Nikon N-STORM Operation Manual
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Nikon N-STORM Operation Manual

Nikon N-STORM Operation Manual

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

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

  • Page 3: Introduction

    • 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.

  • Page 4: Table Of Contents

    Acquiring Images in Continuous Mode ..................16 N-STORM Analysis ..........................22 Terminating the N-STORM System ......................28 Calibration for 3D-STORM ........................29 Chapter The Screens of the N-STORM Software ..................34 N-STORM Control Window ........................34 N-STORM Settings Window........................36 N-STORM Analysis Window ........................37 3.3.1 Tool Bar ............................

  • Page 5: Terminology Used In This Document

    Terminology Used in This Document 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).
  • Page 6 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.
  • Page 7 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.)

  • Page 8: Storm Microscopy Operation

    (Steps from Image Acquisition to Analysis) STORM Microscopy Operation The overall operational steps and their corresponding descriptions in this chapter are as follows: 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 2.2.2 Acquiring Images in...

  • Page 9: Preparation Of The N-Storm System

    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]. Perform safety checks.
  • Page 10 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-(1) Start NIS-Elements AR.
  • Page 11 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.
  • Page 12 Note: Set the cylindrical lens for 3D-STORM to OUT (where it is not in the light path). Set the episcopic illumination to [TIRF]. Put the N-STORM filter cube into the light path. 8-(1) Click [Interlock] on the N-STORM control window to disable the laser interlock.
  • Page 13 256 pixels [Height] 256 pixels Available range The range available for STORM is 256 x 256 pixels only. Configure the settings for the other optical systems for N-STORM. 3D-STORM port LU4A 4-laser unit Electric TIRF illuminator TI-TIRF-E (Ti-E side port)
  • Page 14 Chapter 2 STORM Microscopy Operation STORM slider λ-plate slider ND filter sliders (the two of (The right slider among the three) the three sliders for center and left) Cylindrical lens...

  • Page 15: Acquiring 3D-Storm (2D-Storm) Images

    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.” Click [Settings] in the [N-STORM] control window and then specify the number of frames for each cycle. [Continuous Mode]: Off...
  • Page 16 Chapter 2 STORM Microscopy Operation Finely and properly adjust the laser focal point of the TIRF illuminator. Select the checkbox for the 647-nm laser. 3-(1) Turn on the Perfect Focus System (PFS) in [Ti Pad] 3-(2) and set the focus. Finely adjust the laser focal point of the TIRF 3-(3) illuminator so that the observation target is visible.
  • Page 17 Chapter 2 STORM Microscopy Operation Configure 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...
  • Page 18 Chapter 2 STORM Microscopy Operation 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. 7-(1) Step 1: When the right dialog is displayed, select the imaging laser (647 nm).
  • Page 19 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.

  • Page 20: Acquiring Images In Continuous Mode

    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.
  • Page 21 Chapter 2 STORM Microscopy Operation Finely and properly adjust the laser focal point of the TIRF illuminator. Select the checkbox for the 647-nm laser. 3-(1) Turn on the Perfect Focus System (PFS) in [Ti Pad] 3-(2) and set the focus. Finely adjust the laser focal point of the TIRF 3-(3) illuminator so that the observation target is visible.
  • Page 22 Chapter 2 STORM Microscopy Operation Configure 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 (the figure on the right shows an example when a 405-nm laser is used): 0.2% to 0.3% (values with which proper activation...
  • Page 23 Chapter 2 STORM Microscopy Operation 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. Step 1: 7-(1) When the dialog shown on the right is displayed, select the imaging laser (647 nm).
  • Page 24 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.
  • Page 25 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 in the [Max] box.

