Nikon N-STORM User manual

Super-resolution Microscope
N-STORM
Simple Operation Manual
M605E 12.6.Nx.1

Introduction
i
Introduction
Thank you for purchasing a Nikon product.
This instruction manual is written for users of the Nikon Super-resolution Microscope N-STORM. To ensure correct usage,
read this manual carefully before operating this product.
•No part of this manual may be reproduced or transmitted in any form without prior written permission from Nikon.
•The contents of this manual are subject to change without notice.
•The equipment described in this manual may differ from the actual product in its appearance.
•Although every effort has been made to ensure the accuracy of this manual, errors or inconsistencies may remain. If you
note any points that are unclear or incorrect, please contact your nearest Nikon representative.
•Some of the equipment described in this manual may not be included in the set you have purchased.
•If you intend to use any other equipment with this product, read the manual for that equipment too.
•If this equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be
impaired.
Symbols used in this operation manual
This operation manual uses the following symbols.
Indicates information that should be kept in mind when using this product,or which provides useful hints.
Registered trademark
•Product names and company names used in this document are trademarks or registered trademarks of their respective
companies.
•Trademarks and registered trademarks of their respective companies, as used in this document,are not marked with TM
and ®.

Contents
ii
Contents
Introduction ................................................................................................................................................ i
Terminology Used in This Document........................................................................................... 1
STORM Microscopy Operation .....................................................................................................4
2.1 Preparation of the N-STORM System ...................................................................................................... 5
2.2 Acquiring 3D-STORM (2D-STORM) Images.......................................................................................... 11
2.2.1 Acquiring Images in Normal Mode ...........................................................................................11
2.2.2 Acquiring Images in Continuous Mode..................................................................................... 16
2.3 N-STORM Analysis ................................................................................................................................ 22
2.4 Terminating the N-STORM System........................................................................................................ 28
2.5 Calibration for 3D-STORM ..................................................................................................................... 29
The Screens of the N-STORM Software ..................................................................................... 34
3.1 N-STORM Control Window.................................................................................................................... 34
3.2 N-STORM Settings Window................................................................................................................... 36
3.3 N-STORM Analysis Window .................................................................................................................. 37
3.3.1 Tool Bar .................................................................................................................................... 37
3.3.2 Slider ........................................................................................................................................ 40
3.3.3 Channel Tab ............................................................................................................................. 41
3.3.4 Status Bar................................................................................................................................. 42
Chapter
2
Chapter
3
Chapter
1

1
1
Terminology Used in This Document
N-STORM
System that allows STochastic Optical Reconstruction Microscopy (STORM) with Nikon’s inverted research microscope
Ti-E.
During STORM, some fluorescent probe molecules are randomly stimulated (activated) with relatively weak light to become
activated, after which their images are acquired through an EM-CCD camera (imaging). The series of images that is acquired
through frequent repetition of this process is analyzed and synthesized by software, and formed into a super-resolution
image.
Conventional image
Image of 256 x 256 pixels acquired using an EM-CCD camera. In STORM analysis, a set of sequentially acquired
conventional images is used as material.
STORM image (2D-STORM/3D-STORM image)
Super-resolution image generated as a result of analyzing a dataset of conventional images. It includes information such as
the position, size, and intensity of each individual fluorescent probe molecule.
A STORM image that only has information on the positions in the X- and Y-axis directions is referred to as “2D-STORM
image,” while one that also has information on the positions in the Z-axis direction is referred to as “3D-STORM image.”
Dye pair
Composite dye used for STORM observation, such as the following:
Examples: Alexa405-Alexa647
Cy2-Alexa647
Cy3-Alexa647
Probe
Fluorescent molecule (dye pair or monomolecular dye) used for STORM observation.
Dataset
Set of consecutive frames (conventional images) acquired through an EM-CCD camera. A dataset is saved in ND2 file format
for NIS-Elements.
Frame
Individual conventional image that is acquired through an EM-CCD camera as material to be used to generate a STORM
image.
Activation frame
Frame (image) that is acquired when relatively weak laser light for activation is emitted. This activation causes some
fluorescent probe molecules to become activated. Activation frames are not used for STORM analysis. Note that if images
are to be acquired in continuous mode, there is no difference between the activation and imaging frames because activation
and imaging are performed simultaneously.
Terminology Used in This Document
1
Chapter

