Fluorescence Microscopes

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Luminosa Single Photon Counting Confocal Microscope

Luminosa

Remarkably simple.
Precisely yours.

 

Single Photon Counting Confocal Microscope

Explore new paths in confocal microscopy

Luminosa pairs highest data quality with remarkably simple day-to-day operation. It easily integrates into any researcher’s toolbox and becomes a time-efficient, reliable companion for scientists starting to explore the use of time-resolved fluorescence methodologies as well as for experts wanting to push the limits. Truly a microscopy system that everybody can rely on.

  • Quality and precision you can trust
  • Save time and simply focus on your samples
  • Advanced flexibility

Impressions of Luminosa's capabilities

Remarkably simple. Precisely yours.

PicoQuant has acquired 25 years of experience with time-resolved single photon counting. During this time, PicoQuant has also cultivated strong ties to the scientific community, which we value immensely. We have gathered the extensive knowledge both of our scientific staff and expert scientists, and merged it to create our best time-resolved instrument & software package so far: Luminosa.

QUALITY AND PRECISION YOU CAN TRUST

Optimal performance for single molecule investigations in every measurement context: One-click auto-alignment procedure even without requiring a sample. Galvo scanning (maximum speed) and objective scanning (maximum photon detection efficiency) on the same microscope.

SAVE TIME AND SIMPLY FOCUS ON YOUR SAMPLES

Context-based, intuitive workflows guide you to efficiently harness the full power of smFRET, FCS and FLIM with confidence. Get analysis results with minimal user interaction. GPU-based algorithms provide fast and reliable results.

ADVANCED FLEXIBILITY

Adjust the observation volume to match the dynamics of your FCS and smFRET assays with a single click. An open mode of operation is available for full access to every optomechanical component via software.

What customers say about Luminosa

"Measurements we could not do before."

Dr. Daniel Nettels, University of Zurich, Switzerland

"Extremely valuable."

Prof. Dr. Claus A. M. Seidel, Heinrich Heine University Düsseldorf, Germany

"Absolutely great!"

Prof. Dr. Jörg Enderlein, Georg-August-Universität Göttingen, Germany

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LUMINOSA BROCHURE AND DATASHEET

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ON EXHIBITIONS AND CONFERENCES 

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Benefit from our highly qualified support team

You will profit from the combined expertise of our support team. All supporters hold a PhD degree in the natural sciences. Tap into their years of experience to advance your research!

OUR SUPPORT TEAM OFFERS

  • initial training with hardware and software during system installation, either on-site or remote
  • follow-up training on demand, tailored to your needs
  • application counseling regarding questions from sample preparation and measurement setting optimization to data analysis
  • troubleshooting in case of microscope hardware and software problems
  • system repairs
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DEMONSTRATIONS AND TEST MEASUREMENTS

The team also performs instrument demonstrations and test measurements with your samples in our application lab in Berlin. If you want to find out what Luminosa can reveal about your samples, request a session.

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PicoQuant Support Team

What customers say about our support

"Fantastic support. This is one of the main reasons for me to purchase PicoQuant equipment and recommend it to colleagues! Please keep this service always."

Prof. Dominik Wöll, RWTH Aachen University, Germany

"I'm very glad to have the opportunity to work with PicoQuant instrument and to have the support of a strong experienced team in time-resolved fluorescence to explore new horizons."

Arnaud Spangenberg, Institut des Science de Matériaux de Mulhouse, France

Icon TRMic CourseScientific guidance and user training

Researchers wishing an in-depth introduction to the principles of time-resolved fluorescence microscopy and its applications to the Life Sciences can attend PicoQuant's annual 3-day course including lectures and hands-on training.

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Applications

Dynamic structural biology at the single molecule level

Many protein structures have been solved successfully using methods such as X-ray crystallography or cryo-EM. However, intrinsically disordered proteins or proteins which do not crystallize pose serious problems for these techniques. Moreover, the observation of dynamic conformational changes of proteins as they are performing their tasks in their native environment calls for different approaches.

Here, single molecule fluorescence approaches such as FRET and FCS open new possibilities, as they enable the study of individual molecules at room temperature in solution or even in cells, with the potential to discriminate subpopulations in different locations.

smFRET monitors distance changes between two or more fluorescent labels attached to different protein residues, which provides valuable information about conformational changes, folding or protein interactions. This complements protein structural models and molecular dynamics simulations, allowing researchers to develop a more complete understanding of protein function.

FCS on the nanosecond timescale (nsFCS) reveals the reconfiguration time of the protein chains of intrinsically disordered or folding proteins. Combining this information with polymer physics theory and molecular simulations yields a clearer picture of disordered protein dynamics.

FCS on the microsecond to second timescale measures molecular diffusion behavior, which depending on the experimental context can shed light on the protein mobility, its microenvironment, or on protein oligomerization and interaction.

