Zeiss LSM 880 Bedienungs- und Wartungshandbuch

Zeiss LSM 880 with Airyscan

Confocal Light Microscopy

Basic User Guide

Contents

Brief Description...................................................................................................... 4

Reference ................................................................................................................ 4

Location ................................................................................................................... 4

Suggestion for “Materials and Methods” ............................................................... 5

Information to be added to the acknowledgements section ................................. 5

People and Contacts ............................................................................................... 6

WARNINGS and Advice ........................................................................................... 6

Optics ...................................................................................................................... 7

Laser Lines ................................................................................................................... 7

Detectors ..................................................................................................................... 7

Transmitted Light Techniques ..................................................................................... 7

Incident Light .............................................................................................................. 7

Features and Specifications .................................................................................... 7

Objective ................................................................................................................. 8

Filter-Sets for Incident Light .................................................................................... 9

Additional Equipment ........................................................................................... 10

Computer ...................................................................................................................... 10

Monitor ......................................................................................................................... 10

Potential Techniques ............................................................................................. 10

System Overview ................................................................................................... 11

Know your fluorophores ....................................................................................... 11

Sample Preparation Notes .................................................................................... 11

System Protocols ................................................................................................... 12

Start Up ......................................................................................................................... 12

Shutdown ...................................................................................................................... 13

ZEN 2.3 (black edition) Guidelines ........................................................................ 14

General User Interface .................................................................................................. 14

Sample Localization ...................................................................................................... 15

Image Acquisition .......................................................................................................... 16

Smart Setup ............................................................................................................... 17

Lasers ........................................................................................................................ 18

Imaging Setup ........................................................................................................... 18

Acquisition Mode ...................................................................................................... 21

Channels .................................................................................................................... 22

Definite Focus 2 ............................................................................................................ 23

Multidimensional Acquisition ....................................................................................... 23

Z-Stack ....................................................................................................................... 23

Time Series ................................................................................................................ 24

Tile Scan .................................................................................................................... 24

Positions .................................................................................................................... 25

Regions ...................................................................................................................... 25

Brief Description

Zeiss LSM 880 is an inverted confocal laser scanning microscope with Airyscan, a new

detector concept featuring a 32 channel GaAsP-PMT area detector. The Airyscan detector thus

brings forward simultaneous improvements in spatial resolution and signal-to-noise ratio in

comparison with conventional confocal microscopy. This allows the LSM 880 to resolve small

structures up to 120 nm (XY) and 350 nm (Z) even in thick samples.

The introduction of a Fast module for the Airyscan offers a satisfactory compromise

between perfect optical sections and live cell imaging experiments. The Fast Airyscan mode

reduces photo-bleaching and allows acquisition speeds of up to 19 frames per second at 512 x

512-pixel image resolution.

The LSM 880 with Airyscan highlights the needs of your research for quantitative data in

live cell experiments at superior resolutions.

Reference

Zeiss LSM 880

Location

Room 5.01 (Bacterial Imaging Cluster), 5th floor, Instituto de Tecnologia Química e

Biológica António Xavier (ITQB NOVA)

Suggestion for “Materials and Methods”

Confocal Z-series stacks (step size 200 nm) were acquired on a Zeiss LSM 880 point

scanning confocal microscope using the Airyscan detector, a 63x Plan-Apochromat 1.4NA DIC

oil immersion objective (Zeiss) and the 405 nm, 488 nm and 561 nm laser lines. The Zeiss Zen

2.3 (black edition) software was used to control the microscope, adjust spectral detection for

the emission of 4′,6-diamidino-2-phenylindole (DAPI), Green Fluorescent Protein (GFP) and

tetramethylrhodamine (TMR) SNAP-tag fluorochromes and for processing of the Airyscan raw

images.

Confocal Z-series stacks and tile regions with multiple positions were acquired on a Zeiss

LSM 880 point scanning confocal microscope using photomultiplier tube detectors (PMTs) and

a gallium arsenide phosphide (GaAsP) detector, a 63x Plan-Apochromat 1.4NA DIC oil

immersion objective (Zeiss) and the 405 nm, 488 nm and 561 nm laser lines. The Zeiss Zen 2.3

(black edition) software was used to control the microscope and adjust spectral detection for

the emission of 4′,6-diamidino-2-phenylindole (DAPI), Green Fluorescent Protein (GFP) and

tetramethylrhodamine (TMR) SNAP-tag fluorochromes.

Images were acquired on a Zeiss LSM 880 point scanning confocal microscope controlled

with the Zeiss Zen 2.3 (black edition) software, using a 63x Plan-Apochromat 1.4NA DIC oil

immersion objective (Zeiss), the fluorescence filter sets GFP + TX2 and differential interference

contrast (DIC) optics.

Information to be added to the acknowledgements section

This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-

POCI-01-0145-FEDER-022122) co-funded by national funds from OE - "Orçamento de Estado"

and by european funds from FEDER - "Fundo Europeu de Desenvolvimento Regional”.

People and Contacts

Mariana Ferreira, Bacterial Imaging Cluster (mariana.g.ferreira@itqb.unl.pt)

Adriano O. Henriques, Microbial Development Group ([email protected]l.pt)

Mónica Serrano, Microbial Development Group ([email protected]l.pt)

Pedro Matos Pereira, Bacterial Cell Biology Group ([email protected]l.pt)

Phone +351 214 469 524

Extension 1522/4 or 1566/7

WARNINGS and Advice

 The equipment cannot be used without official training.