Chapter 1 Terminology Used in This Document
2
Imaging frame
Frame (image) that is acquired when laser light for imaging is emitted. In this frame, fluorescent probe molecules fluoresce
only when activation has caused them to become activated. Note that if images are to be acquired in continuous mode, there
is no difference between the activation and imaging frames because activation and imaging are performed simultaneously.
Cycle
Set of frames that consist of an activation frame (normally one frame) and the subsequent imaging frames (normally three
frames) on one channel during image acquisition in normal mode.
Channel
For a multistaining procedure, the cycle in which each dye is observed is called a channel.
Period
Set of cycles that are made up of one cycle of each of the multiple channels on which images are acquired in normal mode.
For example, if images are acquired on two channels, one period consists of two cycles. If images are acquired on only one
channel, a period is the same as a cycle.
Dataset structure in normal mode (example)
Number of image acquisition channels: 2
Number of activation frames per period (per cycle) on one channel: 1
Number of imaging frames per period (per cycle) on one channel: 3
A1: Activation frame on channel 1
I1: Imaging frame on channel 1
A2: Activation frame on channel 2
I2: Imaging frame on channel 2
A
ctivation laser
Channel 1 (405 nm)
A
ctivation lase
r
Channel 2 (561 nm)
Imaging
laser (647 nm)
ON ON
ON
ON ON ON ON ON ON ON ON
Frame
Cycle Cycle
Period
Dataset
(Repeat the same
operation.)
A1 I1 I1 I1 A2 I2 I2 I2 A1 I1 I1

Chapter 1 Terminology Used in This Document
3
Continuous mode
Method of acquiring STORM images using a monomolecular dye that can itself become bright or dark, instead of requiring a
dye pair for activation. Since the activation and imaging laser light are emitted simultaneously to acquire images, acquisition
takes less time than in normal mode. In this case, the definitions of cycle and period are not applied.
Dataset structure in continuous mode (example)
Non-specific activation (NSA)
During image acquisition in normal mode, only those molecules that are detected in the first imaging frame after activation
are classified as being of the relevant channel. Also, those molecules that are not detected in the first imaging frame but in
the second or subsequent frames are classified as being those of a non-specific activation (NSA) channel. Information on
non-specific activation channels is used for crosstalk subtraction. (For details, see step 6 in “2.3 N-STORM Analysis,” in
Chapter 2.)
A
ctivation
laser (405 nm)
Imaging
laser (647 nm)
ON ON ON ON ON ON ON ON
Frame
Dataset
ON ON ON ON ON ON ON ON
(Repeat the same
operation.)

4
2
STORM Microscopy Operation
The overall operational steps and their corresponding descriptions in this chapter are as follows:
The screenshots in this document are presented as an example.
STORM Microscopy Operation
(
Steps from Ima
g
e Acquisition to Anal
y
sis
)
2.1 Preparation of the N-STORM System
2.5 Calibration for 3D-STORM
(only for acquiring new 3D-STORM images)
2.2.1 Acquiring Images in Normal
Mode
2.2.2 Acquiring Images in
Continuous Mode
2.3 N-STORM Analysis
2.4 Terminating the N-STORM System
2
Chapter

