Nikon N-STORM, Operation Manual

Page 1: ...Super resolution Microscope N STORM Simple Operation Manual M605E12 6 Nx 1 ...

Page 2: ......

Page 3: ...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 Sy...

Page 4: ... 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...

Page 5: ...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 f...

Page 6: ...ye 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...

Page 7: ... 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 s...

Page 8: ...ocument are presented as an example STORM Microscopy Operation Steps from Image Acquisition to Analysis 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 ...

Page 9: ...pe 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 ON OFF UNIT1 UNIT2 UNIT3 REVO DSC1 DSC2 SHUTTER1 DC24V IN ERGO JOY MODEL TI HU BC A USB STAGE REMOTE HGFIE TI PS SHUTTER2 5 5 0 3 0 1 This device co...

Page 10: ...f 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 ...

Page 11: ...sec 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 lig...

Page 12: ... 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 checkboxe...

Page 13: ...e 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 1 ND 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 ligh...

Page 14: ...Chapter 2 STORM Microscopy Operation 10 Cylindrical lens STORM slider λ plate slider The right slider among the three ND filter sliders the two of the three sliders for center and left ...

Page 15: ...es 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 Cl...

Page 16: ...ox 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...

Page 17: ...play 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 advanc...

Page 18: ...tify 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 d...

Page 19: ...sition 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...

Page 20: ...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 1 Click Settings in the N STORM control window and then selec...

Page 21: ...g the power slider 2 Make sure that the checkbox for the activation laser the figure on the right shows an example when a 405 nm laser is used 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...

Page 22: ...ted 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...

Page 23: ... 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 start...

Page 24: ... 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 Zoom magnification Molecular position drawing selection Cross Gaussian Gaussian representation ...

Page 25: ...sis 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 in the Max box ...

Page 26: ...s not saved a dialog box is displayed If the currently open molecule list STORM image has not yet been saved the dialog box shown on the right is displayed To save the molecule list select Yes If No is selected the latest molecule list will be discarded If the STORM parameter s in the currently open ND2 file have not yet been saved the dialog box shown on the right is displayed To save the paramet...

Page 27: ...2 Select the darkest bright point of all those to be identified as molecules and then position the mouse pointer to its center Read and write down the value of Peak Height this value is used as the minimum intensity for identifying bright points in the next step 5 Configure the settings for identifying bright points 1 Click Identification Settings 5 1 4 1 Peak Height 4 2 ...

Page 28: ...s Minimum Width nm 200 Maximum Width nm 700 For 2D Maximum Width nm 400 Initial Fit Width nm 300 Max Axial Ratio 2 5 For 2D Max Axial Ratio 1 3 Max Displacement pix 1 Setting Screening The bright point images that are excluded by Screening of Identification Settings are not displayed on any channel Upon the completion of setting click OK 3 Click Start STORM Analysis 4 Select Drift Correction set P...

Page 29: ...s soon as the analysis is complete information on the positions of the molecules is saved in the following two formats Molecule list in binary format File name ND2 file name _list bin Drift correction information File name ND2 file name _drift txt Both the binary file and the text file are saved to the same location as the ND2 file of the dataset Overwriting confirmation dialog If the result of a ...

Page 30: ...low Method of crosstalk subtraction Recommended uses Destination of the molecules exceeding the threshold subtracted molecules Recommended threshold ratio of surrounding molecules Ch A Ch B If the result of imaging is predicted Ch A and Ch B are found to be in clearly separate areas Can be used for 2 channel operation only Ch B 0 51 Ch A NSA If the result of imaging is not predicted NSA 0 49 Ch A ...

Page 31: ...et of conventional images acquired through NIS Elements Parameters for STORM analysis e g Identification Settings Display Options Filter Settings are saved into the ND2 file by selecting the ND2 format in the File Save dialog box of the N STORM analysis window Result of STORM analysis in binary format ND2 file name _list bin STORM image molecule list created through analysis This is automatically ...

Page 32: ...of the 647 nm laser through the GUI VFL software and then turn the power off procedure recommended by the laser maker 1 Set the power to 50 mW click Activate and then wait until the value of Power mW becomes about 50 mW 2 Then set the power to 0 mW click Activate and then wait until the value of Power mW becomes 0 mW 3 Click Off in the Fiber laser window to turn off the 647 nm laser 2 Shut down th...

Page 33: ...irection 1 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 Recommended fluorescent beads TetraSpeck Microspheres 0 1 µm Fluorescent Blue Green Orange Dark Red T 7279 Molecular Probes 2 Acquire images for calibration 1 Click Z Calibration in the N STORM control window If Z Calibration is not displ...

Page 34: ...ion 6 When image acquisition is completed click Cancel in the N STORM Z Calibration window to close the window 3 Click Analysis GUI to display the N STORM analysis window 4 Click File Open A dialog box for selecting a STORM image is displayed 5 Select the image file ND2 file acquired for calibration and then click Open 6 Make sure that the data is appropriate for calibration Check that the bright ...

