N-STORM System ESRIC@IGMM · N-STORM Manual 180215 1 APW ... Check Power, mW is showing 300 mW ......

N-STORM Manual 180215 1 APW

N-STORM System ESRIC@IGMM

To turn on system – see illustrated instructions on the notice board

The 647 laser is high power and does move in TIRF mode do not look down it

directly and do not operate the system without prior training from ESRIC Staff Please do not touch the 100x objective on this system. It has been calibrated for 3D N-STORM experiments

Please report any faults with the equipment to local ESRIC staff (i.e. a person in the IGMM building)

On shelf above system

Exfo Mercury lamp

CoolLED Brighfireld Lamp Stage controller

Nikon Shutter controller

Next to scope

A1 Laserbed (button on LHS system) Turn key

Visible Fibre Laser (big black box on laser bed)

647 laser box (on top of A1 laser controller)

Power switch on

Key-turn horizontal

Wait for white LED light

Launch GUI-VFL software

Press On

Press Activate

Adjust to 300 mW for STORM

Check Power, mW is showing 300 mW

Launch NIS Elements

On the microscope itself it is important that the TIRF concentrator (on the stand) is pushed down for STORM

imaging. (*) in top image

If you are doing 2D storm the cylindrical lens (RHS of scope) needs to be fully out (pushed away from you)

For a 3D experiment the cylindrical lens needs to be in the light path (pulled towards you)

*


In NIS elements

Find sample by eye

The OC panel is used to control the microscope.

NB The epifluorescent shutter is on the side of the

microscope.

Select DAPI / GFP /RFP and find sample by eye.

Switch on the Perfect focus system. (in software or on

microscope stand) to ensure the sample doesn't drift. +

focus via the perfect focus

To visualise on the camera.

Select a configuration from the STORM optical config menu

where it is possible to focus on your fluorophore.

Remove the interlock (RHS menu N-STORM PAD)

Open the shutter (RHS menu)

Activate the camera (green arrow)

(Red box is stop)

Adjust the camera settings so that the

sample is focussed on the camera ensure that z focussing is done using the Perfect focus device

The Exposure time and EM gain may need to be adjusted. In the

camera control window (bottom RHS)

The EM gain should not be above 300

The conversion gain should be set at 3

The TIRF angle may need to be adjusted as required

NB (around 4000 is true TIRF)

This button enables mouse scrolling with the TIRF


To start a STORM experiment:

(1) In the N-STORM window

Check 2D storm or 3D storm as required.

In the bottom menu fill in the number of frames required in the period

count tab

Set the path where the data are to be saved (Data disk Y)

STORM Image and graph are options, they show where

photoswitches from molecules (Blinks) are localised and how many

blinks per frame are being recorded, the graph also shows how quickly

images are being sent to the disk.

For a 1 colour image this should be 85fps

To set up the lasers for the STORM / PALM experiment press Settings.

In the N-STORM-PAD

The Settings dropdown menu will appear

In this window set up the type of experiment, e.g. Single colour

continuous or multicolour continuous

Either set up the fluorophores to be used via the dropdown menu.

Or use the facility presets by pressing the Load key:

The preset menu will appear in the

N_STORM Restore Window

Select the required configuration and press OK

To confirm all settings press OK in the N-STORM settings window

NB It is possible to reduce the camera frame size in the

Camera menu of the N-STORM Settings menu to either 128 x128 or

64 x64 pixels. However this is for smaller / single molecule samples.


(2) Parameters and presets:

It is important to ensure a sparse field of view of photoswitching

molecules. Optimisation of photoswitching buffer will be essential for

this.

With a good buffer use of the 647 laser alone will be sufficient

to collect a dataset. However with a less ideal buffer or a complex

sample the

Blinking fluorophore (e.g.AF647) can be reactivated with 405 nm light.

