 7, AP) so that it is two-thirds * AP*
open.

Once you have adjusted these settings, you can continue with NIS Ele-
7
ments if you want to do transmitted light imaging.

If you have fluorescently labeled samples, switch off the transmitted light (Fig. 3A - 3, Fig. 9E), select the
excitation line and filter for your dye/fluorescent protein (Fig. 3C - 9) by pushing the Fluorescence Filter switch
either towards the wall or towards you and open the Fluo Shutter by pressing on the Fluorescence Filter switch
(Fig. 3C - 9). All this will be done automatically if you select one of the pre-configured fluorescent light paths
(Fig. 11)! The installed fluorescence filters are for DAPI, CFP, GFP/FITC, YFP, RFP/TRITC, and CY5. Check wheth-
er the signal strength is sufficient, find your region of interest and adjust the focal plane if necessary.

If you used the microscope controls to set up fluorescence observation, make sure to close the Fluo Shutter
again (Important!) and continue with NIS Elements.
Nikon Eclipse Ti2 8

NIS Elements Acquisition Setup

Program Interface

All functions are accessible from the Main Menu at the top of the screen (Fig. 8C).

In the right tool area (Fig. 8A) you find the Main Tool Bar (E), the camera settings (Flash 4.0), the OC (Optical
Configuration) panel, which has the pre-configured light paths, the Spectra pad with the LED emission controls,
the microscope control panel, and the LUTs (look-up table) to adjust the image intensity display.

The main screen area (Fig. 8B) is used to display your images and has a top and a side tool bar (Fig. 8D) with dis-
play and annotation options. The icons in the tool bars change depending on your image (Z-stack, multichannel,
etc).

D E

B
8

A - Right Tool Area

B - Main Screen Area
C - Menu Bar
D - Top and Right Display Tool Bars
E - Main Tool Bar
Nikon Eclipse Ti2 9

Basic Image Acquisition

9A
Microscope Settings

Once the software is started, refer to the Optical 9B

Configuration (OC) Panel (Fig. 9A) and the ‘Ti2 Pad’
window (Fig. 9B) to set your light paths: you can
select the objective, camera or ocular observation,
and adjust light intensity. It is recommended to use
the NIS controls during your initial sample setup (see
pages 6-7).
Select the objective and ‘Eyes Brightfield’ (Fig. 9A,
arrow) for your sample setup, all other parameters
are saved with the light paths.

Acquisition
10A

11
10B
In the OC Panel, select ‘Transmitted Light’ to switch to camera observation.
You will see all available pre-configured light paths. Click on the desired light
path to call up all its settings (Fig. 10A, arrow).

Important: Phase Contrast and DIC require specific correction optics for each
objective. They are also not available for all objectives. Therefore, the Phase
and DIC light paths are individually programmed for each objective. If you
select the light path, the microscope will automatically switch to the corre-
sponding objective. Make sure you don’t contaminate the dry objectives with
immersion oil!

Epifluorescence and Fura2 light path settings are also selected in the OC panel
(Fig. 11).

Once you have selected the light path, you need to click on ‘Live’ in the Main
Tool Bar (Fig. 10A). The shutter will open and the transmitted light or LED ex-
citation will be activated. Adjust the exposure time in the drop-down menu in
the camera control panel (Fig. 10B, arrow).
Nikon Eclipse Ti2 10

Since the camera is highly sensitive and has a range

of 65,000 intensity values, you likely need to select
‘Keep Auto Scale’ in the ‘LUTs’ window (Fig. 12, ar-
row) in order to actually see your live image on the
screen (most likely for fluorescence imaging). This
will only change the display of your image on the
screen, the camera settings will not be changed.

Focus your sample and find the visual location (i.e.,

ROI, region of interest).

Once your settings are optimized, select the cam-

era icon (‘Capture’) in the top tool bar (Fig. 10A) to
acquire a single channel image.
12

Advanced Image Acquisition

If you want to take a multichannel image or an image series like a Z-stack, time series, or stitched image, you
need to define these actions in the ND Acquisition setup. Click on the ‘ND’ icon on the left (Fig. 13, circle) or in
the Main Tool Bar (Fig. 10A, 13A) to open the ‘ND Acquisition’ setup window (Fig. 14). You can dock this window
to the bottom centre area or keep it as separate window (Fig 13B).