Page 35: ...ification Settings In the dialog box configure the settings as follows Minimum Height Specify the minimum intensity checked in 2 of the previous step Maximum Height 20000 CCD Baseline 100 Select the 3D checkbox Click to configure the following detailed settings Minimum Width nm 200 Maximum Width nm 700 Initial Fit Width nm 300 Max Axial Ratio 2 5 Max Displacement pix 0 This allows bright points re...

Page 36: ...nts are identified correctly 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 and then perform the test analysis again 12 Click Start STORM Analysis again 13 Click Start to start analysis Analysis of bright points is performed for the enti...

Page 37: ...ay the graph of Wx Wy vs Z if necessary and check that Wx and Wy intersect nearly at 0 on the horizontal axis and that there is no large gap between the plot data distributed points and the fitting data lines Apply the results of calibration to an existing dataset To apply the new calibration result to an existing dataset apply the following procedure 1 Click Save in the Z Calibration dialog box t...

Page 38: ...lects the fluorescent probe to be used 6 Laser checkbox Turns the laser on and off through the AOTF in the laser unit 7 Laser power slider Adjusts the laser power through the AOTF in the laser unit 8 Period Count Specifies the number of periods for the dataset to be acquired 9 Path Specifies the storage location of the dataset to be acquired 10 File Name Specifies the file name of the dataset 11 S...

Page 39: ... be used during image acquisition only 14 Max Specifies the maximum of the adjustment range when the laser power is automatically adjusted using the Auto LP button 15 Run Now button Starts image acquisition according to the settings configured in the N STORM control window 16 Analysis GUI button Displays the N STORM analysis window 17 Advanced button Displays detailed settings 18 Minimum Height Sp...

Page 40: ...This cannot be specified if continuous mode is used to acquire images 3 Continuous Mode Selected when continuous mode is used 4 Conjugated Fluorescence Probe area Specifies the following the fluorescent probe to be used the wavelength of the laser to be used and the display color 5 Activation Probe Name Reporter Probe Name Specifies the names of the activation and reporter fluorescent probes for e...

Page 41: ... include analysis parameters e g Identification Settings Display Options Filter Settings file extension nd2 Molecule List binary files Binary format molecule list STORM image file extension bin Molecule List text files Text format molecule list file extension txt Note An ND2 file only contains parameters for STORM analysis and the results of analysis STORM image are not stored in the file The resu...

Page 42: ...auto adjustment of the intensity for displaying a conventional image If auto scale is enabled while ROI is being used it is adjusted according to the ROI range Switch STORM high resolution image display mode Determines how to display a STORM image None Not displayed Cross Gaussian Gaussian representation Both Mark molecules identified in current frame Marks with a yellow square a molecule identifi...

Page 43: ... rounds of analysis at night Right tool bar Icon Name Description Scale Bar Displays the scale Right clicking displays the property setting dialog Grid Graticules Shows a grid for approximate measurement Right clicking displays the property setting dialog Use ROI Displays ROI The ROI affects the auto scale display of the intensity and histogram among others Use Peak Statistics Displays the peak de...

Page 44: ...f the range for STORM analysis Set As End Period Specifies the end point of the range for STORM analysis 1 Right click Period Slider Select Entire Range Considers the entire dataset to be in the range for STORM analysis 2 F frame Moves between frames If the All channel tab is selected clicking in the first frame or in the last frame of a cycle allows you to move to the last or first frame of the a...

Page 45: ...included in the All tab 2 Fluorescent probe specific channel tab If an image is acquired on multiple channels the channel specific tab is displayed 3 Non Specific Activation In a cycle molecules that are detected not in the first imaging frame but in the second or subsequent imaging frame are classified in a non specific activation NSA channel Information on non specific activation channels is use...

Page 46: ... nm Once STORM analysis is completed the position of the mouse pointer is displayed in nanometers Also during the measurement of distance the interval between two points is displayed as follows dx xxx nm dy xxx nm d xxx nm dx and dy indicate the interval between two points on their respective axes while d indicates the slant distance If the mouse pointer is outside the image the number of currentl...

Page 47: ...rtering i europeiske land Dette symbolet indikerer at produktet skal kildesorteres De nedenstående punktene gjelder for alle europeiske brukere Dette produktet skal kildesorteres og innleveres til dedikerte innsam lingspunkter Må ikke kastes med normalt husholdningsavfall For mer informasjon ta kontakt med din forhandler eller lokale myn digheter Se Symbol för separat upphämtning i euopeiska lände...

Page 48: ...lt Whitman Road Melvile N Y 11747 3064 U S A tel 65 6559 3618 tel 1 631 547 8500 NIKON MALAYSIA SDN BHD NIKON INSTRUMENTS EUROPE B V tel 60 3 7809 3688 Laan van Kronenburg 2 1183 AS Amstelveen The Netherlands tel 31 20 44 96 300 NIKON INSTRUMENTS KOREA CO LTD tel 82 2 2186 8410 NIKON INSTRUMENTS SHANGHAI CO LTD tel 86 21 6841 2050 NIKON INDIA PRIVATE LIMITED tel 91 124 4688500 NIKON SINGAPORE PTE ...