Comfortable and Reliable – Molecular Imaging with bh’s Legendary Lifetime Precision.

This turnkey FLIM microscope is configured to give you the best results out of the box. Equipped with compact and all fiber coupled picosecond pulsed diode-lasers for all existing molecular probes and FRET donors from 375 nm to 785 nm, it provides the results you need, both at university and in the medical lab, at an unbeatable price.

Highlights

Two bh picosecond diode lasers : 60 ps - 120 ps FWHM^(*), 20 MHz, 50 MHz, 80 MHz, CW
High photon rate measurement, IRF ~100 ps^(*): 80 MHz continuous, 120 MHz peak. 4 ps time-channels, ~17 ps jitter. Parallel dual channel acquisition
Wide variety of molecular probes
For any FRET pair, e.g. GFP/YFP, Cerulean/Venus, etc.

^(*)depending on wavelength

Description

Molecular Imaging: What Happens Inside a Cell?

How much Calcium, Sodium, Magnesium is there? And where is it? What is the pH and how is it distributed over the cell? Are two proteins connected to each other? If so, how many of them are connected in pairs and how many are unconnected? Are these parameters changing over the area of the cell? What is the voltage over the cell membrane? These are questions addressed by molecular imaging. There are 'molecular probes' for almost any conceivable cell parameter. These probes are molecules that change their configuration in dependence of their molecular environment. These changes cause changes in the quantum yield and in the fluorescence lifetime. Another probing mechanism is FRET (Förster Resonance Energy transfer). FRET is sensitive to the distance between a donor and an acceptor. Changes in protein binding and protein configuration change the efficiency of FRET, which, in turn, causes changes in the fluorescence lifetime of the donor. Measure the lifetime changes by FLIM, and you know what happens in the cell, and where exactly it happens. Measuring cell parameters by fluorescence lifetime is more reliable than measuring them by fluorescence intesity: In contrast to the intensity the fluorescence lifetime does not depend on the concentration of the probe, the laser intensity, filter characteristics, and other instrumental parameters.

Molecular imaging by FLIM requires accurate mapping of the fluorescence lifetime in cells and tissues. The FLIM technique should be highly sensitive, deliver absolute values for the fluorescence lifetime at a minimum of recorded photons, and reliably resolve multi-exponential decay profiles. Moreover, a sufficient number of excitation wavelengths should be available to provide flexibility in the choice of probes, and beamsplitters and filters should allow the user to select any reasonable detection wavelength. This exactly is the application the bh Molecular FLIM System has been designed for.

Basic Features:

Fully motorized sample stage
4D Z-stack FLIM Video rate recording ready – Express FLIM^(*)
Seamless analysis integration including Phasor Plot +, image segmentation by lifetime, FCS analysis
Area and line scanning modes, as well as true point measurements for correlation measurements.
Fast laser scanning unit for optimal image acquisition
Basic detection filter kit, chosen for your experiment

^(*)Depends on choice of time-tagging unit

Options:

Choice of laser wavelengths
Choice of fast and efficient Becker & Hickl hybrid single photon detectors
Incubator with optional environment control for live cell work.

Specifications

Selected Specifications
Principle	Fast galvo-mirror laser-scanning, de-scanned confocal detection (DC), and bh's multi-dimensional TCSPC FLIM technique
Excitation	ps pulsed lasers, fiber coupled
Scan Rate, Pixel dwell Time	Down to approx. 1 μs/pixel
General Operation Modes	TCSPC FLIM: 2 (multi-) spectral or polarisation channels Time-series, Z-Stack, mosaic (x,y, z, temporal) Excitation-wavelength multiplexed FLITS (fluorescence lifetime-transient scanning) PLIM (phosphorescence lifetime imaging) simultaneously with FLIM, Photon correlation: FCS, FCCS, gated FCS single-point fluorescence and phosphorescence decay
Scan Head
Optical Principle	Fast galvo-mirror laser-scanning
Laser Inputs	Two independent inputs, fiber coupled
Optical Laser Power Control	Continuous ND filter wheel control
Laser Input Requirements	Collimated free-beam, or fiber coupled with 12 mm diameter collimator. 1 to 2 mm beam diameter
Laser Power Regulation, Optical	Continuously variable via neutral-density filter wheels
Outputs to Detectors	Two outputs, detectors are directly attached
Main Beamsplitter Versions	Alignment-free exchangeable dichroics: Longpass, multi-band, wideband, and multiphoton options available
Secondary Beamsplitter Wheel	Three dichroic beamsplitters, polarising beamsplitter, 100% to channel 1, 100% to channel 2
Pinholes	Independent pinhole wheel for each channel
Pinhole Alignment	Electronical, via piezo microstage
Pinhole Size	11 pinholes, from about 0.5 to 10 AU
Emission Filters	Two filter sliders per channel in series
Connection to Microscope	Adapter to left side port or port on top of microscope
Scan Control
Principle	Hardware controlled precision laser-scanning with fast flyback for rapid acquisition
Frame Size	Frame scan 16 x 16 to 4096 x 4096 pixels, line scan 16 to 4096 pixels
X Scan	Continuous or pixel-by-pixel,
Y Scan	Line-by-line
Electrical Laser Power Control	Software control of a laser with analogue modulation input
Laser Multiplexing	Frame-, line-, pixel-, and intra-pixel. Requires software control of laser power.
Beam Blanking	During flyback and when scan is stopped. Requires software control of laser power.
Frame Rate / Scan Speed	Automatic selection of fastest rate or manual selection
Scan Area Definition	Interactive scan region selection, hardware zoom + offsets.
Fast Preview Function	Yes
Beam Park Function	Yes, interactive measurement point selection.
TCSPC System
TCSPC / FLIM Modules	SPC-180NX	SPC-QC-104
Number of Parallel TCSPC / FLIM Channels	Typ. 2, max. 4	Typ. 2, max. 3
Electrical Time Resolution	1.6 ps RMS / 3.5 ps FWHM	16 ps RMS / <39 ps FWHM
Timing Precision /	1.1 ps	11 ps
Minimum Time Channel Width	405 fs	4 ps
Saturated Count Rate	12 MHz	40 MHz, shared among active channels.
Synchronisation with Laser Multiplexing	Up to 4 laser wavelengths
Recording of Multi-Wavelength Data	Simultaneous in 16 channels, via routing function
Experiment Trigger Function	TTL, used for Z-Stack FLIM and microscope-controlled time-series
Operation Modes of TCSPC System	Hardware pre-analysed imaging Photon event stream (FIFO) imaging Point measurements for correlation, long timescale intensity (MCS) Mosaic imaging, time-series imaging multi-detector operation, laser multiplexing operation, cycle and repeat function, autosave function
Software
Data Acquisition Software	bh SPCM, bh LabVIEW for integration of external devices
Scanner Control Software	Integrated in SPCM, bh LabVIEW for integration of external devices
Operation System	Windows 10 / 11 64 bit
Data Analysis Software	bh SPCImage NG
Principle of Data Analysis	MLE fit (GPU assisted processing)
Model of Functions	IRF convoluted single, double, or triple component exponential decay Optional consideration of incomplete decay Shifted component model
IRF Modelling	Synthetic IRF function fit to decay data, auto-extraction of IRF from data, or measured IRF
Excitation Sources
Confocal FLIM	One to four ps diode lasers
Available Wavelength	375 nm to 785 nm
Repetition Rate	20, 50, 80 MHz and CW
Pulse Width	40 ps to 100 ps
Optional	Multiphoton FLIM, free-beam or fiber coupled femtosecond pulsed lasers, single wavelength or tuneable
Detectors
Confocal Detectors	Coupled directly to scan head
Optional	NDD detectors, coupled directly to back port of microscope
Standard Detector	HPM-100-40 hybrid detector with GaAsP cathode, 250 to 720 nm, best for use with ns lifetime dyes
Optional	HPM-100-06 detector with <20 ps FWHM IRF width, 220 to 650 nm, best for ps lifetime autofluorescence studies
Optional	HPM-100-50 detector, 400 to 900 nm, best for long wavelength fluorescence
Optional	MW-FLIM GaAsP multiwavelength detector