Dynamic structural biology

Cellular mechanisms driven by phase separation

Liquid-liquid phase separation is emerging as a key mechanism for regulating the spatial organization of biomolecules in cells. It underlies compartmentalization by condensation of subsets of molecules, which promotes protein interactions. As phase separation is a very dynamic process, it responds quickly to changes in environmental conditions.

Fluorescence methods can be used to study the kinetics of phase separation, to follow the distribution of molecules into different phases, to observe the dynamics of separate phases, and to probe their different properties:

FCS measures the concentration of a labeled species; its extensions FCCS and FLCS can even measure the concentrations of several species simultaneously. Moreover, FCS reports on the diffusion rate of the molecules under study, which can differ between phases.

Fluorescence anisotropy experiments nicely complement FCS by observing the rotational motion of molecules, which depends on the viscosity or rigidity of the local environment.

FLIM of fluorescent sensors can read out environmental parameters such as pH, temperature, membrane tension, or the concentration of various ions in real time. This additional dimension of information can help to interpret the dynamics of different phases and understand their properties from a physical point of view.

Phase separation

Environmental sensing

The properties of a protein's native environment inside a cell, such as molecular crowding, pH fluctuations, or plasma membrane alterations, influence its function. But in many cases, not all of these parameters are known, so they cannot accurately be reproduced in experiments.

In the last few years, a range of fluorescent sensors has been developed that change their fluorescence lifetime in response to a specific environmental change, for example temperature, calcium concentration, glucose concentration or membrane tension. With FLIM, these parameters can be read out quantitatively in real time from live cells in a non-invasive manner, providing context to better understand protein function or regulation.

Environmental sensing

Mapping dynamics and structure of cellular membranes

Cellular membranes are highly complex and dynamic structures. Their molecular composition of lipids and membrane proteins is precisely tuned to obtain the physical and chemical properties necessary for their physiological function. Complementary fluorescence methods can be combined to approach biological membranes from different angels:

FCS of fluorescently labeled lipids or membrane proteins yields diffusion rates and protein mobility. It also reports concentrations of the molecules under study, which can vary across locations.

With anisotropy experiments one obtains the molecular orientation, and changes thereof. Thus, anisotropy imaging can visualize ordered and disordered membrane phases, or monitor the rotational motion of molecules.

FLIM of environmental sensor fluorophores can quantify important physical parameters, for example membrane tension or lipid order.

Mapping dynamics

Core Methodologies

Fluorescence Lifetime Imaging (FLIM)

FLIM is a fluorescence imaging technique that resolves and displays the lifetimes of individual fluorophores rather than their emission spectra. The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon. Since fluorescence lifetime is unrelated to concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity, FLIM is less prone to artifacts than intensity based methods.

Benefits of FLIM:

  • can be used to distinguish between fluorophores
  • provides an additional dimension of information
  • complementary to spectral information
  • technique of choice for many kinds of functional imaging, as a fluorophore’s lifetime can be influenced by environmental parameters such as pH, ion or oxygen concentration, or molecular binding

FLIM-FRET – lifetime-based Förster resonance energy transfer

FRET is a non-radiative process whereby energy from an excited fluorescent molecule (donor) is transferred to a nearby non-excited fluorophore (acceptor) in the range of 2-10 nm. The energy transfer results in donor quenching and leads to changes in the fluorescence intensity of the two fluorophores and a shortening of only the donor lifetime.

In contrast to standard FRET where one measures changes in fluorescence intensity, lifetime-based FRET enables quantitative analysis by using the fluorescence lifetime of the donor molecule as a probe, which is independent on concentration over a broad range. This is crucial since in biological systems like cells the fluorophore concentration often cannot be accurately determined and compared amongst different cells.

Benefits of FLIM-FRET: lifetime-based FRET can identify different sub populations with different FRET efficiencies, where intensity-based FRET would only detect the average value

smFRET – single-molecule Förster resonance energy transfer

In this type of experiments, the FRET process is observed within a single molecule (bearing a donor and acceptor), or between interacting partners that are either freely diffusing through the confocal volume immobilized on a surface.

A technique which can be quite helpful for smFRET is Pulsed Interleaved Excitation (PIE), where several pulsed lasers are synchronized. The laser pulses are separated on a nanosecond time scale to allow simultaneous recording of the temporal behaviour of a sample molecule.

In an smFRET experiment, this allows exciting the donor and acceptor alternately. In this way, the acceptor dye is excited independently of the FRET process to confirm its presence and photoactivity. Molecules lacking an active donor or acceptor are separated from active FRET complexes. This makes it possible to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescing acceptor.

Fluorescence Correlation Spectroscopy (FCS)

Fluorescence Correlation Spectroscopy (FCS) is a correlation analysis of temporal fluctuations of the fluorescence intensity. It offers insights into the photophysics that cause these characteristic fluctuations as well as into the diffusion behavior and absolute concentrations of detected particles. FCS enables the determination of important biochemical parameters such as the concentration and size or shape of the particle (molecule) or viscosity of their environment.

