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)