Chapter 2 STORM Microscopy Operation
5
2.1 Preparation of the N-STORM System
Prepare the microscope, the laser, and other peripheral devices, and then start NIS-Elements AR.
Connecting the piezo Z stage
The controller for the piezo Z stage must be connected to the PC with a USB cable. Remove the analog cable that
directly connects the controller to the microscope. Also, select [Manage devices...] from the [Devices] menu, and
turn off [Ti PiezoZ] under [Nikon Ti].
1 Perform safety checks.
(-> Chapter 3, “Detailed Microscopy Procedure” of the use
r
manual of the TIRF illuminator)
2 Turn the power on.
(1) Turn on the motorized stage and the illumination
light source.
(2) Turn on the piezo Z stage.
(3) Turn on the microscope.
(4) Turn on each laser head. (See the user manuals for
the TIRF illuminator and each laser head.)
(5) Turn on the LU4A laser unit.
(6) Turn on the PC.
POWER
Motorized stage power supply
Diascopic illumination power supply
HG precentered fiber illuminator
POWER
ONOFF
UNIT1 UNIT2 UNIT3
REVO
DSC1 DSC2 SHUTTER1
DC24V IN
ERGO/JOY
MODEL TI-HUBC/A
USB STAGE REMOTE HGFIE
TI-PS SHUTTER2
5 5 0 3 0 1
Thisdevice complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions:
(1)this device may not cause harmful interference, and (2) this device must accept any interference
receivedincluding interference that may cause undesired operation.
ThisClass A digital apparatus complies with Canadian ICES-003.
Cetappareil numØrique de la classe A est conforme  la norme NMB-003 du Canada.
MADE IN JAPAN
INTERLOCK
PIEZO
PFS
OFFSET
Microscope
LU4A
2-(1)
2-(1)
2-(1)
2-(3)
2-(5)

Chapter 2 STORM Microscopy Operation
6
When MPB Communications Inc.’s 647-nm laser is
used
(1) Start the laser control software GUI-VFL.
(2) Click the [On] button in the window to turn on
the 647-nm laser.
The value of [SHG temp.] is displayed.
(3) Follow the procedure below to gradually
increase the output of the 647-nm laser through
GUI-VFL (procedure recommended by the
laser maker).
Set the power to 50 mW, click [Activate], and
then wait until the value of [Power, mW]
becomes about 50 mW.
Then, set the power to 200 mW, click [Activate],
and wait until the value of [Power, mW] reaches
about 200 mW. The value of [Power, mW] may
change by a few percent, but this does not
affect the acquisition of STORM images.
After the completion of this procedure, use
NIS-Elements to adjust the laser power. It is not
necessary to use GUI-VFL for adjustment.
(For details on the procedure for turning off this laser,
see step 1 in “2.4 Terminating the N-STORM
System.”)
3 Start NIS-Elements AR.
(1) Start NIS-Elements AR by double-clicking the
corresponding icon.
(2) When the camera driver selection dialog is
displayed, select [ANDOR with N-STORM]. (To
change the camera driver after NIS-Elements is
started, select [Select Driver…] from the [Acquire]
menu.)
(3) Display the [N-STORM] control window and the
[DU-897 Settings] (camera) control window.(Select
[Acquisition Controls] from the [View] menu and then
select the control windows.)
3-(1)
3-(2)
(1)
(2)
(3)
(3)

Chapter 2 STORM Microscopy Operation
7
4 Place the imaging target in the visual field.
Set a specimen, direct the light path to the binocular part to
perform epi-fluorescence microscopy, and then put the
STORM imaging target into the visual field.
(For details on the procedure for epi-fluorescence
microscopy, see the user manual for the microscope.)
5 Configure camera settings.
In the [DU-897 Settings] control window (EM-CCD camera
settings), configure the settings as follows.
[Format For Live]: No Binning
[Format For Capture]: No Binning
Exposure time: Any setting (50 msec or 1
frame recommended)
[Readout Mode]: EM Gain 10 MHz 14-bit
[EM Gain Multiplier]: 30
[Conversion Gain]: Maximum available value
[Desired Temperature]
(Commands -> Advanced
Camera Settings): -70°C
6 Wait until the temperature of the camera stabilizes at
about -70°C and [Desired temp. differs!] disappears.
It takes a few minutes for the temperature of the camera to
stabilize.
7 Put the objectives for STORM into the light path.
Objectives: CFI Apo TIRF 100x oil (NA1.49) or
CFI Plan Apo VC 100x oil (NA1.40)
Use Nikon immersion oil Type B or Type NF for oil
immersion of the objectives.
When using CFI Apo TIRF 100x oil, adjust the correction
ring to suit the cover glass. If a cover glass No. 1-S
(No.1.5) is used, it is recommended that the position of the
correction ring be 0.165 mm.
5