Important: ‘ND Acquire’ in the Main Tool Bar (Figs. 10A, 13A) represents only a selected number of options with
basic settings. Select the ‘ND acquisition’ on the left for full capability. Therefore, all acquisition steps are
explained based on the windows for the left ‘ND acquisition’ icon (Fig. 13, circle). The simplified ‘ND Acquisition
Wizard’ (Fig. 14) is always shown for reference.

A C

13
Nikon Eclipse Ti2 11

The options for ND Acquisition appear in

the main ‘ND Acquisition Wizard’ window
(Fig. 14) and at the top of the ‘ND Acquisi-
tion’ window (Figs 15-19) if selected.
They are:
Timelapse - Multipoint - Z-stack -
Multichannel - Large Image (left ND icon
only and separate button in the Main Tool
Bar (Fig. 10A). 14

Multichannel

Fluorescent signals can be captured sequentially and merged into one overlay image. Transmitted light channels
can also be included in the sequence.
Multichannel imaging is perfectly represented in the ND Acquisition Wizard on the Main Tool Bar.

B D
E
C

F
15

• Select the ‘lambda’ tab (or ‘Multichannel’) in the ND

Acquisition window (Fig. 15A) and check-mark the box
to activate this protocol.
• Select those channels you want to acquire (‘...’ and
drop-down menu, Fig. 15B). Select the fluorescence
channels in the order from longest to shortest wavelengths to minimize interferences!
• Make sure the box beside each channel you want to acquire is checked! (under optical configuration, Fig.
15C).
• Select ‘Save to File’ and define the path and folder where to save your image (Fig. 15D). This option is
found under ‘Acquisition Parameters’ in the ND Acquisition Wizard.
• Define the file name (Fig. 15E) and press ‘Run now’ (Fig. 15F).
Nikon Eclipse Ti2 12

Z-Stack

A series of images from different focal planes in a sample can be acquired and, e.g., converted to a 3D model of
the specimen.

A
B
D
E
C

F
16

• Select the ‘Z’ tab (or ‘Z-stack’) in the ND Acquisition

window (Fig. 16A) and check-mark the box to activate
this protocol.
• Select how to define the range of your Z-Stack (Fig.
16B, there are 3 options - see below).
• Define the step size (Fig. 16C) - the system will auto-
matically assign a predefined step size depending on
the objective used. Recommended step sizes are listed in the table on page 4.
• Select ‘Save to File’ and define the path and folder where to save your image (Fig. 16D). This option is
found under ‘Acquisition Parameters’ in the ND Acquisition Wizard.
• Define the file name (Fig. 16E) and press ‘Run now’ (Fig. 16F).

Define range by setting top and bottom plane

- Select ‘Live Stream’, focus and adjust exposure
- Move the Z drive to the top position of your stack and press the ‘Top’ button
- Move the Z drive to the bottom position of your stack and press the ‘Bottom’ button

Define range by symmetric distance from the current (= Home) position

- Select ‘Live Stream’, focus and adjust exposure
- Press the ‘Home’ button to define the home position
- Specify the Z-range in µm
- The software will acquire images in equal distances to the home position

Define range by asymmetric distance from the current (= Home) position

- Select ‘Live Stream’, focus and adjust exposure
- Press the ‘Home’ button to define the home position
- Specify the Z-range with 2 values: one for ‘Below’ and one for ‘Above’ in µm
- The software will acquire images in those distances to the home position
Nikon Eclipse Ti2 13

Large Image

With the Large Image option, you can acquire a number of individual images (tiles) and merge them into a
large image if the ROI exceeds the camera field of view. To do this, it is possible to define tile regions and posi-
tions.

A
B
D
E
C

F
17

• Select the ‘Large Image’ tab in the ND Acquisition window (Fig. 17A)
and check-mark the box to activate this protocol.
• Select how to define the area of your Large Image (Fig. 17B, there are
3 options - details see below).
• Define how the tiles are stitched together (Fig. 17C); a 10-15% overlap
between the tiles is recommended.
• Select ‘Save to File’ and define the path and folder where to save
your image (Fig. 17D).
• Define the file name (Fig. 17E) and press ‘Run now’ (Fig. 17F).