The ratio of imaging and bleaching (647 laser) to activation (405 laser)

can either be manually controlled in the experiment or a desired

number of photoswitching events can be preset in the software by

activation of the AutoLP button. The 405 laser needs to be checked 'on'

for this in the N-STORM pad

Alternatively the maximum amount of photoswitching

events can be specified, the default is 70%

In the Advanced tab the Identification Parameters for on the

fly analysis of photoswitching events can be specified

(3) To Start a STORM experiment (AF647)

i. With all shutters open and the camera live turn the Imaging laser (e.g. 647) up to 100%, this will

convert all the fluorophores into the off state

ii. A sparse population of blinking fluorophores will appear

iii. Press Run now this will start the STORM acquisition and open the (A) ND Acquisition window with

the live image, (B) the ND progress window, allows pausing and aborting (C) The N-STORM image

window showing on the fly rendering and (D) the N-STORM Graph showing number of

photoswitching events NB changing the Identification Parameters (*) in the N-Storm pad can change

this, the computer may run slow when acquiring if the ID parameters are set to find too many

events. In which case increase the minimum height parameter.

*

A B

C

D


Analysing a STORM experiment: To analyse the data (can be done on analysis PC, Holy Cow in the IGMM)

Check Applications

and select

Define/RUN N-

STORM Analysis

Select Analysis Gui from the bottom of the N-STORM Pad

In the GUI using the folder icon find the raw data

Select the required file and load the associated Molecule list

(If data are moved the raw data ND2 file and the .bin and .txt

document which contain info about molecule position and

drift must be moved together. Otherwise the localisations

will be lost.)

Press Open

In the STORM analysis GUI, The raw image is opened

N-STORM GUI: Quick guide to useful icons

(1) Identification Parameters (blue cog) defines

what a particle is or isn't

(2) Red arrows = Start STORM analysis

(3) Display settings are white rectangle with arrow

(4) Filter settings is the seive

(5) Capture image snapshot for easy export

(6) Resolution / localisation accuracy, right click and select

Properties

1 2 3 4 5


The 2D fitting is centroid fitting like QuickPALM (2)

When analysing a Storm experiment the ID settings need to be carefully set up. Press

the blue cog to open the Identification settings GUI

It is recommended that the ROI is autofitted. To define the

parameters for autofitting press >> (*)

Expands showing the Screening menu.

This allows definition of the minimum and maximum width

expected of a fluorophore blink

The initial fit width (i.e. how large the expected diffraction

limited blink is) and the expected discrepancy in the ratio in

the x and y plane (fit tolerance).

The centroid fitting cannot cope with overlapping blinks

from 2 or more fluorophores and rejects them.

If the structure is filamentous or densely labelled the

centroid fitting will not work. Instead overlapping pixels can

be fitted by checking the box. This is multiple emitter fit

code written by the Zhuang lab (3) . This is slower.

Once the ID settings have been set up they can be tested to see how

well they perform by pressing Test in the Start STORM analysis GUI

It is recommended that a period (i.e. frame) is chosen in the middle of

the experiment as there will be quite a few images which are too

saturated with blinks at the beginning to localise.

An example of localisations is shown (right)

False positives are indicated. (*)

These can be removed by adjustment of the Identification

setting parameters

False negatives (i.e. blinks which aren’t identified by the

system – can be corrected for by refining the ID settings

N.B. To remove the yellow squares the icon on the LHS menu

with a yellow square around it can be pressed.

The data to be analysed can be cropped in the

analysis window by right clicking at the bottom of

the image on the frame and selecting the start

and end period to analyse

*

*

*


To start the STORM analysis once the experiment is complete

press start in the Storm Analysis gui

Post processing the 2D STORM image The processed STORM image (right) consists of dots, these

indicate blinks and their size and shape depend on the

localisation accuracy which can be adjusted.