  • Page 26: N-Storm Analysis

    [Open]. Saving open data In the N-STORM analysis window, it is not possible to open two or more ND2 files at once. For this reason, if a new file is opened, the currently open file is closed. If, at this time, the file contains any information that is not saved, a dialog box is displayed.
  • Page 27 Chapter 2 STORM Microscopy Operation Check the minimum intensity of the bright points to be identified as molecules. To check the intensity, click [Use Peak 4-(1) Statistics]. Select the darkest bright point of all those to be identified as molecules, and then position the mouse pointer to its center.
  • Page 28 Chapter 2 STORM Microscopy Operation In the dialog box, configure the settings as follows. 5-(2) [Minimum Height]: Specify the minimum intensity of the bright points, which was checked in the previous step. [Maximum Height]: 20000 [CCD Baseline]: 100 [3D]: On For 2D ...
  • Page 29 Chapter 2 STORM Microscopy Operation Upon the completion of the test analysis, the confirmation dialog is displayed. Click [OK]. Make sure that the bright points are identified correctly. 5-(5) If the intended result is not achieved If bright points of noise are detected or if bright points of signals are not detected, reconfigure the parameters for identifying bright points in [Identification Settings]...
  • Page 30 Chapter 2 STORM Microscopy Operation Subtract crosstalk. (Only in normal mode. The crosstalk subtraction function is not used for images acquired in continuous mode.) Click [Cross-Talk Subtraction]. For [Source Channel (Ch A)], select the channel for 6-(4) 6-(2) which crosstalk is to be subtracted. Select the method and the threshold for crosstalk subtraction.
  • Page 31 [File Save] to save the STORM parameters. The parameters for STORM analysis are saved to the ND2 file. Type of file used by N-STORM The file formats used by the N-STORM software are as follows. File type File name Description Set of conventional images acquired through NIS-Elements.

  • Page 32: Terminating The N-Storm System

    Chapter 2 STORM Microscopy Operation Terminating the N-STORM System Upon completion of the necessary operation, terminate NIS-Elements AR and turn off the microscope, the laser, and other peripheral devices. Terminate NIS-Elements AR. When MPB Communications Inc.’s 647-nm laser is being used Follow the procedure below to gradually decrease the output of the 647-nm laser through the GUI-VFL software, and then turn the power off (procedure recommended by the laser maker).

  • Page 33: Calibration For 3D-Storm

    Z-axis direction with the ellipticity ratio of an unfocused bright point image, allowing the analysis of positions in the Z-axis direction. Make preparations for acquiring 3D-STORM images according to “2.1 Preparation of the N-STORM System.” A fluorescent bead specimen is used for calibration.
  • Page 34 (Z stack) • 20 frames at the focal position When image acquisition is completed, click [Cancel] in the [N-STORM Z-Calibration] window to close the window. Click [Analysis GUI] to display the N-STORM analysis window.
  • Page 35 Chapter 2 STORM Microscopy Operation Check the minimum intensity of bright points Click [Use Peak Statistics]. 7-(1) Select the darkest bright point of all those to be identified as molecules, and move the mouse pointer onto its center. Read and write down the value of [Peak Height].
  • Page 36 Chapter 2 STORM Microscopy Operation Click [Start STORM Analysis]. Select [Drift Correction], set [Periods] to [1], and then click [Test]. Test analysis is performed for the currently displayed period. Upon completion of the analysis, the confirmation dialog is displayed. Click [OK]. Make sure that the bright points are identified correctly.
  • Page 37 Click [Auto Calibrate]. 15-(1) The calibration results (coefficients of the calibration curve function) obtained through the procedure above are automatically saved as internal settings in the N-STORM software, and subsequently are automatically applied to 3D-STORM analysis. 15-(2) Check the results of calibration on the graph Clicking [Graph] in the [Z Calibration] dialog box displays a graph showing the results of calibration.