Depending on your sample and the size of your large image, it is recommended to create a focus surface. To
do this, select ‘Use Focus Surface’ in the ND Acquisition Window (Fig. 17, arrow). Then define several positions
across your large image region and focus the image there. Select ‘Add’ and they will be added with XYZ position
information. The software will extrapolate these data to create a focus surface over the entire region.
Important: Large Image is not represented in the ND Acquisition Wizard - it has it’s own icon, the ‘Scanning Wiz-
ard’ in the Main Tool Bar (Fig. 10C).

Define area by number of tiles in X and Y direction

Find the ROI - select the centre area – define the number of tiles in X and Y direction – this is simple and quick,
but it is very general.

Define area by size

Find the ROI - select the centre area – define the area in millimetres - this is also simple and quick, but very
general.

Define area by a specific pattern

Find the ROI - select the centre area – select the ‘Pattern’ button in the ND Acquisition window - click on the
‘Browse’ button - select one of the saved Large Image patterns.
Nikon Eclipse Ti2 14

Time Lapse

A sequence of images can be captured over a period of time to create a time lapse series.

A C
D
B
E

F
18

• Select the ‘Time’ tab (or ‘Timelapse’) in the ND Ac-

quisition window (Fig. 18A) and check-mark the box
to activate this protocol.
• Define the interval and duration of your image acqui-
sition; make sure the box beside each time phase you
want to acquire is checked (Fig. 18B).
• You can combine consecutive time acquisition proto-
cols with different intervals and durations by clicking ‘Add’ (Fig. 18C).
• Select ‘Save to File’ and define the path and folder where to save your image (Fig. 18D). This option is
found under ‘Acquisition Parameters’ in the ND Acquisition Wizard.
• Define the file name (Fig. 18E) and press ‘Run now’ (Fig. 18F).

Multipoint Imaging
A
B
D
E

C G

F
19

Images from different XY coordinates in your sample can

be acquired, e.g. if you work with samples in well plates.
Nikon Eclipse Ti2 15

• Select the ‘XY’ tab (or ‘Multipoint’) in the ND Acquisition window (Fig. 19A) and check-mark the box to
activate this protocol.
• Define your multiple positions - there are several options, see details below.
• Select whether to include the Z position (Fig. 19C) - it is recommended to include the Z position, especial-
ly in live cell imaging, as the focal planes might differ slightly.
• Select ‘Save to File’ and define the path and folder where to save your image (Fig. 19D). This option is
found under ‘Acquisition Parameters’ in the ND Acquisition Wizard.
• Define the file name (Fig. 19E) and press ‘Run now’ (Fig. 19F).

Define points manually

Move the stage to the positions of interest, focus if necessary, and click ‘Add’ (Fig. 19B).

Define points randomly (Fig. 20A)

Click on the ‘Custom’ button (Fig. 19G) - select the ‘Random’ tab - select the
size and shape of the area (rectangle or circle) to be filled with random points
and define the reference XY coordinates needed - specify the number of ran-
dom points - click ‘Finish’ .
20A
Define points in a carrier (Fig. 20B)
If your sample is in a wellplate, select ‘Custom’ (Fig. 19G), in the new window,
select ‘Manual’ (if you know the distance between the wells) or ‘Interactive’.
This method lets you specify the number of fields to be scanned and starts a
wizard in which you will define the top-left and bottom-right corner of the
20B wellplate. The distances between the wells are then calculated automatically.

Working with carriers

NIS Elements has a large number of templates for commercially available carriers (Fig. 21A) that can be ac-
cessed through the Main Menu: ‘View’ - ‘Acquisition Controls’ - ‘Wellplate and Slide Navigation’. Once you have
selected the carrier, you can navigate within this template (Fig. 21B).

21A 21B
Nikon Eclipse Ti2 16

Combined ND Acquisition

Two or more of the ND Acquisition Dimensions can be com-

bined. A typical example is Fura-2 imaging over a specific
time. A

If you use the left ‘ND’ icon:

B

• Define all ND Acquisition protocols you want to include

and make sure the checkbox in each tab is selected
(Fig. 22, arrow).
• Based on those settings, an overview shows how the
.nd2 file structure will look like (Fig. 22, A).
• Select ‘Order of Experiment’ to select the item which
defines the desired order of loops in the .nd2 file (Fig.
22, B). 22 C
• Select ‘Save to File’, specify path and file name as described above (Fig. 22, B).
• Click ‘Run now’ (Fig 22, C).