Chapter 2 STORM Microscopy Operation
8
8 Perform an observation with TIRF illumination.
(1) Direct the light path to the EM-CCD camera.
Note: Set the cylindrical lens for 3D-STORM to OUT
(where it is not in the light path).
(2) Set the episcopic illumination to [TIRF].
(3) Put the N-STORM filter cube into the light path.
(4) Click [Interlock] on the N-STORM control window to
disable the laser interlock.
If the interlock cannot be disabled
If clicking [Interlock] does not disable the interlock, the
light path may not be set to the side port, or the laser
safety cover of the stage may not be secured properly.
Make sure that the light path is set to the side port (the
EM-CCD camera for STORM),and that the laser safety
cover is secured. Then, click the [Interlock] button again.
(5) Click [Live] of NIS-Elements to display the live
image on the screen.
(6) Select the checkbox for the 647-nm laser and set
the power to about 5% to 10%. (If the power is too
strong, photo-bleaching further occurs.) Deselect
the checkboxes for the other lasers.
(7) Set the shutter of the laser to OPEN in the
N-STORM control window.
(8) Set the laser position to the TIRF position (about
4200). (For details on how to adjust the TIRF, see
the user manual for the TIRF illuminator.)
(9) After TIRF observation is completed, temporarily
deselect the checkbox for the 647-nm laser on the
N-STORM control window.
8-(2)
8-(6), (9)
8-(4)
8-(8)
8-(7)
8-(1)

Chapter 2 STORM Microscopy Operation
9
9 Configure camera ROI settings.
Select [Camera ROI] -> [Define ROI] from the [Acquire]
menu of NIS-Elements, and then configure the settings as
follows.
[Left] 128 pixels
[Top] 128 pixels
[Width] 256 pixels
[Height] 256 pixels
Available range
The range available for STORM is 256 x 256 pixels only.
10 Configure the settings for the other optical systems
for N-STORM.
LU4A 4-laser unit Electric TIRF illuminator TI-TIRF-E 3D-STORM port
(Ti-E side port)
Cylindrical lens
ND filter slider STORM
slider
λ-plate
slider
ND
filter slider
N-SIM/N-STORM
switching port is
used
2D-STORM IN *1 IN IN OUT OUT 1x
3D-STORM IN *1 IN IN OUT IN *2 STORM *2
IN: Included in the light path, OUT: Not included in the light path
*1ND filter slider of the LU4A 4-laser unit
L2 position: Two ND32s are put into the light path.
L3 position: Both ND2 and ND4 are put into the light path.
L4 position: Two ND32s are put into the light path.
*2Cylindrical lens
When the cylindrical lens is put into the light path, the image will be slightly blurred.
Fix the laser unit with
four blocks during
transportation.
ND filter sliders of the LU4A
4-laser unit

Chapter 2 STORM Microscopy Operation
10
Cylindrical lens
STORM slide
r
λ-plate slider
(The right slider among the three)
ND filter sliders
(the two of
the three sliders
for center and left)