(1) Correct system drift: Drift can be corrected by pressing

the drift correction button (its not entirely clear how this

works if there are no fiducial marks in the image, use with

care)

The drift correction can be applied or removed once

calculated using the snake icon in the LHS menu

(2) Remove false positive localisations:

This can be refined using the filter settings button

Here the relative frame range can be set. The number of localisations can

be refined by adjusting the Density parameters. The radius is the search

radius for a localisation to be considered 'real', setting this to 5x the

resolution can help. Also altering the count which is the number of blinks

found within the search radius can be adjusted. This means that only areas

where there is a large amount of blinking, (e.g. an epitope which has

bound to an antibody with a dye on it / a.k.a. Real staining) will be

localised. The number of molecules shown in the final image vs blinks

identified is shown in the menu at the bottom of the window.

Antibodies and dye molecules can stick to glass and dirt / crud on the

coverslip can also cause spurious switching. Adjusting these parameters

reduces the number of identified molecules (blinks) shown. This is acceptable as

some are false positives. The number of localisations shown is indicated

at the bottom of the results GUI

(3) Determine localisation precision (standard error of fitting) to do this the photons per count should be

entered into this box. They can be calculated the following information:

Photons per count calibration (filter settings STORM GUI) Use Andor DU897 camera settings:

EM gain 17MHz at 16-bit

Conversion gain of 3

Photons per count = 4.85/EM gain

*


(4) Export all the information about localisations right click in the STORM imaging analysis menu

It is essential when images are reported in the literature that they contain not only a scale bar but

also information about the error of localisation as this depends on how much light is collected. This

data also should be kept in case of editors questions

NB Error of precision must not be used until the calibration for photons per intensity count has

been entered into the filter settings menu as an incorrect value will invalidate the equation (see

Betzig Science 2006 for more info about how the maths works)

The information about the STORM experiment can be saved by right clicking

in the STORM image and choosing properties.

Information about the Experiment, the positions of

the molecules and statistics is kept here. This file

should be saved for each experiment.

To find out what the localisation precision

(Standard error of fitting is the technical term)

Right click in the STORM image and choose ROI

statistics

The menu with ROI statistics will appear. Ensure

that the Molecule list is exported if data are for

publication.

Alternatively the grey wedge in the LHS menu

will also show this data

RHS icons which may be useful for localisation precision and image display

Top shows or hides the scale bar

Next down puts a grid on the image

The next one hides the grid

The yellow graph shows the intensity of a blink in raw and reconstructed images


(5) Enhance the rendered data if required.

The STORM display options can improve the look of the rendered image

The LHS menu icon grey fuzzy dot (LR) toggles the diffraction image on or off

The red dot and cross icon shows the Higher Resolution image as either a cross,

Gaussian blob, none or both.

The icon with the yellow box toggles on and off localised particles

The red zigzag arrow toggles on and off the drift correction

The STORM display options

allow more in depth control of the rendered data.

The GUI is self explanatory.

Advanced menus top allows for control over data of which

precision accuracy is shown. The dimmer the intensity of the data

the larger the standard error of fitting there will be.

The bottom menu is used for 3D storm Data fitting. The data range

shows which part of the 3D image is shown (discussed later)

(6) Export the analysed higher precision image. For this there should be a (i) Low resolution

diffraction limited image, (ii) A high resolution improved image and (iii) an overlay.

Once the output image is satisfactory it should be converted into a standard ND2

file and saved as a .tif. The camera icon can do this.

NB any data in the STORM analysis GUI can't be opened normally in Fiji for conversion into a

publication quality figure, it needs to be exported first

References:

(1) Rust MJ1, Bates M, Zhuang X. Nat Methods. 2006 Oct;3(10):793-5 DOI: 10.1038/nmeth929

(2) R. Henriques, M. Mhlanga Nature Methods 7, 339–340(2010) doi:10.1038/nmeth0510-339

(3) https://github.com/ZhuangLab/matlab-storm/blob/master/Functions/Analysis/GPUmultifit.m

(4) E. Betzig, H. Hess, et al Science 2006. 313 no. 5793 pp. 1642-1645 DOI: 0.1126/science.1127344


Multicolour STORM – acquisition

The 100x lens needs to be very clean for this. Please use the isopropanol, colour registration may need to be

measured by ESRIC staff using 100nm TetraSPECK beads. The correction collar on the 100x may need to be

adjusted. Please do not do this yourself.

