Laboratory for Biophysics, Institute of Physics Belgrade

Laboratory / facilities & equipment

Nonlinear Microscopy setup

Our homemade experimental setup for nonlinear laser scanning microscopy (NLSM) have both, reflection and transmission detection arms, and consists of following imaging modalities:

  • Two Photon Excitation Fluroescence - TPEF
    • excitation: 720-930nm and 1040nm
    • detection: selection of broad and narrow band filters in VIS region
  • Second Harmonic Generation - SHG
    • polarization resolved
    • excitation: 840nm, 900nm, 1040nm
    • detection: 420nm, 450nm and 520nm
  • Third Harmonic Generation - THG
    • excitation: 1040nm
    • detection: 347nm

In addition, there is possibility for sample processing: cutting, inscription of arbitrary patterns, selective photo bleaching.

More about the NLSM setup


Laser sources used for excitation in NLSM:

  • Kerr lens mode locked Ti:Sa laser
    • wavelength: 700-1000nm; pulse duration: 160 fs; repetition rate: 76 MHz; average power: 2 W
  • Pulse picker for Ti:Sa laser
    • repetition rate: single shot to 4 MHz
  • Regenerative amplifier for Ti:Sa laser
    • pulse energy 6 µJ, repetition rate: 250 kHz; wavelength 800 nm; pulse duration: 200 fs
  • SESAM mode locked Yb:KGW laser
    • wavelength: 1040nm; pulse duration: 200fs; repetition rate: 83MHz; average power: 2W
More about the lasers

Optical autocorrelator

Our homemade optical autocorrelator enables measurements of ultrashort pulse durations in the range 10 fs - 100 ps.

More about the autocorrelator

Cooled CCD spectrometer

Portable, fiber coupled, USB spectrometer, coupled with NLSM setup, for very weak fluorescence signal measurements and spectral analysis upon the excitation in the arbitrary chosen point on the image.

More about the spectrometer

In progress

We are currently developing two more microscopic techniques:

  1. Fluorescence correlation spectroscopy (FCS) - quantitative technique with single molecule sensitivity (through Hemmaginero project)

  2. Further reading of FCS:
  3. Structured Illumination Microscopy (SIM) - imaging technique with super resolution (through Move For The Science - #PokreniNauku project)

  4. Further readings on SIM:

Nonlinear Microscopy setup

General. TPEF and SHG modalities are fully operational in back reflection arm, while the transmission arm for both THG and SHG is under development through the HEMMAGINERO project. Also, it is possible to perform polarization SHG imaging in backreflection with control of both, polarization of incident laser beam and polarization of SHG signal (analyzer).

Two Photon Excitation Fluroescence - TPEF is the most common effect/signal that is detected in NLSM. It is analogous to the single photon excitation that is utilized in confocal and epifluroescent microscopy, but in TPEF the fluorescent molecule is excited by absorbing two photons simultaneously. Upon the two photon excitation, the fluorescence occurs in the same manner as in the single photon excitation.

NLSM set up with lasers

Objective lenses

Type Magnification NA (immersion) Working distance Max field of view
Zeiss 12.5 0.3 551
Zeiss Plan-APOCHROMAT 20 0.8 0.55 233.9
Zeiss EC Plan-NEOFLUAR 40 1.3 (oil) 0.21 115.4
Zeiss 40 0.65
Zeiss W Plan-Apochromat 2.5 40 1.0 (phys) 2.5 114.5
Olympus 100 1.2 (oil) 49.8
Zeiss 100 1.25 (oil)

* (for 3.75x beam expander)

Sample mounting and positioning. The sample holder is for standard microscope deck glass dimensions on manual XY (lateral) table, whilst Z-axis (axial) is motorized with step size 0.3 um.

Detection and acquisition. For detection in back reflection arm an analogue PMT is used, while in transmission arm a PMT with photon counting module is planned. Maximal recording speed is approximately 1s for 1024x4024 pixels image without averaging, which is limited by the sample rate (1.2 Msamples/s) of the NI-639 acquisition card.

Sample processing. In addition to imaging mode, there is an processing mode available which enables controllable sample cutting and selective photo bleaching with arbitrary patterns (images). It is possible to control the speed of the focal spot across the sample and the dwell time in one point. The galvo scanning mirrors are synchronized with shutter, so the beam does not burn the sample between the slices.

Arbitrary patterns inscribed in the butterfly wing scales by selective photo bleaching

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Mira 900, Coherent Inc - Kerr lens mode locked Ti:Sa laser
  • wavelength - tunable 700-1000nm (from 930-1000nm nitrogen purging is necessary)
  • pulse duration - 160fs
  • repetition rate - 76MHz
  • maximal average power (@800nm) - 2W
  • powered by VERDI V10 - CW frequency doubled diode pumped Nd:YVO4 laser, wavelength 532 nm, max. power 10 W

Pulse picker

The acousto-optic Bragg cell single-pass pulse picker for pulse train from Mira 900
  • repetition rate - single shot to 4 MHz
  • Bragg cell material: SiO2
  • diffraction efficiency - 50%

Regenerative amplifier

RegA 9000, Coherent Inc, Ti:Sa regenerative amplifier for pulses from Mira 900
  • wavelength - 800nm (fixed)
  • pulse duration - 200fs after compression
  • repetition rate - single shot to 250 kHz
  • maximal pulse energy - 6 µJ
  • Powered by VERDI G12 - CW frequency doubled diode laser, wavelength 532 nm, max. power 12 W


GLX-Yb, Time-bandwidth producs AG - SESAM mode locked Yb KGW laser wavelength - 1040nm
  • pulse duration - 200 fs
  • repetition rate - 83 MHz
  • maximal average power - 2 W

Optical autocorrelator:

Home-made optical autocorrelator for ultrashort pulse duration measurements. Built up through the bilateral projects with DESY.
  • Michelson interferometer with retroreflctor (corner cube prism) in the arm with motorized stage
  • second order (intensity) autocorrelation using BBO crystal
  • pulse durations: 10 fs - 100 ps
  • computer controled

Optical autocorrelator set up

Control software inteface and autocorrelation trace for: (left) fs pulse from Mira 900; and (right) uncompressed ps pulse from RegA 9000

Cooled CCD spectrometer for weak signal measurements

Sensitive, fiber coupled USB spectrometer with high SNR suitable for the detection and analysis of weak fluorescence originating from the biological samples.
  • Glacier X, bz BWTEK
  • Coupled with NLSM setup
  • Spectral analysis of the fluorescence signal upon the excitation in the arbitrary chosen point on the image
  • Resolution: 4 nm
  • Range: 350-150 nm

Purchased through the project "Move For The Science" funded by Filip Moriss.