If you use the ‘ND Acquisition wizard’ in the main tool bar:

• Under ‘Acquisition Parameters, check all ND Acquisition protocols (Timelapse, Multipoint, Z-stack, Multi-
channel) that you want to include (Fig. 14).
• Based on those settings, the relevant tabs will appear on the left of the window - you need to select each
tab and set the parameters as described above
• Select ‘Save to File’, specify path and file name as described above (Fig. 14).
• Click ‘Run’ (Fig 14).

In order to combine the ‘Scanning Wizard’ and the ‘ND Acquisition Wizard’ you need to select ‘Addons’ in the
Main Menu (Fig. 8C). There, you’ll find the options:

Large Images: Scanning Wizard (= icon in Main Tool Bar)

Scanning Wizard EDF (combines the ‘Scanning Wizard’ with the option to take Z-Stacks)
ND Acquisition: ND Acquisition Wizard (=icon in Main Tool Bar)
ND Acquisition Wizard + Large Images (Timelapse, Z-Stack, Multichannel in the ‘ND
Wizard’ can be combined with the ‘Scanning Wizard’)

For complex and long acquisition protocols an ‘ND Progress Window’ will appear which displays the overall prog-
ress, time elapsed and time remaining in the experiment as well as more details on status, remaining disk space,
and the option to pause the acquisition.
Nikon Eclipse Ti2 17

Perfect Focus System (PFS)

The PFS is a focus maintenance system that automatically corrects focus shift of the specimen or the stage due
to temperature. It is most useful in long-term imaging sessions.
The PSF uses a mismatch in the refractive index to measure, track, and maintain the distance between the ob-
jective and the sample surface (i.e., cover glass, well plate bottom, etc.).
It has an optical offset function, which allows for manual adjustment of the focal plane position within the spe-
cific range for each objective.
With the exception of the 4X objective, all objectives are compatible with the PFS. The system will give an error
message on the display of the controller when the objective can’t be used with PSF.
Perfect Focus will be disabled when Z-Stack images are acquired!

The Perfect Focus System is not very useful with ...

• Fixed, mounted specimen and oil immersion objectives, since the mounting medium mostly has the
same refractive index as glass and immersion oil.
• Thick samples or samples that generally strongly scatter light.
• Samples in plastic dishes other than the ones recommended.
• Samples which have the cover glass or well plate bottom contaminated with dust, oil, etc.

Starting the PSF

• Start focusing your sample. When the objective comes into the PSF range, you will hear several short
beeps and the PSF indicator flashes.
• Continue to focus until you have the correct focal plane.
• If the PSF indicator flashes, press the PSF button either on the microscope focus wheel or on the Nikon
controller box (Fig. 3A,C - 10, Fig. 5F).
• This PSF reference point is different for each objective.

The status of the PSF is displayed on the PSF indicator on the front panel of the microscope:
Indicator light

On PSF on - perfect focusing is in progress

Flashing in slow intervals PFS on - waiting for interface detection
Flashing at fast intervals PSF off - perfect focusing is off but position is within range
Off PSF off - perfect focusing is off, position is out of range

Offset adjustment
To account for different focal planes within your sample (e.g., when imaging several wells of a plate), you can
further focus your sample while the indicator light is on, i.e., the PSF is on and in progress. When reaching the
upper limit of the allowable range, a beep tone sounds. If you leave this range, the PSF will remain on but the
indicator will switch to slow flashing.
The offset range depends on the objective.
Nikon Eclipse Ti2 18

Viewing Options

Depending on your image (Multichannel, Large Image, Z-Stack) you can change how the data of your image are
displayed in the tool bars at the top and right side of the centre area (Fig. 23). The options will vary depending
on your image data. Some of the defaults are shown here.

From top to bottom

Show Probe (=small ROI to show intensity)
From left to right Show Background Probe
Enable LUTs Show Grid
Show LUTs Window Show Scale
Keep Auto Scale LUTs Show Frame
Auto Scale Turn ROI On/Off
Reset LUTs Show Intensity Profile
Pixel Saturation Indication View LUT Intensity
Enable Dynamic Threshold Show Annotations
View Binary
View Colour
View Overlay (Colour & Binary)
From left to right
Fit to Screen
Best Fit
1:1 Zoom
Zoom In / Zoom Out
Numerical Zoom
Minimize Window
Display All Open Images
Close Image 23

If you right-click on the icons, or click on the arrow beside them, you will open the drop-down menu with more
options for and properties of each tool.

