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MICA MICROHUB (LEICA MICROSYSTEMS) WIDEFIELD AND CONFOCAL MICROSCOPE

The MICA MicroHub widefield and confocal microscope at Centro de Química da Madeira (CQM, University of Madeira) main objective is to support research at the Center. It is routinely used as a powerful and advanced imaging technique for acquiring 2D and 3D sharp fluorescent images of biological and non-biological specimens, by researchers at CQM. This includes researchers from the following national R&D projects; Post-Doc, PhD and Biochemistry (BSc and MSc) students. Furthermore, the CQM MICA MicroHub is open to external service for both private and other public institutions.

Microscopy

Microscopy allows the visualization and analysis of structures and features of samples that are not visible to the naked eye. This is only possible by directing light or electrons through or onto a specimen, which will interact with the samples and change or originate new signals. An image of the sample is then magnified and focused by a system of lenses or electromagnetic fields being, afterward, directed to the detector. This technique enables the study of cells, tissues, microorganisms, and materials at high magnification and resolution, providing critical insights into their structure, composition, and function.

The resolution of a microscope is the ability to distinguish two distinct points of a sample. Is the minimum distance at which these two points can be visualized as separate entities. For photon-based microscopy, such as widefield and confocal, the maximum resolution limit is around 200 nm. The resolution depends on the numerical aperture (NA), wavelength of light (λ, namely half of the wavelength of the light used), refractive index of the mounting medium (n) and angle of the cone of light (θ).

Widefield fluorescence and confocal microscopy

WIDEFIELD MICROSCOPY

Widefield microscopy is one of the basic and widely used microscopy techniques that enables rapid observation of samples by illuminating the entire sample in field of view simultaneously (in and out-of-focus planes). The excited sample then emits fluorescence that reaches the detector, generating an image of the stained sample with one or more target-specific fluorescent molecules. It is particularly suitable for thin samples and fast image acquisition, and is commonly applied in cell biology studies, fluorescence labeling, and high-throughput assays. Its simplicity and speed make it a versatile tool for routine imaging applications. However, the actual resolution achieved by a widefield microscopy is exacerbated by background signal, due to the out-of-focus fluorescence. This leads to an overall decrease in the signal-to-noise ratio and a subsequent decrease in achievable resolution and contrast. Improvement of image quality and resolution is possible through computational clearing or deconvolution software.

WF	ICC	LVCC	SVCC
* WF – widefield fluorescence; ICC – Instant Computational Clearing; LVCC – Large Volume Computational Clearing; SVCC – Small Volume Computational Clearing.

CONFOCAL MICROSCOPY

Confocal microscopy, also relying on fluorescence, is based on point-to-point illumination, where a pinhole hardware component rejects the out-of-focus fluorescence emitted from labeled sample, blocking background signals of reaching the detectors. This results in sharply focused images with higher contrast and overall resolution. The main advantage is that it allows optical sectioning and three-dimensional reconstruction of the sample, since most of the out-of-focus background fluorescence is removed through the microscope’s hardware. Thus, this technique is well suited for the detailed analysis of thick specimens, tissues, and complex subcellular structures.




REFERENCES

LEICA MICROSYSTEMS (https://www.leica-microsystems.com)

MICA MicroHub equipment (Leica Microsystems)

The equipment is installed in CQM/UMa under its strategic funding project FCT/CQM, project references: UIDP/00674/2020 and UIDB/00674/2020.

Supervisor: João Rodrigues (PI), Helena Tomás (PI)

Unit Manager: Rita Castro (PhD)

Autorized User (UT): Rita Castro (PhD), Mara Gonçalves (PhD)

Applicant: Any researcher, research group or Entity (public or private)