Chapter 2 STORM Microscopy Operation
11
2.2 Acquiring 3D-STORM (2D-STORM) Images
To perform STORM analysis, automatically repeat light stimulation and image acquisition with the appropriate laser power to
create a dataset. There are two types of mode for acquiring images: normal mode and continuous mode. In normal mode,
activation and imaging are performed in separate frames. In continuous mode, they are performed simultaneously.
The following basically describes the procedure for acquiring 3D-STORM images. Any differences from the procedure for
acquiring 2D-STORM images are noted together with the symbol “”.
2.2.1 Acquiring Images in Normal Mode
In the example below, the following two types of probe are used.
• Channel 1: Alexa405-Alexa647 dye (Alexa647 activated by Alexa405)
• Channel 2: Cy3-Alexa647 dye (Alexa647 activated by Cy3)
Calibration for 3D-STORM
Before the first 3D-STORM image is acquired, it is necessary to perform calibration. See “2.5 Calibration for
3D-STORM.”
1 Click [Settings] in the [N-STORM] control window and
then specify the number of frames for each cycle.
[Continuous Mode]: Off
[Activation Cycle] (number of activation frames): 1
[Reporter Cycle] (number of reporter frames): 3
After making this setting, click [OK].
2 Select [3D-STORM] from the list at the top left of the
[N-STORM] control window.
 To acquire a 2D-STORM image, select
[2D-STORM].
1
2

Chapter 2 STORM Microscopy Operation
12
3Finely and properly adjust the laser focal point of the
TIRF illuminator.
(1) Select the checkbox for the 647-nm laser.
(2) Turn on the Perfect Focus System (PFS) in [Ti Pad]
and set the focus.
(3) Finely adjust the laser focal point of the TIRF
illuminator so that the observation target is visible.
4Check activation.
(1) Make sure that the checkbox for the reporter laser
(647 nm) is selected, and then set the power to
100% by adjusting the power slider.
(2) Make sure that the checkbox for the activation laser
(405 nm, 457 nm, or 561 nm) is deselected, and
then set the value to about 0.3% by adjusting the
power slider.
(3) Select the checkbox for the activation laser so that it
emits laser light for about a second, and then
immediately deselect it.
Check that the live image becomes bright once and
then returns to the previous state as a result of
activation causing the fluorescent probe to
fluoresce.
When using multiple activation lasers, check this for
each activation laser.
If activation cannot be checked
If emission of the activation laser does not make the live
image bright, this can be improved by replacing the
buffer solution of the specimen with a fresh solution.
3-(1)
3-(3)
3-(2)
4-(2)
4-(1)
4-(3)

Chapter 2 STORM Microscopy Operation
13
5Configure the N-STORM acquisition settings.
The settings are as follows.
Fluorescent probe checkbox:
Turn on the probe to be used.
Output of the activation laser (405 nm, 457 nm, 561
nm):
0.2% to 0.3% (values with which proper activation
is performed as shown in step 4 above)
Output of the imaging laser (647 nm):
100%
[Period Count] (number of periods for image
acquisition):
Any setting (normally 5000 to 20000 *1)
[Path], [File Name] (folder to which the file is to be
saved and the file name):
Any setting
[STORM Image] (whether to display the STORM
image during image acquisition):
If this checkbox is selected, the STORM image is
previewed during acquisition. Normally, this
should be selected.
[Graph] (whether to display a graph during image
acquisition):
If this checkbox is selected, a graph showing the
number of bright points in the image is displayed.
Normally, this should be selected.
[Minimum Height] (minimum intensity):
300 *2
*1 The above number of periods for image acquisition
is a reference value. Adjust it according to the
structure or coloring status of the specimen. Even
when it is set to 20000 periods in advance, if it is
confirmed as being large enough on the real time
analysis (preview) screen, it is possible to halt the
acquisition (for example, about 10000 periods) by
clicking [Finish].
*2 The above-mentioned minimum height is a
reference value. Adjust it according to the amount of
background light for the specimen, etc.
6 Click [Run Now].
Disabling large image mode
If large image mode is enabled with the ND Acquisition
function of NIS-Elements, images cannot be acquired.
Disable the acquisition of large images before clicking
[Run Now].
5
6