Adding a scale bar to your image (Fig. 24)

Select the ‘Show Scale’ icon in the right panel. A scale bar
will appear in your image. Click and hold the scale bar to
move it around, right-click on the ‘Show Scale’ icon and
select ‘Properties’ to change the length and appearance of
the scale bar.

Switch view between the individual channels (Fig. 25)

The default display of a multichannel image is
the overlay. Below your overlay image are the 24
‘Channel tabs’. By selecting one of them, only
this channel will be displayed.
25
Nikon Eclipse Ti2 19

LUTs - Look-Up Tables (Fig. 26)

LUTs is a tool for image colour and brightness modifica- A B F
tions. You can enhance the image for observation without C
changing any of the image data. If you select a specific LUT
setting, it is saved along with the image file.

The best starting point is to select ‘Keep Auto Scale’ (Fig.

D
26A, applies auto scale permanently, also to live images) or
‘Auto Scale’ (Fig. 26B, adjusts the right slider position of all
channels automatically). E
But you may want to change the LUTs manually to equalize
several images for observation:

Select ‘ Enable LUTs’ (Fig. 26C)

26
The main parameters to change are
Gamma Parameter - move the centre point of the gamma curve up or down (Fig. 26D) - although this is
generally not recommended
Intensity Range - move the sliders from the edges of the range towards the centre (Fig. 26E)
You can reset those values with a double-click on the graph or the ‘Reset LUTs’ button (Fig. 26F).
Up to 4 LUTs can be displayed simultaneously.

ND views
There are several ways to display your ND images. The most common options are listed below:

27

• Split Image View (Figs. 27, 28A) displays the individual channel images side by side in the display

• Slices View (Fig. 28B) - displays orthogonal XY, XZ, and YZ projections of the images sequence (Z-Stack or
Time Lapse, then it’s XY, XT, YT)

• Volume View (Figs. 28C, 29) - creates 3D model of the acquired object (Z-Stack)

• Tiled View (Fig. 28D) - displays individual frames of the selected dimension beside each other; one or two
dimensions can be viewed at a time (Z-Stack, Time Lapse, or Multipoints)

• Maximum/Minimum Intensity Projection View (Fig. 28E) - analyzes all frames of one dimension and picks
pixels with the maximum/minimum intensity values to be displayed in the resulting image (Z-Stack, Time
Lapse)
Nikon Eclipse Ti2 20

A B C D E

28

Volume Viewer
This module allows you to view and move your Z-Stack as a 3D model and navigate through it from all angles and
sides (Fig. 29).
Select the ‘Volume Viewer’ icon in the top display tool bar, or
In the Main Menu, select ‘View’ - ‘Image’ - ‘ND View’ - ‘Volume View’
The current Z-Stack is displayed as 3D model.
The top Display Window Toolbar will now show several options relating to viewing this model (Fig 29A: Free Ro-
tation Control, X-, Y-, Z-Axis Rotation Control, View Plane, Blending options, 3D settings, etc.) and a new control
panel with more functions will pop up, called ‘Volume Options’ (Fig. 29B).
You can also create an .avi movie of the 3D model.

29
Nikon Eclipse Ti2 21

Image Processing

NIS Elements has a multitude of analysis options for individual images and high content batch processing. Some
basic and quick examples are described below. Please refer to the online help or contact Michaela if you need a
specific function not listed here.

Clarify.ai and Denoise.ai

Clarify.ai is an AI-based image processing function that, by means of a neural

network, has learned the characteristic components of fluorescent light emit-
ted from out-of-focus planes. It can remove “blurred light” from fluorescence
images, providing clear, high-contrast images and it processes images faster
than conventional deconvolution algorithms. The application is simple - the
parameters are obtained from the image metadata - just select ‘Clarify.ai’ in
the main menu, check ‘Create new document’ and mark the channel(s) you
want to improve - then select ‘OK’ (Fig. xxx).