Downloads

Documents

The bh TCSPC Handbook
10th edition, September 2023

View

Datasheets

Molecular-Interaction-Imager.pdf

Molecular Imaging by FLIM

Principle

When a molecule is in the excited state it can return to the ground state by emitting a photon, by internally converting the energy into heat, or by exchanging energy with another molecule. Consequently, the time the molecule stays in the excited state - or the fluorescence lifetime - depends on the molecular environment of a fluorophore. Accurate measurement of the fluorescence lifetimes or, more exactly, the fluorescence decay functions in the pixels of a FLIM image can therefore be used to obtain reliable information on biological systems. An inherent advantage of FLIM in this respect is that the fluorescence lifetime, within reasonable limits, does not depend of the concentration of the fluorophore, the laser power, the detector gain, or other experimental or instrumental details. Measurement of molecular parameters by FLIM is therefore more reliable than by intensity measurement.

Requirements to a Molecular FLIM Technique

Molecular Imaging experiments have, of course, to be performed in live cells or live tissue. This implies that the photon rates available from the sample are very limited. High sensitivity and high photon efficiency, i.e. maximum signal-to-noise ratio for a limited number of detected photons, are therefore important. Other requirements are high time resolution, high timing stability, and capability to record and resolve multi-exponential decay profiles. Helpful are also optical sectioning capability, absence of lateral crosstalk, and the capability to record in several wavelength intervals simultaneously. Exactly these requirements are inherent features of the bh TCSPC FLIM technique and the bh TCSPC FLIM systems.

Molecular Parameters Measured by FLIM

In most instances molecular imaging is performed by incubating or transfecting the samples with a fluorophore or several fluorophores that show the desired dependence of the molecular parameter of interest. There is a wide variety of such 'molecular probes', please see W. Becker (ed.), Advanced TCSPC Applications, Springer 2015.

Molecular parameters that can be favourably measured by FLIM are pH, concentrations of Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, Hg⁺⁺, Cl^-, Glucose, membrane potential, local viscosity, local temperature, and a wide variety of other cell parameters for which molecular probes are available.

FRET Imaging

Förster Resonance Energy Transfer, or FRET, is an interaction of two molecules in which the emission band of one molecule overlaps the absorption band of the other. In this case the energy from the first molecule, the donor, can transfer into the second one, the acceptor. FRET can result in an extremely efficient quenching of the donor fluorescence and, consequently, in a considerable decrease of the donor lifetime.

The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance. Therefore it is noticeable only at distances shorter than 10 nm. FRET is used as a tool to investigate protein-protein interaction and protein conformation. In the first case, different proteins are labelled with the donor and the acceptor, and FRET is used as an indicator of the binding between these proteins. In the second case, donor and acceptor are attached to the same protein. The intensity of FRET is then an indicator of the protein conformation. Correct FRET measurement requires double-exponential decay analysis and thus cannot be performed with FLIM systems which do not record the full decay curves, please see 'A Common Mistake in Lifetime-Based FRET Measurement'. Double-exponential FTET measurement is not only free of external calibration, it also delivers the classic FRET efficiency, the FRET efficiency of the interacting donor, the fraction of interacting donor, and the donor-acceptor distance from a single FLIM measurement. Please see 'Double-Exponential FLIM-FRET Approach is Free of Calibration'.

Molecular Imaging System