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The compact instrument for quantitative cell biology with single-molecule detection

Q2 is a laser scanning nanoscope (LSN) that incorporates several measurement modalities for experimental quantitative biology and material sciences applications requiring the single molecule detection sensitivity.

Key Features of Q2 Laser Scanning Nanoscope:

  • Single- and multi-photon excitation on three separate input ports
  • Two channel acquisition
  • One, computer-controlled-aperture, pinhole
  • Fast scanning mirrors
  • Powered by VistaVision, a user-friendly software package for the acquisition of confocal images, FLIM/ FRET, RICS, and FFS (FCS, PCH, scanning FCS, N&B)

Excitation Modality
Three separate input ports allow for simultaneous alignment of one-photon excitation (laser diodes and supercontinuum laser) or multi-photon measurements (fiber laser, Ti:Sapphire laser).

Microscopes
Q2 can be interfaced with most commercial epifluorescence and upright microscopes.

Detectors
GaAs detectors, hybrid photomultiplier tubes, avalanche photodiodes.

Specifications for Q2

Microscope and Coupling
  • Olympus, Nikon, Zeiss and Leica
  • Inverted and upright
  • Left side port and back port
1p Excitation1
  • ISS laser launcher (models for 3, 4, and 6 laser diodes), wavelengths available from 375 nm to 640 nm; pulse interleave excitation (PIE)
  • Super-coninuum lasers, wavelength from 400 to 700 nm
2p Excitation1
  • Ultra fast femtosecond pulse Ti:Sapphire lasers
  • Ultra fast femtosecond pulse fiber lasers
Data Acquisition Unit
  • FastFLIM (Digital Frequency Domain FLIM)
    • Supports all above listed applications except particle tracking and nanoimaging
  • TCSPC card (Time Domain FLIM)
Detectors
  • GaAs PMT (Hamamatsu H7421 and H7422P models)
  • Hybrid PMTs (Hamamatsu R10467U models)
  • APDs
Positioning Controls2
  • ISS 3-axis control unit
  • ISS XY galvo scanning mirrors control unit
  • ISS Z-piezo control unit
  • Microscope built-in focusing control module
  • Automatic XY stages (ASI, Prior)
Software
  • VistaVision (Windows 7, 64-bit)
Computer & Monitor
  • 3GHz Processor, 8GB RAM, 200GB hard drive for 64-bit VistaVision
  • 27" 1280 x 1024 resolution monitor
  • Windows 7 64-bit Professional
Power Requirements
  • Universal power input: 110-240 V, 50/60 Hz, 400 VAC
  1. Q2 was fully evaluated and validated for using ISS laser launcher, Fianium SC-400 laser and Toptica FemtoFiber Pro 2p laser.
  2. VistaVision provides utilities for measurements in spectroscopy mode (at a single point), raster or orbit scan mode (2D XY), optical sectioning mode (3D), time-lapse mode, stage scan mode for multi wells, or a combination of them.

Schematic Diagram of Q2

Measurement Examples for Q2

Confocal Imaging

Confocal image of Convallaria (Lily of the valley). Laser excitation wavelength is 561nm (supercontinuum laser). The fluorescence is collected through a bandpass filter centered at 600 nm with 37 nm bandpass. The objective is an Olympus 20X water immersion (NA = 0.54). The pinhole aperture is 2 Airy units. The image size is 1024 x 1024 pixels (200 µm x 200 µm).

Fluorescence Lifetime Imaging (FLIM)

The sample is Convallaria (Lily of the valley). The laser excitation wavelength is 488nm from a supercontinuum laser (40 MHz repetition rate, 6 ps pulsewidth). The emission on Ch1 is acquired through a 600/37 nm filter; on Ch2 the emission is acquired through a 525/50nm filter. The objective is Olympus 60X (water immersion; NA = 1.35). The pinhole is 1 Airy unit.

2p FLIM-FRET

mTurquoise-5aa-Amber (T5A) expressed in fixed cells (donor-alone control) mTurquoise-5aa-Venus (T5V) expressed in fixed cells (FRET standard) Laser excitation: 780nm, 80MHz; Emission: donor channel: 475/35nm; Objective: Olympus 60X / 1.35NA;

2p FLIM imaging of HE stained pig intestine tissue

Laser excitation: 780nm, 80MHz; Emission: 500 - 650 nm; Objective: Olympus 20X / 0.54NA; Image Size:1024x1024 (200 µm x 200 µm)

2p FCS

Fluorescein in HPLC water PH 7.4.
FFS comprises a whole family of application tools that reveal the inner molecular dynamics upon the detection of fluctuations of molecules due to thermal motion. They include
  • FCS, Fluorescence correlation spectroscopy
  • FCCS Fluorescence cross-correlation spectroscopy
  • PCH, photon counting histogram

Measurements for Q2

  • Intensity and Lifetime Imaging (single plane and z-stack)
    • 1p or 2p confocal images
    • FLIM in frequency-domain or in TCSPC
    • Phosphorescence Lifetime Imaging (PLIM)
    • Polarization images
  • Single Point Measurements
    • Intensity, polarization, kinetics, lifetime
  • Fluorescence Fluctuations Spectroscopy (FFS)
    • Fluorescence Correlation Spectroscopy (FCS)
    • Fluorescence Cross-Correlation Spectroscopy (FCCS)
    • Photon Counting Histogram (PCH)
    • Fluorescence Lifetime Correlation Spectroscopy (FLCS)
    • Scanning FCS
    • Number & Brightness (N&B)
    • RICS (raster imaging correlation spectroscopy)
  • Superresolution
    • 3D particle tracking trajectories
    • Nanoimaging reconstruction with 20 nm resolution
  • Single Molecule Analysis
    • Burst Analysis
    • FRET and Correlation methods
    • PIE-FRET methods