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39 – LSM 880 Training Guide

Using AiryScan

FAST

Once our light path has been designed, we can set up our baseline settings for the raw AiryScan data. We start by deciding how

we want to balance resolution with speed using the sampling frequency.

From the Acquisition toolbar, expand the blue dialog labeled

‘Acquisition Mode’.

Choose sampling rate.

Note: The sampling rate is broken down into 4 options:

– 2x sampling (Nyquist) – choose this mode for the best

possible resolution

OPT

– 1x sampling (equivalent to confocal) – choose this option

for image quality comparable to confocal

FLX

– 0.7x undersampling for speed > resolution

– 0.5x undersampling for speed >> resolution

Set Speed to Max.

Note the system defaults to bi-directional scanning mode. Leave it in

bidi mode for highest speed.

Note: The AiryScan processing modes have a correction factor built in

for any bi-directional scanning artifacts.

Содержание LSM 880 with AiryScan FAST

Страница 1: ...Training Guide for Carl Zeiss LSM 880 with AiryScan FAST ZEN 2 3 Optical Imaging Vital Microscopy Core Baylor College of Medicine 2018...

Страница 2: ...n ON both Systems PC and Components switches By default the Lasers key located on the side of the remote paddle should always be in the ON state but please verify 3 Turn ON HXP 120V epi fluorescence l...

Страница 3: ...uble click ZEN Black icon to start software Note Be sure to use Zen Black instead of ZEN Blue Icon above is for Black version 6 When software has started you will be presented with a login screen Sele...

Страница 4: ...es of tools for sample observation image acquisition image processing and system maintenance Image Display This area is where each newly captured image will appear Settings here allow the user to cont...

Страница 5: ...5 LSM 880 Training Guide Getting Started with ZEN 2 3...

Страница 6: ...ue toolbar labeled Laser and select Argon from the laser list 3 Toggle Power button from Off to On Note This laser requires a 5 minute warm up time Once you select On the system will count down from 3...

Страница 7: ...visualization in the eyepieces Green generic green filter set Red generic red filter set Blue generic blue filter set TL DIC transmitted light All Closed turn off all light sources Note These shortcut...

Страница 8: ...Microscope 4 Via the microscope eyepieces visualize your sample and adjust the focus Z and stage position X Y accordingly 5 Once a suitable location is found click ALL CLOSED from shortcuts 3 6 Select...

Страница 9: ...ode is the fastest approach many fluorophore combinations will exhibit enough spectral overlap to be able to detect unwanted signal from neighboring fluorophores using this method This phenomenon is k...

Страница 10: ...cks apart from laser line and detector choice All other settings must remain the same This limits you to a maximum of 4 fluorescence channels plus one transmission image with this mode 2 Frame track s...

Страница 11: ...elength fluorophore Alexa 568 in this case on Track 1 Note By default ZEN will start with one track Track 1 To add additional tracks click the button next to the track list 3 Click Visible Light to op...

Страница 12: ...velength fluorophore In our example we will be using a sequential scan with Alexa 568 and Alexa 488 so Ch1 or ChS1 will be used for the Alexa 488 signal and Ch2 will collect the Alexa 568 8 Define upp...

Страница 13: ...ling frequency can be quickly selected with the Optimal button Scan Speed overall speed of the scan during acquisition Reports pixel dwell time and total scan time Increasing dwell time slowing down t...

Страница 14: ...thickness Reports section thickness and pinhole diameter Gain Master controls the analog amplification voltage for the PMT in Volts The higher this value the more sensitive the detector becomes to the...

Страница 15: ...here your signal is the highest but your axial resolution is at its lowest As you reduce the aperture diameter you are increasing your Z resolution but reducing your signal level This is a reasonable...

Страница 16: ...Range Indicator place a check in the box The Range Indicator LUT assigns red pixels to areas in the image that are overexposed and blue pixels to background areas that are underexposed Note The Range...

Страница 17: ...mately prevent any unnecessary photobleaching of other channels 2 Click Live to start scanning 3 Turn on Range Indicator LUT as shown on the previous page 4 Deselect laser checkbox for the active trac...

Страница 18: ...trast have the brightest areas fall just below overexposure red and the background areas fall just above underexposure limit blue In a properly balanced image you should see no red or blue pixels with...