Multicolour STORM images are acquired in exactly the same way as single colour images. However in the N-

STORM settings GUI the Multicolour continuous mode must be selected. The parameters are either Loaded

(if preset) or manually entered as for single colour STORM.

The lasers are activated in the order 1,2,3. Very short imaging times for the cameras and excellent staining /

brightness is required here. Otherwise the blinking events will occur after imaging.

For PALM/STORM it is recommended that the STORM experiment is carried out first but the 405nm laser

isn't used and the imaging buffer is optimised. The PALM imaging (using Eos ideally( can be carried out

second. This can be altered dependant on the imaging.

Multicolour STORM - analysis

To reconstruct the data everything is the same as for single colour imaging except that the spectral

registration of the colours needs to be carried out.

In the STORM analysis GUI the spectral registration can be carried out by using the spectral alignment GUI

When the three crosses are separate there image is not registered. At

the last calibration chromatic aberration of 100nm on this system was

noted.

Click the icon and all of the crosses move together, the image is now

spectrally re-registered.


3D STORM

Image collection

This is only suitable for certain applications. Denser or more complex samples won't work because of out of

focus light. Please speak to one of the ESRIC managers about your sample before starting.

IMPORTANT: Ensure the Cylindrical lens is inserted in front of the camera before imaging

is started and the perfect focus is on (Small silver lever, RHS of microscope)

3D STORM is carried out in widefield using very intense laser light.

To visualise the sample

(i) Use STORM HiLO settings

(2) Adjust the TIRF slider in the Tipad so the image

is not illuminated in TIRF

(3) In the N-STORM GUI (RHS TOP) set 3D STORM

The image is then acquired as a single or multicolour

STORM image would be. (see 2D acquisition instructions on page

4)

3D Analysis.

Astigmatism in the image alters the point spread function and allows capture of a 800nm

section, providing the FOV is sparse. 400nm above the focal plane the Point Spread

Function (PSF which means image of a perfectly round sphere below the resolution limit

of the instrument) will appear stretched in the x axis. 400nm below the focal plane it will

appear stretched in the y axis (see figure, right)

The analysis software can identify these differently shaped Point spread functions and

assign their localisation spatially. However this can only be done because a calibration

dataset has been generated using perfectly round beads. For the calibration the 100x

objective position is optimised. This is why it is essential not to alter the calibration

settings and also not to adjust the 100x objective


To ensure the stretched PSF is fitted the Identification Settings need to be adjusted for 3D

images. Open the Identification settings in the N-STORM analysis pad

To analyse the data in 3D it is important to have the Autofit ROI on and 3D

checked

The parameters the software uses to recognise a blink also need to be

modified using the >> button (*)

In the ‘Screening’ settings which pop out:

(i) Maximum fit must include the elongated blink of dyes above / below the

focal plane. (This can be measured, 1000 is a rule of thumb).

(ii) The axial ratio of the x:y axis in a blink will be different and a higher

tolerance e.g. 3 or more is needed

Press OK to analyse the image

NB if you think the Z calibration has been altered you can reload a present setting, see ESRIC facility staff

Post processing the 3D multicolour

STORM image

The output image shows the Z information which

was included and excluded

LHS Icons:

For depth coding the 3D image,

3D drift correction

Shows specific parts of the 3D rendering

and 3D colour coding

Colour correction of the 3D image

It is essential to set the localisation precision in

Filter Settings in the same was as a 2D image

(see page 7)

A resolution limit of 50nm in z is expected. The

number of rejected blinks in x.y and z is shown

3D display of the STORM image can be

visualised by pressing the 3D volume icon

(top menu)

In the 3D viewer you can control the display of the rendered

image. The camera icon allows the output to be stored.