Chapter 2 STORM Microscopy Operation
14
7 Measure the gap between the boundary surface of the
cover glass and the position of the observation target
(Z-axis direction) using the method below.
 This step is not required for 2D-STORM.
(1) Step 1:
When the right dialog is displayed, select the
imaging laser (647 nm). Set the focus to the
boundary between the cover glass and the
specimen by using the PFS offset controller. (Adjust
the power to about 50% to 100% using the slider.)
Set the focus to the boundary surface of the cove
r
glass.
When setting the focus, move the XY stage and view, as
boundary marks, the dye molecules that are
non-specifically adsorbed onto the boundary surface of
the cover glass to identify the boundary surface.
After setting the focus, click [OK]. The XY stage
automatically returns to its original position.
(2) Step 2:
When the dialog shown on the right is displayed, set
the focus to the target imaging position by using the
PFS offset controller.
After setting the focus, click [OK]. The measured
gap (Z-axis direction) is automatically saved and
used in analysis.
When acquisition is started, the dialog box shown on the
right is displayed. Do not perform any operation using this
dialog box until image acquisition has been completed.
Clicking [Events...], [Pause], or [Refocus] prevents the
acquisition of appropriate images for STORM analysis.
To halt the acquisition, click [Finish] or [Abort]. (If [Finish] is
clicked, the acquired images are saved. Be careful
because, if [Abort] is clicked, the acquired images will be
discarded.)
7-(1)
7-(2)
Cover glass
Boundary surface of
cover glass
Cover glass
Imaging target surface

Chapter 2 STORM Microscopy Operation
15
8Adjust the power of the activation laser, if necessary,
during real time analysis (preview) of the acquired
images.
Image acquisition is started. The result of real time
analysis (preview) and a graph are displayed.
Recommended number of bright points
The recommended data for STORM analysis is as
follows: the density of bright points is not too low but not
so high that they do not overlap each other. For
example, a structure that covers the entire screen (such
as a microtubule in a cell) should be provided with about
100 to 200 bright points per frame in a visual field with
256 x 256 pixels. The number of bright points to be
activated can be adjusted by changing the power of the
activation laser.
Real time analysis (preview) screen during image acquisition
Details of real time analysis
During real time analysis, the value of [Minimum Height] on the N-STORM control window is used as a threshold to
analyze bright points. Real time analysis does not include drift correction.
Graphic screen during image acquisition
The number of bright points per frame of
imaging probe
Graph of the number of bright points for each
channel (for some latest periods)
Graph of the number of bright points
for each channel (plot only the first
imaging frame of each period)
Display width (period count) setting
A
utomatic scroll on/off
Scale setting
: Maximum, minimum, scale width
(Note that the scale width is adjusted
so that it does not overlap with
numbers in the graph.)
Zoom magnification
Molecular position
drawing selection
[Cross]/[Gaussian]
(Gaussian representation)
The thickness of cross
Channel display color
Delete any Gaussian
representation/cross
that has already been
drawn from the
display. (Molecule
position data remains
undeleted.)
Period number
Period number

Chapter 2 STORM Microscopy Operation
16
After the completion of acquisition, click [x] in the windows
for real time analysis (preview) and graph to close the
windows.
After the power of the activation laser is adjusted, clicking
[Auto LP] in the [N-STORM] control window automatically
adjusts the laser power so that the number of bright points
is maintained. Also, the upper limit on the power for
automatic adjustment(%) can be specified on the [Max]
box.
2.2.2 Acquiring Images in Continuous Mode
In the example below, the following probe is used.
• Alexa647 dye (Alexa647 activated by Alexa405)
Calibration for 3D-STORM
Before the first 3D-STORM image is acquired, it is necessary to perform calibration. See “2.5 Calibration for
3D-STORM.”
1Click [Settings] in the [N-STORM] control window and
then select [Continuous Mode].
After making this setting, click [OK].
2 Select [3D-STORM] from the list at the top left of the
[N-STORM] control window.
 To acquire a 2D-STORM image, select
[2D-STORM].
1
2