 Before your first use contact Mariana Ferreira to schedule

training

 Save all data into your own storage device as we routinely clean

the computer’s data

 Register your utilization in the logbook

 Airyscan raw data reconstruction must be done in the

microscope computer (book slots accordingly). For all

downstream image analysis image analysis a free software

license is available for ZEN lite at ZEISS website. Alternatively,

you can use FIJI.

 Contact the responsible people in case of any doubt or any

issue with the equipment.

Optics

Laser Lines

 405 nm (30 mW)

 Multi-line - 458/488/514 nm (25 mW)

 561 nm (20 mW)

 633 nm (5 mW)

Detectors

 2 Photo-Multiplier Tube (PMT)

 1 Gallium Arsenide Phosphide (GaAsP)

 1 Airyscan (32 GaAsP)

 1 Transmitted Photo-Multiplier Tube (T-PMT)

Transmitted Light Techniques

 Differential interference contrast (DIC)

 Phase Contrast

 Bright Field

Incident Light

 Fluorescence (HXP UV lamp)

Features and Specifications

 Axio Observer 7 SP Inverted Microscope Stand

 Motorized Stage 130 x 100 STEP

 Stage Controller XY STEP SMC 2009

 Z-Piezo WSB 500

 Incident Light Source: HXP 120 V illuminator (w/ 200 W mercury short-arc reflector

lamp)

 Motorized Condenser w/ six positions (w/ DIC)

 Definite Focus 2 w/ ACR (Automatic Component Recognition) motorized nosepiece

 Eyepieces: PL 10x/23 Br. Foc. (High eyepoint and Focusing)

Objectives

Magnification1 10x 20x 63x

ZEISS System EC Plan-NEOFLUAR Plan-APOCHROMAT Plan-APOCHROMAT

Class

Colour and Chromatic

Correction

Colour and Chromatic

Correction

Colour and Chromatic

Correction

Aperture (A) 0.3 0.8 1.4

Immersion Air Air Oil

Free Working Distance

(mm)

5.2 0.55 0.19

Cover Glass (mm) 0.17 0.17 0.17

Contrasting Methods Phase Contrast (PH1)

Differential Interference

Contrast (DIC II)

Differential

Interference Contrast

(DIC III)

Lateral Resolution (XY)

@525 nm (µm)2 0.7 0.2625 0.15

Nyquist Sampling (XY)

@525 nm (µm)3

0.28 0.105 0.06

Zoom to Nyquist

Sampling (XY) @525 nm

with 512x512 scan

format

Number of

field/(magnification*zo

om)

= Nyquist sampling

Axial Resolution (Z)

@525 nm (µm)2

0.735

0.09 1.4 × 0.525 × 1.338

0.64 1.4 × 0.525 × 1.515

1.96

Nyquist Sampling (Z)

@525 nm (µm)3

3.2667 0.6146 0.2273

1Notice that the microscope is equipped with two Eyepieces: HC Plan S 10x/23 Br. M

2  = . 

 and  = .  × 

 from Olympus website: Resolution and Contrast in

Confocal Microscopy

3  =   

.

Filter-Sets for Incident Light

Filter Cube Excitation Filter Dichromatic Mirror Emission Filter Emission Colour

DAPI G 365 395 BP 445/50 Blue

GFP BP 470/40 495 BP 525/50 Green

RFP BP 565/30 585 BP 620/60 Red

BP - bandpass filter

These have predefined Buttons in the Locate Tab.

Emission Filters for Airyscan and Fast Airyscan

Position Wavelengths Emission Colours Optimal Fluorophores

2 BP 420-480 + BP 495-550 Blue + Green to Yellow DAPI + GFP, FITC, YFP

3 BP 420-480 + BP 495-620 Blue + Green to Red

DAPI + GFP, FITC, YFP,

Rhodamine, RFP

4 BP 465-505 + LP 525 Cyan + From Yellow

CFP + YFP, Rhodamine,

Cy5

5 BP 495-550 + LP 570 Green to Yellow + From Red

GFP, FITC, YFP + RFP,

Cy5

6 BP 570-620 + LP 645 Red + Far Red RFP + Cy5

BP – Band Pass filter

LP – Long Pass filter

Additional Equipment

Computer

 Type: Desktop

 Brand: HP®

 Model: HP 840

 Features: 2x Intel® Xeon® E5-2623 v3 3.0 GHz (quad-core); 64GB DDR4-2133 MHz

ECC RAM (12 free slots); 4x 2TB 4 TB RAID 10; NVIDIA Quadro K2200 4GB

 OS: Windows 7 Ultimate Embedded x64

 Available Software: Zeiss Zen 2.3

Monitor

 Brand: HP®

 Model: DreamColor Z32x

 Diagonal: 32”

 Native Resolution: 4K UHD (3840 x 2160 @ 60 Hz)

Potential Techniques

 High Resolution microscopy

 Photobleaching

 FRAP

 FRET

 FLIM (please contact Mariana Pinho, mgpinho@itqb.unl.pt)

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