Microscopy > Components > Zeiss, FCS and FLIM

Zeiss, FCS and FLIM

The Zeiss LSM710, LSM780, LSM880 confocal microscopes series can be easily upgraded to Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) capability using photon counting detection. Data are acquired using one of two modalities:

  • the FastFLIM (digital frequency domain); or
  • the TCSPC (time-domain).

FastFLIM (proprietary technology) is the new digital frequency domain (DFD) approach to FLIM measurements. Its advantages, when compared to the TCSPC are twofold:

a) the short time required for data acquisition; and
b) the higher sensitivity of the technique (due to the 100% duty-cycle).

Alternatively, FLIM data can be acquired using the TCSPC method, or both methodologies can be implemented on the same instrument.

The Zeiss, FCS and FLIM upgrade package includes the following items:

FastFLIM Unit or TCSPC Unit It accepts the output (via BNC) from up to four PMTs of the confocal unit. The synchronization signal from the Zeiss confocal head is connected to the unit.
Detectors 2-detectors coupled to the descanned port confocal head of the LSM microscope; or to the non-descanned port (for multiphoton instruments)
Detectors fast PMTs
Laser Launcher Available for 3-, 4- and 6-lasers. The lasers beams are superimposed and the output of the laser launcher is connected to the microscope by using a fiber optic.
Computer Running VistaVision by ISS A separate computer, with a 27" flat monitor, 2556x1440 resolution

Acquisition and Analysis Software
Fluorescence Fluctuations Spectroscopy (FFS) Measurements
  • Fluorescence Correlation Spectroscopy (auto- and cross-correlation)
  • Photon Counting Histogram (PCH)
  • FFS measurements at target XYZ locations in an image
  • FLCS, Fluorescence Lifetime Correlation Spectroscopy
  • Number & Brightness (N&B)
Imaging Module Measurements
  • Single-point (intensity, polarization, lifetime)
  • Single plan and z-stack (polarization images, Ratiometric, FLIM)
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
FLIM images time-domain (single plane and z-stack)
  • Acquired in time-correlated single photon counting (TCSPC)
Single Molecule Module
  • Burst Analysis
  • FRET and Correlation Methods
  • PIE-FRET Methods
Light Sources
  • Laser diodes: 370, 405, 440, 473, 488, 635 nm
  • Single-photon pulsed lasers
  • Multi-photon lasers
Laser Launcher
  • Models for 3, 4, 6 laser diodes. Light is delivered to the microscope through a single-mode fiber optic.
Input Channels
  • Two
Detectors
  • GaAs PMT (Model H7422P)
  • Hybrid PMTs (Model R10467U)
CLK
  • Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
  • 6 µs/pixel
Unit Control
  • USB
Computer
  • 3 GHz, 8GB RAM, 200 GB hard drive, 27" monitor
  • Windows 10, 64-bit

Figure 1 below is a schematic of the units making the LSM 710/LSM 780 instrument and the upgrade package provided by ISS.

Figure 1. Schematics of the upgrade package for the Zeiss LSM instrument. The part to the right includes the instrument components (PC, control electronics, scanner and laser launcher). The left part of the schematics includes the components provided by ISS with the upgrade package.

Below is a list of selected publications featuring the Zeiss, FCS and FLIM upgrade from ISS.

Millisecond Spatiotemporal Dynamics of FRET Biosensors by the Pair Correlation Function and the Phasor Approach to FLIM
Hinde, E., Digman, M.A., Hahn, K.M., Gratton, E.
PNAS, 2013, 110, 135-140.
Metabolic Trajectory of Cellular Differentiation in Small Intestine by Phasor Fluorescence Lifetime Microscopy of NADH
Stringari, C., Edwards, R.A., Pate, K.T., Waterman, M.L., Donovan, P.J., Gratton, E.
Scientific Reports, 2012, 2, 568.
Biosensor FRET Detection by the Phasor Approach to Fluorescence Lifetime Imaging Microscopy (FLIM)
Hinde, E., Digman, M.A., Welch, C., Hahn, K.M., Gratton, E.
Microsc. Res. Tech., 2012, 75, 271-281.
Label-free Separation of Human Embryonic Stem Cells and Their Differentiating Progenies by Phasor Fluorescence Lifetime Microscopy
Stringari, C., Sierra, R., Donovan, P.J., Gratton, E.
J. of Biomedical Optics, 2012, 17(4), 046012.