Zeiss Axio Observer with Apotome Plus

Inverted Widefield Microscope

LED lines

385 nm; 423 nm; 469 nm; 555 nm; 591 nm; 631 nm; 735 nm

Camera

Monochrome CMOS camera, 20 MP, pixel size 2.74 x 2.74 um, diagonal chip 17.4 mm

Transmitted Light

Bright Field; Phase Contrast

Available Techniques

Z-stacks; Multiple Positions; Time series; Tile scan

Optical Sectioning with Apotome

Training is required before use

An equipment guide is available in our Guides section

Contact BIC for training

System Booking

Location: 5.01

Warnings

The equipment cannot be used without official training
Before your first use contact the responsible people 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
Apotome 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

Booking Rules

Booking is done online through Agendo. Please mind the following rules when booking the microscope:

Priority booking of afternoon slots is given to users with live cell experiments that require growth in the same day. If you have fixed or time insensitive samples, please try to book other times;
Maximum 2 slots per user per week (one slot = one morning or one afternoon);
These rules are applicable until the previous Friday. If you need more than two slots in a week and they are still available on the previous Friday, at 4pm, feel free to book them.

Know Your Fluorophores

Use resources such as the FPBase or the Thermofisher SpectraViewers to explore your fluorophores. Learn their excitation and emission wavelengths, and what are their optimal LED lines and filters.

Brief Description

The ZEISS Axio Observer inverted widefield microscope equipped with the Apotome optical sectioning system is a high-performance imaging platform designed for advanced fluorescence microscopy of biological samples. The system enables high-resolution multichannel, time-lapse, and three-dimensional imaging of samples mounted on slides, multiwell plates, and live-cell imaging chambers. Typical applications include cell biology and tissue imaging, protein localization studies, live-cell dynamics, multi-fluorophore experiments, and quantitative fluorescence analysis.

Suggestion for “Materials and Methods”

Images were acquired using a ZEISS Axio Observer inverted widefield microscope equipped with an Apotome structured illumination module for optical sectioning. A 20 MP monochrome Axiocam 820 camera, (pixel size 2.74 μm) and a [objective model, e.g., Plan-Apochromat 63×/1.4 NA oil] objective were used. Multichannel fluorescence imaging was performed using [single/multiband] filter sets optimized for [fluorophore(s), e.g., DAPI, GFP, Alexa Fluor 594].

The excitation was provided by a Viluma 7 LED light source, using the following channels: [LED wavelengths used, e.g., 385, 469, 555 nm]. Image acquisition parameters — exposure time ([e.g., 100 ms]), LED intensity ([e.g., 15%]), binning ([e.g., 2×2]), and gain ([e.g., 1×]) — were kept constant across all samples to allow quantitative comparisons.

For 3D imaging, Z-stacks were acquired with a step size of [e.g., 0.3 μm], and optical sectioning was performed using the Apotome with [e.g., 5] grid phases. Time-lapse and tile scan acquisitions were controlled using ZEN software (ZEISS), with hardware autofocus enabled and environmental control maintained at [e.g., 37 °C, 5% CO2, [O2]%, and humidified atmosphere], where applicable.

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. The acquisition of the imaging system used in this work was supported by the project "Infraestruturas Integradas de Investigação no ITQB NOVA" (Integrated Research Infrastructures at ITQB NOVA), co-funded by FEDER via the "Programa Operacional Regional Lisboa 2030", with the reference LISBOA2030-FEDER-01318100.

Objectives

*Magnification¹*	10x	20x	40x	63x	100x	100x
ZEISS System	EC Plan-Neofluar	Plan-Apochromat	Plan-Apochromat	Plan-Apochromat	Plan-Apochromat	Plan-Apochromat
Class	Colour and Chromatic Correction	Colour and Chromatic Correction	Colour and Chromatic Correction	Colour and Chromatic Correction	Colour and Chromatic Correction	Colour and Chromatic Correction
Numerical Aperture (NA)	0.3	0.8	0.95	1.4	1.4	1.4
Immersion	Air	Air	Air	Oil	Oil	Oil
Free Working Distance (mm)	5.2	0.25	0.25	0.19	0.17	0.17
Cover Glass (mm)	0.17	0.17	0.17	0.17	0.17	0.17
Contrasting Methods	Phase Contrast (Ph)	Bright Field	Bright Field	Bright Field	Bright Field	Phase Contrast (Ph)

¹Notice that the microscope is equipped with two Eyepieces: HC Plan S 10x/25 Br. M