Denoise.ai is another AI-based image processiong tool that improves the sig-
nal-to-noise ratio in your image, especially in samples with low signal inten-
sity. Just like clarify.ai, the tool is simple to use and obtains the paramaters
from the metadata of your images. Select ‘Denoise.ai’ in the top menu, check
‘Create new document’ and mark the channel(s) you want to improve - then
select ‘OK’ (Fig. xxx).

Original Image Clarify.ai Denoise.ai

Nikon Eclipse Ti2 22

ND2 Files

You will find the options for ND2 file processing in the Main Menu under ‘Image’ - ‘ND Processing’. Most of the
image processing commands can be applied to one frame, all frames, or selected dimensions in the .nd2 file. You
will select this in the command dialog window.

When an .nd2 file is opened, its structure is pictured at the bottom of the image window. There is a time line
with all captured images indicated by gray markers. The blue-highlighted marker indicates the currently ob-
served image.

The time dimension has a time line, loops of the other dimensions (e.g., number of channels or Z-stack planes)
are shown as rectangles. The current selection in the loops/timeline is highlighted green. Browse the .nd2 file by
clicking inside the timeline/loops.

You can play the selected sequence or go through the sequence frame by frame.

If you right-click the selection, a submenu will come up and you can adjust, delete, or crop your selection.

Examples

Thresholding
Specifying correct threshold limits is crucial in automated image analysis. NIS Elements has modes for threshold-
ing available: RGB - HSI (hue, saturation, intensity) - Per channel - Intensity (Fig. 30A).
The mode depends on the type of image!
For Large Images, you have the option to display the threshold preview only on a limited area of your image to
speed up the adjustment process - check the box ‘Preview on selected area’.

• Open your image

• In the Main Menu select ‘View’ - ‘Analysis Controls’ - ‘Thresholding’ A
• The ‘Thresholding’ Control Panel will come up (Fig. 30)
B
• Select one of the Thresholding modes
• Define the thresholding limits:
Select one of the pixel picking tools (Fig. 30B) and start clicking on
objects in the image that you want to be detected. You can also
directly define the thresholding limits by moving the vertical lines
within the histogram (Fig. 30, arrows). D

All pixels with numeric values within the threshold limits will be saved in C
the resulting binary layer.
If there are misdetected objects you may be able to eliminate them by 30
morphology or size - these functions are available at the bottom of the control panel (Fig. 30C).

Basic operations on the binary layer can be performed before it is displayed on the screen (Fig. 30D):
Clean (removes all small objects from the binary image)
Smooth (smoothes the binary image contours)
Fill Holes (fills holes with binary objects)
Separate (separates objects)
Nikon Eclipse Ti2 23

If you want to threshold for circular objects of similar size, you can select ‘Spot Detection’.
In the Main Menu select ‘Binary’ - ‘Spot Detection’
Select the method how the spots are detected (bright or dark, clustered or different sizes).
The ‘Spot Detection’ dialog window will come up (Fig. 31).
Select the channel on which you want to detect the objects (Fig. 31A).
You can display your objects either as circular areas, or as spots:
• Select ‘Circular Area’ (Fig. 31B).
• Check if you can see objects in the
preview (if not, ‘Reset’ and start
over). A
B E
• Adjust the values for ‘Typical Di- C
ameter’ and ‘Contrast’ to fit your D
objects tightly into the displayed
circle and to remove unwanted 31
spots (Fig. 31C).
• Choose where to apply the current settings (Fig. 31D) and click ‘OK’.
• Save the new binary layer together with your file.
OR
• Select ‘Spot’ (Fig. 31B).
• Adjust the pixel size of the spots (Fig. 31E).
• Check if you can see objects in the preview (if not, ‘Reset’ and start over).
• Choose where to apply the current settings (Fig. 31D) and click ‘OK’.
• Save the new binary layer together with your file.

Create an EDF (Extended Depth of Focus) image

If you have a Z-Stack, the EDF function combines this Z-Stack into one focused image A B
by picking the focused regions from each frame and display the resulting data. You
32
may want to create an EDF for your image analysis.
Open your image and select the EDF icon in the Top Display Tool Bar (Fig. 32). You have the choice to view the
regular 2D image (Fig. 32A) or a 3D surface of this image (Fig. 32B).