  • Page 38: The Screens Of The N-Storm Software

    Description 2D-STORM/3D-STORM Switches between the 2D-STORM/3D-STORM modes. switching button [Settings] button Displays the [N-STORM Settings] dialog box, in which the number of frames for each cycle and the type of fluorescent probe are specified. Help button Displays help. [Shutter] button Collectively opens or closes laser shutters.
  • Page 39 Chapter 3 The Screens of the N-STORM Software Name Description [Graph] checkbox Selects whether to display a screen of graph of the number of bright points during acquisition of the dataset. To display the graph, select this checkbox before clicking [Run Now]. Also, when using Auto LP, select this checkbox.

  • Page 40: N-Storm Settings Window

    Chapter 3 The Screens of the N-STORM Software N-STORM Settings Window Name Description [Activation Cycle] Specifies the number of activation frames for each cycle. (This cannot be specified if continuous mode is used to acquire images.) [Reporter Cycle] Specifies the number of imaging frames (frames that capture the light emission of the reporter) for each cycle.

  • Page 41: N-Storm Analysis Window

    Chapter 3 The Screens of the N-STORM Software N-STORM Analysis Window Window that is used to perform STORM analysis on an acquired dataset. 3.3.1 Tool Bar Top tool bar Icon Name Description [File Open] Opens a dataset (file extension: nd2) or a binary-format molecule list (file extension: bin).
  • Page 42 Chapter 3 The Screens of the N-STORM Software Icon Name Description [Start STORM Analysis] Starts STORM analysis and drift correction. [Apply Drift Correction] Only performs drift correction. [Display 3D STORM Displays a 3D image (3D-STORM only). Image] [Display Options] Determines how to display an image.
  • Page 43 Chapter 3 The Screens of the N-STORM Software Icon Name Description [Height map] Performs color-coding on a STORM image according to the position in the Z direction (3D-STORM only). [Batch STORM Analysis] Continuously performs multiple rounds of STORM analysis. Used to automatically perform multiple rounds of analysis at night.

  • Page 44: Slider

    Chapter 3 The Screens of the N-STORM Software 3.3.2 Slider A slider is used to move between the frames of conventional images. Name Description Moves to a frame that is placed in the same ordinal position as (period) the current position in any of the adjoining periods. (Moves to the third frame of the previous or next period if the third frame is currently displayed.)

  • Page 45: Channel Tab

    Chapter 3 The Screens of the N-STORM Software 3.3.3 Channel Tab A channel tab is used to change the display of multiple channels included in a dataset. Right-clicking a channel tab allows you to set the property of the relevant channel. It is also possible to select whether to display the [Non Specific Activation] channel and the [Z Rejected] channel.

  • Page 46: Status Bar

    Chapter 3 The Screens of the N-STORM Software 3.3.4 Status Bar Left side Description (Normal) [P: current-period/number-of-all-periods-for-the-dataset, Fr: current-frame/number-of-all-frames-for-the-current-cycle, Fa: current-frame/number-of-all-frames-for-the-dataset] If the current frame is an activation frame, [(A)] is displayed. Cycle A cycle is a set of frames for one channel that are included in one period.
  • Page 47 Symbol for separate collection applicable in European countries Symbol for kildesortering i europeiske land This symbol indicates that this product is to be collected separately. Dette symbolet indikerer at produktet skal kildesorteres. The following apply only to users in European countries. De nedenstående punktene gjelder for alle europeiske brukere.
  • Page 48 NIIKON INSTRUMENTS INC. 〒532-0003 1300 Walt Whitman Road, Melvile, N.Y. 11747-3064, U.S.A. 大阪市淀川区宮原 3-3-31（上村ニッセイビル 16F） tel. +1-631-547-8500 電話：（06）6394-8801 （バイオサイエンス） NIKON INSTRUMENTS EUROPE B.V. 九 州 支 店 Laan van Kronenburg 2, 1183 AS Amstelveen, The Netherlands 〒913-0034 tel. +31-20-44-96-300 福岡市東区多の津 1-4-1 電話：（092）611-1111 （バイオサイエンス・産業機器）...

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