Nikon N-STORM Manuale utente

Super-resolution Microscope

N-STORM

Simple Operation Manual

M605E 12.6.Nx.1

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

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

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

Chapter

Chapter 1 Terminology Used in This Document

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

ctivation laser

Channel 1 (405 nm)

ctivation lase

Channel 2 (561 nm)

Imaging

laser (647 nm)

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

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.)

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.)

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

e Acquisition to Anal

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

Chapter

Chapter 2 STORM Microscopy Operation

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

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-(3)

2-(5)

Chapter 2 STORM Microscopy Operation

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)

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