Create ROIs
ROIs can be created either in the Main Menu: select ‘ROI’, or by clicking on the
‘Turn ROI On/Off’ icon in the right Display Tool Bar.
You can change the properties of a selected ROI by right-clicking on it and
select an option in the submenu that comes up. Many ROI controls can also be
accessed through ‘Ctrl’- Mouseclick in the image (Fig. 33).
If an .nd2 file has a time dimension, the ROI can be defined as ‘Global’ or
‘Changing over time’.
If an .nd2 file has a multipoint dimension, the ROI can be defined ‘Per Multi 33
Point’; this means the ROIs in each point are entirely independent from the
ROIs in other points. If the ROI is defined as ‘Global’ it will be applied to all points.
Nikon Eclipse Ti2 24

Deconvolution
Deconvolution might be necessary for some image stacks. To be able to do this, you need the PSFs (Point Spread
Functions) for the corresponding objectives. Each objective has its own PSF for each channel. The software can
automatically calculate the PSFs for each objective and wavelength.
Two deconvolution modules are available in the Main Menu:
2D Deconvolution for standard 2D images (one focal plane)
3D Deconvolution for Z-Stacks

• Open your image/image series and in the Main Menu select ‘Deconvolution’ and either ‘2D Deconvolu-
tion’ or ‘3D Deconvolution’ (Fig. 34A).
• In the control panel that comes up, check that the parameters (objective, wavelengths, NA, etc.) are
correct - if not, change them (Fig. 34B).
• Select which channels to deconvolve (Fig. 34C) and click ‘Deconvolve’ (Fig. 34D).
• Depending on your image, this process might take quite a while. You can select to do the deconvolution
only on a specific ROI (Fig. 34E).

E D
34
Nikon Eclipse Ti2 25

Colocalization
The Colocalization function under Main Menu - ‘View’ - ‘Analysis Controls’ - ‘Colocalization’ opens a new window
(Fig. 35A) which automatically shows the scatter plot and Pearson Correlation for 2 channels in the entire image
or for selected ROIs (Fig. 35B, C).
If you have more than 2 channels in your image, you need to select 2 of them in the drop down menues (Fig. 35,
arrow). You can define one or more ROIs in your image, if you select all of them, the data for all ROIs will be dis-
played in the table below the scatter plot. You can then export the data into an Excel spread sheet or as report
(Fig. 35D and see below, Data Management).

A D

35

Measurements
Rough measurements can be performed utilizing graticules.
• To activate the graticules, press the ‘Show Graticule’ button (Fig. 23)
• Select the graticule type (grid, circle, cross, ruler) in the drop-down menu of this icon
• Define the graticule properties (shape, colour, line width)
• Define the graticule density (= closest distance between two line intersections)
• Measure

A binary layer can be created by ‘Copy Graticules to Binary’

A graticule mask can be created, stored and loaded by ‘Save Graticule Mask As’ and ‘Open Graticule Mask’
The following list describes all features that can be measured within NIS Elements:
Object features - measured within a binary layer (binary objects)
ROI features - measured within ROIs
Field features - measured over the entire image area
Manual - features available in manual measurement
EDF features - measured in an EDF (Extended Depth of Focus) image
3D features - measured on 3D binary objects within a thresholded Z-Stack data set)
Tracking features - measured on tracked binary objects or ROIs
Nikon Eclipse Ti2 26

Manual measurements like length, length 3D, angle, area, radius can be
measured over an image. The results are being recorded to a simple statistics
table, which can be exported. The data can also be presented as graph.
• In the Main Menu select ‘View’ - ‘Analysis Controls’ - ‘Annotations
and Measurements’
• A new window will pop up (Fig. 36)
• Select a tool for your measurements
• Measure the objects in the image using the mouse
• Select where to export the results
• Export (see page 26)

36
Automated measurements can be performed in combination with user-defined macros.
The key procedures are:
Optical system calibration (only necessary in specific cases, the system is calibrated)
Image acquisition
Define threshold, create the binary layer
Selection of measured features
Perform the automated measurement
Result presentation

The binary layer and the colour image can be used in the automated measurement. The binary layer is typically
used for shape and size measurements while in the colour layer intensity or hue measurements are carried out.
Measurements can be performed over the entire frame, in a measurement frame, or in a specific ROI.
NIS Elements distinguishes two types of automated measurements: Object Measurement and Field Measurement.