The camcorder allows a movie to be taken.

The crosses for localisation can be viewed.

Different planes (X, Y or Z can be visualised using the View

tab).

*


Changing the way that the 3D data is displayed using the Display Options tab.

Here Height map can be selected/ By pressing the Advanced tab the look up table for the

height map

The advanced tab in height map allows the range of particles shown to be adjusted in the Min Max tabs

By changing the colours in the box the 3D rendering colours alter and a scale coding depth intensity (LHS)

appears

The z rejected blinks (because they were a funny shape) are shown or hidden here and the height map can

be toggled on and off.

Data should be exported in the same way as a 2D imaging

The 3D height map can also be visualised by pressing the icon with the graph in colour on the LHS menu

Thuderstorm and SOFI can be used to analyse STQRM data and are newer algorthims than used here. Sam

Hess’s PALM algorithms are useful for PALM analysis


Facility Only

TIRF Alignment

Set the TIRF angle in the software via the TIRF up optical config (widefield illumination). Ensure STORM

concentrating lens is in. Remove Cylindrical lens. Focus on a chroma slide (red), set 674 laser power to 1%

and view live image to the camera. From the image options select the cross-hair graticule. If laser in not

centred then the laser fibre may need centering. Where the fibre enters the TIRF unit is a cylindrical mount

with two grub screws. Take this off to reveal 4 grub screws which will adjust the fibre, north, south, east

and west. Ensure the laser spot is centred on the graticule

Remove the chroma slide and switch on all lasers. Push back the transmitted light illumination pillar and

ensure all wavelengths colocalise on the cross on the ceiling. Adjust the verniers and and undo the locking

screw to make adjustments

3D STORM Calibration

Remove STORM lens from TIRF illuminator

Insert Cylindrical lens

The Z piezo needs to be on the system for the so the SIM needs to be booked as well.

Plug piezo via USB into the USB ports on the back of the PC not the front ports.

In Elements go to Devices>Device Manager>Disconnect Nanodrive

Use 100nm tetraspeck beads in PBS using the 8 well labtek2 chambers.

A field which is sparse but has at least 10-20 well distributed beads is needed for this.

Leave the beads to settle for a few minutes

Use 1:500 or 1:1000 (?) dilution of tetraspeck beads (I.e what the manufacturers

recommend and then dilute this 1:5)

Set Optical config to Widefield position. Ensure the image of the beads is not saturated

in any channel and has good SnR ratio. Use PFS. Check each channel individually before

enabling all lasers. The correction collar position should be optimised

In the Identification settings window click Z Calibration. The calibration performs

multiple Z stacks, moving above and below focus and records the astigmatism of the

beads at different distances from the coverglass (201 images per channel captured)

Once complete open the saved dataset in the STORM analysis gui. In the ID settings window select 3D, this

changes the default screening parameters. Autofit ROI should also be selected. 10 good beads need to be

identified, so you may need to adjust the minimum height setting and run a test identification on a few

frames. Run the STORM analysis on the dataset.

When this is finished, go to ID settings, Z calibration, Auto-Calibrate. Common errors include bad SNR

image, clumpy beads or not enough beads.

Check that beads are being identified in every channel.

When analysis has finished use the cross hair view to judge where the bead has been localised in each

channel. Check the position when chromatic correction has been applied.


2D Warp Calibration (variation in chromatic aberration)

Use 100nm tetraspeck as for the 3D calibration. In ID settings uncheck 3D and click XY Warp.

*Do not remove 100x lens, filters or camera (detection light path). If so calibrations have to be

redone.

N-STORM System ESRIC@IGMM · N-STORM Manual 180215 1 APW ... Check Power, mW is showing 300 mW ......

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Transcript of N-STORM System ESRIC@IGMM · N-STORM Manual 180215 1 APW ... Check Power, mW is showing 300 mW ......