Microscopy > Components > Nikon, FCS and FLIM

Nikon, FCS and FLIM

The Nikon A1 and C2 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 Nikon, 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 Nikon 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

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 nm - 1000 nm
  • Single-photon pulsed lasers
  • Multi-photon lasers
Laser Launcher
  • Models for 3-, 4-, 6-laser. Light is delivered to the microscope through a single-mode fiber optic.
Input Channels
  • Two
  • GaAs PMT (Model H7422P)
  • Hybrid PMTs (Model R10467U)
  • Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
  • 6 µs/pixel
Unit Control
  • USB
  • 3 GHz, 16GB RAM, 27" monitor 2556 x 1440 resolution
  • Windows 10, 64-bit

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

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

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

Photoluminescence Enhancement and High Accuracy Patterning of Lead Halide Perovskite Single Crystals by MeV Ion Beam Irradiation
Palei, M., Motapothulab, M, Ray A., Abdelhadya, A.L., Lanzanod, L., Prato, M., Panda, J.K., Scarpellini, A., Pellegrinif, V., Primetzhofer, D., Petralanda, U., Manna, L., Dang, Z.
J. Mater. Chem. C, 2020,8, 9923-9930
Chromatin nanoscale compaction in live cells visualized by acceptor-to-donor ratio corrected Förster resonance energy transfer between DNA dyes.
Pelicci, S., Diaspro, A., Lanzanò, L.
J Biophotonics. 2019 Jul 31:e201900164. doi: 10.1002/jbio.201900164. [Epub ahead of print]