Technical Specifications

x = included, o = optional, - = not available

Name	Description	Mica CORE
Illumination and Detection
Transmitted light contrast	Integrated modulation contrast (IMC) automatically adjusted and brightfield contrast in RGB or gray scale mode	x
Incident fluorescence illumination	4 x LEDs: 365 nm, 470 nm, 555 nm and 625 nm	x
Widefield detector type	5 MP CMOS FluoSyncTM detector enabling 4  label simultaneous imaging	x
Confocal illumination	4 x Laser diodes: 405 nm, 488 nm, 561 nm and  638 nm	x
Confocal detector type	HyD FS FluoSyncTM detector enabling 4 label simultaneous imaging	x
Environmental Control
Live cell package	Temp. (to 45°C), CO2 (0 - 10%), Humidity	-
Hypoxia upgrade	Temp. (to 45°C), O2 (1% to air), CO2 (0 – 10%), Humidity	-
Objectives
Included objectives	HC PL FLUOTAR 1.6x/0.05 HC PL FLUOTAR 10x/0.32	x
Recommended objectives	HC PL APO 20x/0.75 CS2 HC PL APO 40x/1.10 W CS2 motCORR HC PL APO 63x/1.20 W CS2 motCORR	x
Adaptive Immersion
Immersion dispension (closed loop water dispenser)	Forming and maintaining water immersion for one objective is feedback controlled and does not require any interaction	x
Microscope Focus
Autofocus	Reflection-based Adaptive Focus Control (AFC) and image-based Autofocus for transmitted light and fluorescence images (can be combined with AFC).	x
Image enhancement methods
THUNDER	Including all THUNDER methods: Instant Computational Clearing (ICC) Small Volume Computational Clearing (SVCC) Large Volume Computational Clearing (LVCC)	x
LIGHTNING Basic		x
LIGHTNING Expert		o
Vibration isolation
Anti-vibration table	Isolates the imaging against vibrational disturbances in the environment	x

 

Core Functionalities of Mica

x = included, o = optional, - = not available

Name	Description	Mica CORE
FluoSyncTM	FluoSyncTM detection hardware with fully integrated digital spectral hybrid unmixing for separation of up to 4 labels during simultaneous imaging in either widefield or confocal mode.	x
OneTouch	Sets all technical excitation and detection parameters according to the experimental demands. This can either be done automatically or with a single click on-demand.	x
Focusing	Keeps the sample in focus throughout the experiment with a simple selection from a choice of 3 focus strategies.	x
3D Imaging	Allows acquisition of 3D volumes in widefield and confocal mode.	x
Mixed TL & WF	Combines transmitted light with widefield imaging.	x
Mixed TL & CLSM	Combines transmitted light with confocal imaging.	x
Sample Finder	Quickly and automatically generates an in-focus overview of the relevant sample areas.	x
Navigator	Powerful package including Assay Editor, Stitching and Mark and Find license. Uses overview for navigation and for defining positions and regions in any shape. Displays all acquired images in spatial relation to all other images.	x
Objective Collision Prevention	Prevents the objective from colliding with a microtiter plate to protect the objective and sample.	x
Learn & Results	Aivia-powered pixel classifier: easy to train, it generates fast and reproducible image segmentation results. The software generates beautifully visualized results with full traceability of the data points to the source in the image.	x

Objectives

- N PLAN 2.5 x / 0.12 Dry (used for overviews of sample)
- HC Plan Apo 10 x / 0.40 CS2 Dry
- HC Plan Apo 20 x / 0.75 CS2 Dry
- HC PL APO 63 x / 1.20 CS2 Water (motCorr), (Automatic water pump)

 

Thunder Algorithms

- 3 dedicated algorithms are available that remove out-of-focus light in real-time, thus providing fast optical sectioning capabilities:
- Instant Computational Clearing (ICC): Removal of background and out-of-focus light.
- Small Volume Computational Clearing (SVCC): ICC and deconvolution algorithm for thin samples (e.g. cells, yeast)
- Large Volume Computational clearing (LVCC): ICC and deconvolution algorithm for thick samples (e.g. tissue sections, embryos, organoids).

Management of the equipment and organization - Responsabilities

Unit Manager (UM)

- Responsible for equipment management, approval of analysis/reports/quotations and consumables acquisition.
- Responsible for equipment operation and maintenance.

Autorized User (AU)

- Responsible for equipment operation.
- Make annual reports on the functioning of the Unit. Send to Unit Manager.
- Responsible for the preparation of the equipment reservation/operation time according to the norms of operation of the center.
- Make recommendations to UM about updating or purchasing material in order to ensure an efficient service.

Applicant (AP)

- Responsible for time reservation of the equipment and services cost.
- Accurately fill the sample registration form with all the necessary informations and the registration logbook in accordance with the Center's operating rules.

