Microscopy > Components > Leica, FCS and FLIM


Leica, FCS and FLIM

The Leica SP8 confocal microscope 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 Leica, 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 Leica confocal head is connected to the unit.
Detectors HyD detectors
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

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
  • Supercontinuum laser
Laser Launcher
  • Models for 3, 4, 6 laser diodes. Light is delivered to the microscope through a single-mode fiber optic.
Input Channels
  • Two (up to four)
Detectors
  • Leica HyD detectors
CLK
  • Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
  • 6 µs/pixel
Unit Control
  • USB
Computer
  • 3 GHz, 8GB RAM, 27" monitor
  • Windows 10, 64-bit

Figure 1. Schematics of the upgrade package for the Leica Frequency-Domain Confocal Microscope. 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.

Figure 2. Schematics of the upgrade package for the Leica Time-Domain Confocal Microscope. 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.