Страница 19: ...ion but increases signal level reported to the detector Closing this aperture will reduce the signal level but increase axial resolution Laser Intensity The intensity of the laser illumination source...

Страница 20: ...erture to 1 AU You can certainly close the confocal aperture below this value and continue to improve resolution just understand that below 1 AU you will lose signal level at an exponential rate Conve...

Страница 21: ...unforeseen effects on the sample For most lasers on this microscope we recommend laser power settings between 1 and 5 to start It is recommended to start as low as you can possibly go and still get a...

Страница 22: ...s here but they are readily apparent when using a gain value 500V Below 500V it is very difficult to detect much noise at faster scan speeds however when using the detector above this value you will f...

Страница 23: ...system has averaging functions in the Acquisition Mode tab page 14 that can be used to clean up the image Simply select the rate of averaging 1 2 4 8 or 16x and rescan Note Averaging signal will incre...

Страница 24: ...dialog click Set First to mark the start position 5 While continuing to scan focus the drive in the opposite direction until your sample comes back into focus then continue focusing until it goes out...

Страница 25: ...idimensional Acquisition settings 2 To calculate the required number of cycles divide the duration you require for the series by the interval For example if we wish to collect for 6 hours in total wit...

Страница 26: ...stage position Here you can manually define the number of tiles in X and Y along with the Overlap to define the tile area Bounding grid creates a tile scan by defining the boundaries of the section Co...

Страница 27: ...m the list of open images on the right hand side of the screen 2 Click to save the image 3 Navigate to the local D and select the folder with your name If there is not one please create one 4 Choose t...

Страница 28: ...ose ZEN 2 3 application Note When prompted turn off the lasers change the state of each laser to OFF 3 Shut down Windows operating system Note Please wait for operating system to shut down completely...

Страница 29: ...the blue dialog labeled Imaging Setup 2 Begin with the longest wavelength fluorophore Alexa 568 in this case on Track 1 Note By default ZEN will start with one track Track 1 To add additional tracks c...

Страница 30: ...h can slow the system down When choosing this filter try to choose the best filter that matches two of your fluorophores In our example we would want to choose a filter for both the Alexa 488 and the...

Страница 31: ...olbar expand the blue Channels dialog 2 Click SR airyscan mode Note This mode will automatically set our pinhole and ensure our Optimal features are set appropriately 3 Start Live scan 4 Turn on range...

Страница 32: ...o to Acquisition Mode and under Frame Size click Optimal Note Using the Optimal feature is essential for sampling the field of view correctly for AiryScan The raw data needs to be sampled at Nyquist 2...

Страница 33: ...re the image has enough detail and contrast please do not attempt to use channels where the fluorescence is diffuse 1 Go to Maintain tab in the main toolbar and undock the Airyscan dialog Place this s...

Страница 34: ...34 LSM 880 Training Guide Aligning AiryScan...

Страница 35: ...AiryScan detector view shows evenly illuminated array as shown in example image Note Initially you may see the quality and status readout Out of Range or Bad while the alignment is running These are...

Страница 36: ...it to generate the final image 1 Go to Processing tab and select Airyscan Processing from method list 2 Select previously scanned image as Input image 3 To start select strength to Auto and select 2D...

Страница 37: ...To add additional tracks click the button next to the track list 4 Click Visible Light to open the laser dialog 5 Select the laser line you wish to use for this track 6 Set laser power slider this is...

Страница 38: ...which can slow the system down When choosing this filter try to choose the best filter that matches two of your fluorophores In our example we would want to choose a filter for both the Alexa 488 and...

Страница 39: ...Note The sampling rate is broken down into 4 options SR 2x sampling Nyquist choose this mode for the best possible resolution OPT 1x sampling equivalent to confocal choose this option for image quali...

Страница 40: ...base LSM The first value is the percentage laser power for the FAST mode and is equivalent to the second value in the LSM mode For example 2 of 561nm in FAST mode is equivalent to 0 25 in LSM mode 3...

Страница 41: ...41 LSM 880 Training Guide Using AiryScan FAST 9 Snap Image 10 Processing AiryScan FAST data see page 36 for procedure...

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