Fluorescence Lifetime Correlation Spectroscopy (FLCS)

Fluorescence Lifetime Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS contributions.
FLCS is of particular advantage when using spectrally inseperable fluorophores that differ in their lifetime for Fluorescence Cross-Correlation Spectroscopy (FCCS) because it offers elimination of spectral cross talk and background. It also offers a way around detector afterpulsing artifacts.

Fluorescence Cross-Correlation Spectroscopy (FCCS)

Fluorescence Cross-Correlation Spectroscopy (FCCS) discriminates FCS signals from two different species based on their different emission spectra.

Anisotropy imaging

Measurement of steady-state and particularly time-resolved fluorescence anisotropy offers fascinating possibilities to study molecular orientation and mobility as well as processes that affect them. In general, anisotropy does not depend on the concentration of fluorophores, i.e. is independent of detected signal intensity. This is of particular importance for the interpretation of smFRET measurements where fluorophore mobility might affect the resonance transfer efficiency. Time-resolved anisotropy measurements are more informative, as steady-state ones only report time averaged values, without direct insight into the dynamics of the process.


Software

The Luminosa software includes single molecule detection, FCS, and time-resolved imaging methods, as well as the possibility to flexibly define custom measurement and analysis modes. Its design permits quick and easy workflows for experiments, which allow the users to focus on their samples. Clearly arranged interfaces showing only parameters relevant for each application promote reproducible measurements yielding consistent datasets.

Simply select the fluorophores in use, and the software automatically configures excitation lasers, the beampath and the detection. It takes care of parameters important for time-resolved measurements, such as laser repetition rate, and pulsed interleaved excitation. Several online previews of the data during acquisition enable the users to instantly check the sample and data quality, saving precious instrument time. Users can explore their data immediately thanks to fast GPU-based analysis routines.

Luminosa Software Start

LumiFinder • FRETcompass • InstaFCS • InstaFLIM

Sample-free auto-alignment • CalEx • VarPSF

Feature highlights

LumiFinder:
Automated detection and measurement of immobilised emitters

FRETcompass:
Automated determination of correction factors used in single molecule FRET experiments based on the benchmark study published in 2018

InstaFCS:
Online FCS fitting with automatic suggestions for model and parameters

InstaFLIM:

  • Provides first suggestions for FLIM species separation without user interaction
  • Suggested parameters can be fine-tuned
  • Simultaneous TCSPC and phasor analysis options for ROI determination

Sample-free auto-alignment:
Ensures optimal performance for every measurement, even the most challenging ones

CalEx:

  • Excitation laser power calibration allows setting and displaying excitation intensity in μW
  • No external power meter required

VarPSF:
Switch between diffraction limited and larger observation volume

Luminosa software Laser settings FLIM screenshot

System Highlights

Luminosa Single Photon Counting Confocal Microscope

  • Software-controlled confocal system based on an inverted microscope
  • Versatile excitation system with laser wavelengths from 375 to 1064 nm
  • VarPSF: fine-tuned observation volume for FCS and single molecule FRET experiments
  • Motorized positioning table for “tiling and stitching” in transmission and FLIM mode
  • Scanning options: FLIMBee galvo scanner and piezo objective scanning
  • Up to 6 truly parallel detection channels with SPAD and/or Hybrid PMTs
  • < 700 ps dead time per channel and 5 ps time bins
  • One-click auto-alignment for consistent optimal performance
  • Fast results with minimal user interaction thanks to GPU accelerated algorithms and context-based workflows for FCS, FLIM, and single molecule detection

Download brochure and datasheet

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Next Events with Luminosa

We will demonstrate Luminosa at the following events. Contact us for options to meet our experts there.

Cell Bio 2022, an ASCB / EMBO Meeting
December 3 - 7, 2022 | Washington, D.C., USA

Cell Bio 2022, the joint meeting of the American Society for Cell Biology (ASCB) and European Molecular Biology Organization (EMBO), will showcase a diverse global community of the brightest minds in cell biology in person. The unique meeting focuses on cell biology as the fundamental basis of biology as well as sessions on emerging interdisciplinary topics.

Cell Bio

SPIE Photonics West 2023
January 28 - February 2, 2023 | San Francisco, CA, USA

The world’s largest photonics technologies event where scientists discuss research in biomedical optics, biophotonics, industrial lasers, optoelectronics, microfabrication, MOEMS-MEMS, displays, quantum technologies, including quantum 2.0, and other similar topics with a focused, engaged audience.

SPIE Photonics West 2023

BPS Biophysical Society Annual Meeting 2023
February 18-22, 2023 | San Diego, CA, USA

The Biophysical Society Annual Meeting continues its long-held reputation for bringing together leading scientists from the all over the world who work at the interface of the life, physical, and computational sciences. The dynamic five-day meeting provides attendees with opportunities to share their latest unpublished findings and learn the newest emerging techniques and applications.

BPS Biophysical Society Annual Meeting 2023

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