Microscopy > Components > Olympus, FCS and FLIM

Olympus, FCS and FLIM Upgrade

The FV1000 and FV1200 confocal microscopes 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).

The Olympus 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 Olympus confocal head is connected to the unit.
Detectors Options of:
  • Internal detectors
  • 2-detector unit coupled to the fiber optic port of the FV1000/FV1200
  • 2-detectors coupled to the confocal head of the FV1000/FV1200
Detectors fast PMTs or SPADs.
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) Module 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
  • Scanning FCS
  • Number & Brightness (N&B)
Imaging Module Measurements
  • Single-point (intensity, polarization, lifetime)
  • Single plan and z-stack (polarization images, ratiometric, FLIM)
Superresolution Module
  • Particle tracking
  • Nanoimaging
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
  • Ti:Sapphire 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
  • Internal
    • (original) PMTs
  • External
    • GaAs PMT (Model H7422P)
    • Hybrid PMT (Model R10467U)
CLK
  • Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
  • 4 µs/pixel
Unit Control
  • USB
Computer
  • 3 GHz, 8GB RAM, 200 GB hard drive, 27" monitor
  • Windows 7, 64-bit

The upgrade can be carried out following one of two modalities:

a) By adding two external PMTs (2-detector unit); the fluorescence signal is collected through the fiber port of the microscope and diverted to the 2-detector unit (Figure 1)
b) Utilize the internal PMTs of the FV1000 (Figure 2)

Figure 1. Schematics of the upgrade package for the Olympus 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 Olympus FV1000. The part to the right includes the instrument components (PC, control electronics, scanner and laser launcher). The signal is collected from the internal detectors of the microscope and diverted to FastFLIM unit (left part) of the schematics which includes the components provided by ISS with the upgrade package.