Regulation of operation of the MICA MicroHub microscope equipment

General Guidelines

These Guidelines are intended to be a guide to the autorized users and applicants that uses the CQM MICA equipment and as such must be followed by all. Otherwise, access to use the microscope will be denied.
• The user should keep the equipment in good working order.
• The user needs to provide the guarantees, when required, to safeguard the damage and/or faults in the equipment;
• The user is responsible for damage and/or malfunctions in the equipment that is affected by it, if not covered by the warranty and insurance;
• The right to use the equipment, complying with the established rules, in accordance with the terms approved by the Center.
• THE OPERATION OF THE MICA EQUIPMENT IS ONLY AUTHORIZED TO THE RESPONSIBLE RESEARCHERS, AFOREMENTIONED.
• Only authorized users will be granted access to the microscope. In order to be authorized to use the equipment, EVERYONE must first receive training even if you are already familiar with the technique.
• ALWAYS register your work in the Logbook.
• Report on the Logbook about any problem with the equipment or software and inform the UT as soon as possible.
• The applicants should submit an analysis request by filling out online the sample registration form, as detailed as possible, to proceed with the MICA sample analysis, upon approval by the Unit Manager. Be advised that your supervisor must be informed of your MICA registration in advance.
• Please be responsible and careful. Curiosity is good, but not with an expensive equipment.
• Remember that your supervisor WILL PAY for your usage time and possible damages.
• The final cost of each analysis takes into account the: maintenance costs, analysis type, total user time and user type (academic or industrial). For details about prices and charges applied, check our price table.
• If there is a reservation made YOU HAVE TO RESPECT IT, even if it is an emergency. Don't start an analysis unless there is enough time to finish it before the next person. In the same manner, DO NOT schedule an analysis just moments before you need, plan your time properly or wait for an opening. For more information on time reservation, please contact
• Samples must be properly labeled according to the submitted form and should be removed from the MICA sample holder after the analysis is finished. Unlabeled samples will be disposed of.

MICA Widefield and Confocal Services

Analytical services

• Brightfield microscopy (BF) and Integrated Modulation Contrast (IMC).
• Widefield fluorescence microscopy with 4 simultaneous fluorescence channels.
• Possibility of THUNDER imaging with 3 different algorithms:
- Instant Computational Clearing (ICC);
- Small Volume Computational Clearing (SVCC);
- Large Volume Computational clearing (LVCC)
• Confocal microscope, with possibility of LIGHTNING deconvolution.
• Z-stacks
• Time-lapse (without CO2 and temperature control incubator).

The user fees are intended to cover CQM MICA expenses, which include analysis consumables, parts, and maintenance. There will be a minimum usage charge of 1 hour after which, use time will be rounded up to the nearest 30 mins. Samples must be prepared according to required conditions for requested observations and as submitted in the registration form.
Choose the class that is best suited to your case or consult us using the address: .

The price of services provided is divided into three classes, namely:

1. CQM Research groups
2. FCT National Centers and National Public Laboratories
3. Companies and private Research Centers

Analytical Services (€/h)*	Class 1	Class 2	Class 3
Service cost (€/h) ǂ	30	50	80
Widefield (brightfield and/or 1 fluorescence channel) #	20	40	60
Confocal (1 channel) #	30	50	70
ǂ minimum service cost. # to be added to service cost. When combining both brightfield/widefield and confocal, the price will be based on the higher-priced item).
ADDITIONALS
Additional channels (up to 4)	5	10	20
Custom Em/Ex channel	5	10	20
Observation with 63x objective (water immersion)	10	20	30
Image Modalities
3D-stacking	10	20	30
Time-lapse	15	30	40
Multiple stage points	5	10	20
THUNDER (ICC, LVCC, SVCC. for each)	5	10	20
Lightning	5	10	20
Special requirements	Under consultation

(prices: valid from January 2026; VAT not included, please add tax at rate of 22%)

Schedule MICA widefield and confocal services

To access the MICA widefield and confocal Service, please fill the sample registration form. If you are not a CQM member and would like to perform a sample analysis, please contact us via and check the pricing list.

Submit sample here (exclusively for CQM members)

After the form submission, you will receive in your mail box an email confirming the successful sample registration and the filled form.