Object counts (Fig. 37):

Automated object detection can be performed after thresholding the image (see above).
Object count can also be performed on the Live image
In the Main Menu, select ‘View’ - ‘Analysis Controls’ - ‘Object Count’
The general workflow for object count is:

Thresholding Use restrictions Review the results - Export

37
Nikon Eclipse Ti2 27

Macros

A macro is an executable sequence of commands for image analysis that you can define using the NIS Elements
Macro Recorder (recommended) or write yourself (programming knowledge required!!).

Recording and saving a macro

• In the Main Menu, select ‘Macro’ - ‘Record’
• Perform the series of actions you would like to record
• Finish the recording by selecting again ‘Macro’ - ‘Stop Recording’
• Check the macro in the Macro Editor before you save it: ‘Macro’ - ‘Edit’
• Save the macro via ‘Macro’ - ‘Save As’

Running a macro
If you just created your macro and it is currently loaded in NIS Elements, select ‘Macro’ - ‘Run’
If you want to run a saved macro, select ‘Macro’ - ‘Run from File’

The macro execution can be stopped anytime by pressing the ‘Ctrl’ + ‘Break’ key combination.

Data Management

File format
By default, none of the single snapped images is automatically saved!
All snapped images are kept open in the image window (you see tabs of the acquired images lined up at the top
of the window) and they need to be saved as described below.
For the ND Acquisition, you will probably select the option ‘Save File As’. The ND2 (= multi-dimensional) files
contain a sequence of images.

Image layers
Each image can be saved with several layers
Annotation Layer - stores vector objects like measurements, text labels and other annotations
Binary Layer - is created when thresholding or spot detection was performed
ROI Layer - stores created ROIs
Colour Layer - contains image data captured by the camera
Nikon Eclipse Ti2 28

Saving images
Some of the most common image file format are: TIFF, JPEG2000, JPEG, LIM, ome.TIFF, ND2, AVI, MP4
There are several ways to save your individual images:
In the main menu:
‘File’ - ‘Save’ or
‘File’ - ‘Save As’ (automatically called up for new images)

In the main toolbar:

• Select the ‘Disk’ icon to save your files as above.
• You can select the file format (tif, avi, etc.) in the drop-down menu.
• Check the ‘Save Color Image’ option if you want to save the image colours into your file!
• Check the ‘Save Annotations’ option if you want the scale bar or other annotations saved into your image
file!
• When saving a multi-channel image as TIFF file, select ‘Convert to RGB’.

To save an image with the scale bar, right-click on the ‘Show Scale’ icon (Fig. 23), select ‘Burn Scale’. This will
automatically convert the image to an RGB tif or jpeg format with the scale bar saved in it - a warning appears
because some information of your Metadata may be lost - it’s best to previously save your image in the original
.nd2 or LIM format!!

Save (export) image sequences as .avi or .mov

Under the ‘File’ - ‘Save As’ function, .nd2 files can be exported as .avi or .mov files. If it’s a time lapse, you can
define the playback time (frame rate) and which video compression is applied.
If your nd2 file includeds several dimensions, you need to select the one that will be used to create the movie
(‘Use Time Loop’ - ‘Use Z-stack Loop’ - ‘Use Z-stack and Time Loop’).
XY Points: when the ND document contains multiple points, you can decide how these are saved. Either only the
current point can be save into the file, or all points can be saved as separate files. The different points can’t be
exported into one single file.

Export analysis data

Measurements and other analysis data can be exported from NIS Elements to an Excel spreadsheet or a tab de-
limited text file.
In the Main Menu, select ‘Export’ and select the desired file type.

Create reports
Report Creator allows you to create a print-ready document which can contain measured data, images, graphics,
etc.
Open the Report Creator under ‘File’ - ‘Report’ - ‘New Blank Report’
A graphical editor appears, it works very much like any text editor - you can insert text fields, simple shapes,
images, tables, or graphs.
Nikon Eclipse Ti2 29

Shutdown

1. Lower the nosepiece, remove your sample, clean objective lenses with lens paper

2. Save your data, close NIS Elements, transfer your files to OneDrive (preferred) or the server

3. Switch off microscope

4. Switch off camera

5. Shut down computer

6. Cover the microscope

2-Hour Rule
If someone else has booked the microscope within the next 2 hours, please don’t switch off the com-

puter, microscope and camera. Don’t cover the microscope.