ALMF-IIT is a core facility for light microscopy that incorporate a Nikon Imaging Centre developed in partnership between Fondazione Istituto Italiano di Tecnologia and Nikon Instruments Italy.

The ALMF-IIT aims to provide to a wide community of scientists and professionals throughout Italy, Europe and the Rest of the World, with the support of companies, e.g. Nikon Instruments Italy, a large number of up-to-date imaging methodologies to monitor the living cell activity at high spatial and temporal rate. The main expertise of the ALMF-IIT is related to Super resolution and multiphoton microscopy, and it is developed in the unique multidisciplinary environment of IIT. The ALMF-IIT supports in-house scientists and visitors in using light microscopy methods for their research. The ALMF-IIT also regularly organises in-house and international courses to teach basic and advanced light microscopy methods (https://mix.iit.it).
The ALMF currently manages 15 advanced microscope systems, providing users with several workstations for image analysis with a total usage of the facility exceeding 10,000 hours per year.

Our staff can support users at different levels:

• Project planning, sample preparation, microscope selection and use, image processing and visualization
• Support of advanced microscopy techniques e.g. FLIM, FRAP, FRET, FCS and super-resolution
• Developing accessory software and microscopy equipment, co-developments with industrial partners, pre-evaluation of commercial equipment
• Image and data analysis for light microscopy

ALMF facility staff also supports users to access other IIT facilities services that can be useful to realize or complete their experiments. 

In the era of incredible advances in optical microscopy we can state that a new paradigm was born. ALMF-IIT is our Aleph.

An Aleph is one of the points in space that contains all other points. The only place on earth where all places are – seen from every angle, each standing clear, without any confusion or blending. If all places in the universe are in the Aleph, then all stars, all lamps, all sources of light are in it, too. It is the microcosm of the alchemists and Kabbalists…the “multum in parvo!” (freely adapted from El Aleph, 1945 JL Borges).

Platforms available for access through Instruct-ITALIA

• Optical nanoscopy
• Advanced Three-dimensional optical microscopy
• Atomic Force Microscopy (AFM)
• Correlative microscopy

Instrumentation

• Nikon time-lapse wide field microscope. Eclipse Ti-E, a powerful inverted microscope system with Okolab incubation system. Stable Time-lapse Imaging with Automatic Focus Correction System
• Nikon spinning disk confocal microscope. Yokogawa Head on TiE Inverted Microscope with Okolab incubation system and Andor camera iXon 897
• Nikon confocal microscope. A1R Resonant Confocal System on TiE Inverted Microscope
• Nikon confocal and multiphoton resonant scanner microscope, with ISS fast-FLIM module. A1r MP Multiphoton Confocal System on TiE Inverted Microscope with Okolab incubation system and FLIM capability
• Nikon super resolution system. N-STORM Super-Resolution System on TiE Inverted Microscope and Andor camera iXon 89
• Nikon super resolution system. N-SIM Super-Resolution System on TiE Inverted Microscope with cage incubator system and Andor camera iXon 897
• Olympus Confocal inverted microscope. FV1000 Confocal microscope
• Leica Spectral confocal, multiphoton and STED inverted microscope. Leica TCS STED-CW gated and super-continuum excitation
• Custom adapted FLIM and MP-STED CW microscope. On Leica TCS STED-CW gated
• Custom adapted atomic force microscope (AFM) and STED-CW gated microscopy. A JPK on a confocal, MP and STED Leica TCS STED-CW gated
• Custom made confocal, STED-CW gated, STED-FCS microscope
• Custom made MP and SW 2PE-STED microscope
• Custom made confocal and 2colors 3D STED microscope based on supercontinuum white light
• Custom made IML-SPIM microscope
• Custom made iSPIM microscope
• GI (Genoa Instruments) ISM microscope
• Nanoscale expansion microscopy

Techniques

Optical nanoscopy

Optical nanoscopy describes several techniques, which allow imaging at a spatial resolution higher than the conventional optical one imposed by the diffraction limit. The Abbe law defines the ability to distinguish two objects at a distance d as d = λ/(2 N.A.) where λ is the wavelength of light and N.A. the numerical aperture of the lens. Late in the 40s the Italian physicist Giuliano Toraldo di Francia introduced the concept of super-resolution. The concept is simple: one cannot violate physical laws – this means that using a lens and visible light d=200 nm – however, if some a priori or additional information can be added it can results in the formation of an image with details beyond the diffraction barrier limit. Optics and probes approaches can be used. Probe approaches, i.e. fluorescence-based, offered a large number of possibilities that culminated in the so-called super-resolved fluorescence microscopy approach, awarded with the Nobel Prize in Chemistry in 2014. I particular, Eric Betzig, W.E. Moerner and Stefan Hell brought "optical microscopy into the nano dimension". Depending on the practical implementation, we can divide optical nanoscopy into two major groups of methods:
1. Deterministic super-resolution: based on beam scanning, e.g., stimulated emission depletion (STED), ground-state depletion (GSD), reversible saturable optical fluorescence transitions (RESOLFT), or structure illumination,e.g., saturated structured illumination microscopy (SSIM).
2. Stochastic super-resolution: based on the localization of single fluorescent molecules, e.g., fluorescence photoactivatable light microscopy (FPALM), stochastic optical reconstruction microscopy(STORM), Super-resolution optical fluctuation imaging (SOFI) .
However, one has other chances to improve resolution. As main cases we manage: Structured Illumination Microscopy (SIM) that employs a physical (rotated patterns) and computation (inverse problem in the Fourier domain); Expansion microscopy that takes advantage of a smart material and chemical combination enlarging in a physical way the sample, following polymer cross-link with fluorescent molecules, Image Scanning Microscopy(ISM) and pixel reassignment that uses, in our case, a SPAD array detector to collect photons and a computational approach to reassign information at the appropriate location getting an improved resolution and extracting spectroscopic data.

Advanced Three-dimensional optical microscopy

Confocal laser scanning microscopy and computational optical sectioning microscopy are integrated, as 3D methods, with light sheet optical microscopy, a view displaced 90 degrees apart of selected planes of illumination (SPIM) able to remove undesired information plane by plane, and two-photon excitation (2PE) microscopy, allowing a physical selection of the imaged details in the three-dimensional space due to the non-linear process of excitation of fluorescence and on the consequent red-shift offering a better penetration depth. Both approaches reduce overall photobleaching of the specimen and can take advantage of the super-resolution approaches, namely: IML-SPIM (IML - Individual Molecule Localization SPIM) and 2PE-STED.

Atomic Force Microscopy (AFM)

Atomic force microscopy allows getting information at the molecular level with the constraint of being an a-specific and surface microscopy technique. However, information about the physical properties of the sample, for example, local elasticity, can be collected beyond molecular and atomic imaging at room temperature or in liquid. The coupling with optical nanoscopy methods widens in a significative way its utilization in life sciences, biophysics and nanoscale biophysics.

Correlative microscopy

Due to its function, optical nanoscopy can often be used with other high-resolution methods. The resolution of both electron and atomic force microscopy is even better than optical nanoscopy resolution, but by combining E.M. or AFM with optical super-resolution, it’s possible to achieve chemical sensitivity and specificity at unprecedented resolution. Nonetheless, all the quantitative information collectable by advanced fluorescence methods applied to live specimens can be correlated with nanoscale morphological imaging of non-optical techniques. The correlative approach opens an essential window on the interpretation of super-resolved fluorescence microscopy images.