Comfortable, Label-Free and Near-Instantaneous Two-Photon Metabolic Imaging with B&H’s Legendary Lifetime Precision.

This turnkey FLIM microscope is configured to give you the best results out of the box. Equipped with TOPTICA’s newest turn-key femtosecond pulsed lasers for NADH and FAD excitation it provides the results you need both at university and in the clinic, at an unbeatable price.

Highlights

2x TOPTICA FemtoFibre Ultra (780 nm & 920 nm): ~1 W, <100 fs, 80 MHz, AOM + GDD; Wavelength multiplexed excitation
Ultra-precise lifetime measurements, IRF ~20 ps: Time-channels down to 213 fs, 3 ps jitter. Parallel dual channel acquisition
Detection max. 250 nm – 720 nm: NADH: 420 nm – 480 nm, FAD: 500 nm – 550 nm

Description

Healthy Cells or Tumor? Determine the Metabolic State by Fluorescence of NAD(P)H and FAD

The bh Multiphoton Metabolic FLIM System determines the state of the metabolism of live cells and tissues by precision recording of NAD(P)H and FAD decay functions. Different than in early NADH experiments, the state of the tissue is not characterised by the lifetimes of NAD(P)H or FAD but by the amplitudes of the components of their double-exponential decay profiles. Near-perfect separation of the two fluorescence signals by laser multiplexing adds to the reliability of the results. Multiphoton excitation by femtosecond fibre lasers reaches deep tissue layers and is friendly to live cells. In combination with advanced features of bh's SPCImage NG data analysis quantitative information of the metabolic state is obtained.

In addition to single metabolic-state images, the system is able to record z stacks of tissue samples, and time series of the metabolic parameters over seconds, hours or even days. Although the system is optimised for metabolic FLIM it is not restricted to this application. With its two excitation wavelengths it also records FLIM data from a wide variety of molecular probes, delivering high quality pH images, calcium images, chloride images, FRET images, and SHG images.

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 different single photon hybrid detectors
Incubator with optional environment control for live cell work.

Specifications

Selected Specifications
Principle	Fast galvo-mirror laser-scanning excitation, non-descanned detection (NDD), and bh's multi-dimensional TCSPC FLIM technique
Excitation	fs pulsed laser, free-beam or 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 excitation
Laser Inputs	Two independent inputs, fiber coupled or free-beam
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
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 or control of a separate AOM.
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
Multiphoton FLIM	Free-beam or fiber coupled femtosecond pulsed lasers, single wavelength or tuneable
Optional	Confocal FLIM, one additional BDS-SM ps diode laser can be coupled in the system.
Detectors
NDD Detectors	Coupled directly to back port of microscope
Optional	Confocal detectors, coupled directly to scan head
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

Multiphoton-Metabolic-Imager.pdf

The bh Metabolic Imaging Technique

Spectroscopic Background

NAD(P)H (nicotinamide adenine (pyridine) dinucleotide) and FAD (flavin adenine dinucleotide) are coenzymes involved in the cell metabolism. Both NAD(P)H and FAD are fluorescent. FAD and, especially, NAD(P)H are unique in the sense that their fluorescence intensities and fluorescence decay functions bear direct information on the metabolic state of the cells: The fluorescence lifetimes of NAD(P)H and FAD depend on the binding to proteins. Unbound NAD(P)H has a fluorescence lifetime of about 0.3 to 0.4 ns. Bound NAD(P)H has a lifetime of about 1.2 ns. For FAD the effect of binding is opposite: Bound FAD has a lifetime of a few 100 ps, unbound FAD of a few ns. Interestingly, the bound/unbound ratios of NAD(P)H and FAD change with the type of the metabolism. Cells that run preferentially oxidative phosphorylation (healthy cells) have more bound NAD(P)H and FAD, cells running preferentially Glycolysis (tumor cells) have more unbound NAD(P)H and FAD. Please see page Applications, Metabolic Imaging and 'The bh TCSPC Handbook', chapter Label-Free FLIM of Cells and Tissues.

Earlier Attempts to Determine the Metabolic State

For almost two decades it has been attempted to derive metabolic information from simple fluorescence lifetimes of cells or tissues. Please see The bh TCSPC Handbook for references. These attempts failed for two reasons. First, the fluorescence signal of NAD(P)H and FAD were not clearly separated. However, metabolism-induced lifetime changes for NAD(P)H and FAD go in opposite directions. The resulting changes in the net lifetime are therefore unpredictable, and cannot be used as an indicator of changes in the metabolic state. Second, the lifetimes of the decay components depend on cell parameters which are unrelated to the metabolic state. Therefore, the net fluorescence lifetime of cells and tissues is not a quantitative indicator of the metabolic state.

bh's Metabolic FLIM Technique

bh's metabolic FLIM technique differs from the earlier approaches by

cleanly separating the fluorescence signals of NAD(P)H and FAD. Separation is obtained by combining excitation-wavelength multiplexing on the excitation side and spectral filtering on the detection side.
using the amplitudes of the decay components of NAD(P)H and FAD decay curves instead of the lifetimes. The amplitudes of the decay components, a1 and a2, represent the fractions of bound and unbound NAD(P)H and FAD. They are not influenced by environment-dependent component lifetimes.

It turns out that the component amplitudes (or the ratios of the amplitudes) quantitatively represent the metabolic state. The results are largely independent of the cell type. Healthy cells have an a1 below 0.7, tumor cells an a1 above 0.7. Please see 'The bh TCSPC Handbook', chapter Label-Free FLIM of Cells and Tissues.

Making Metabolic FLIM Work

Extracting the amplitudes of the decay components requires accurate recording of the decay data in the pixels of the image, and reliable FLIM data analysis. Both problems have been solved by bh. The timing stability of the bh TCSPC FLIM modules is better than a few picoseconds, and the decay curves are resolved into a large number of time bins. (We recommend 1024 for metabolic FLIM) The HPM-100 hybrid detectors have a clean IRF without bumps and secondary pulses. The detectors are free of afterpulsing, resulting in an exceptionally low recording background. These features greatly improve the accuracy of FLIM data analysis. Data analysis is performed by SPCImage NG. The MLE fit of SPCImage yields highly accurate component lifetimes and amplitudes even at low photon numbers. The accuracy is further enhanced by intelligent binning, image segmentation functions, and the availability of a Global Fit. An example of an NADH a1 image is shown below.

Two-Photon Excitation

It should not be concealed that metabolic FLIM can also be performed also with the standard DCS-120 confocal FLIM System. The only requirement is that it has the right lasers installed. Please see (Appnote). However, there is a problem. NAD(P)H needs excitation at 375 nm or shorter. A wavelength this short does not go far into biological tissue. This restricts imaging to cells or single-cell layers. The bh Metabolic FLIM Imager therefore uses two-photon excitation. The excitation wavelengths is then 780 nm for NAD(P)H and 920 nm for FAD. These wavelengths penetrate as far as 100 micrometers into tissue. Fluorescence is detected through a non-descanned detection beam path so that photons are efficiently detected even if they get scattered on the way out of the tissue.

Metabolic Imaging System