۳D Scanning Laser Spectrometer RAMOS CARS

Spatial XYZ resolution of CARS signals (Objective lens: 60х, NA=1.2, watter immersion):< 0.7 μm
Spatial resolution of Raman signals (excitation wavelength 633 nm; objective lens: 60х, NA=1.2, watter immersion):XY: < 300 nm Z:   < 700 nm
Spectral detectable range:CARS signals:            985 – 5000 cm-1 Raman signals:          75 – 6000 cm-1
Spectral resolution:CARS signals:                7-8 cm-1 Raman signals:              0.25 cm-1(grating 75 l/mm Echelle); 0.6 cm-1 (grating 1800 g/mm)
Scanning range (fast scanning mode, 60x lens)XY: 225 х 225 μm Z:   80 μm
Control and automation:Fully motorized

 

Optical microscope

Type:inverted
Model:Nikon Ti-U
Stage:motorized
– travel range:114 х 75 mm
– accuracy (for 1 mm traveling):0.06 μm
– XY repeatability:± 1 μm
– minimal step:0.02 μm
Micro- objective lens:60 х NA-1.2 water immersion 20 x NA-0.45
Z-scanner: 
– type:piezo scanner
– lens movement range:80 μm
– minimal step:50 nm
– Reapetability:< 6 nm
Illuminator for reflection mode:Halogen lamp 100 W
Illuminator for transmission mode:LED
Laser input port:motorized triple turret
High resolution digital video camera: 
– type:digital color CCD camera
– sensor:1/2″, 2048 x 1536 pixeles
– ADC:10 bit,  12 frames per min

 

CARS excitation laser system

Single module optical parametric laser system consisting of: 
Picosecond solit state Nd:YVO4 laser 
Output wavelength:1064 nm
Pulse duration:6.5 ps
Output power:6 W
Pulse repetition rate:85 MHz
Mode composition:TEM00
Beam quality (М2):< 1.5
Beam diameter:2 mm
Tunable SOPO (Synchronously pumped Optical Parametrical Oscillator) 
Wavelength tuning range:690 – 990 nm
Output power:> 400 mW (at 800 nm)
Pulse duration:5 – 6 ps
Pulse repetition rate:85 MHz
Polarization:horizontal
Mode composition:TEM00
Beam quality (М2):< 1.2
Beam diameter:2 mm
Built-in delay tuning optical line 

 

Laser for Raman spectroscopy

Laser type:cw HeNe
Wavelength:632.8 nm
Average output power:> 10 mW
Polarization:linear
Beam diameter:0.65 mm
Beam divergence:< 1.24 mrad

 

Laser beam convergence and appointment unit

Laser shutter:3 pieces (Nd:YVO4 laser, SOPO and HeNe laser)
Polarizer:2 pieces (Nd:YVO4 laser and SOPO)
Wavelength plate λ/22 pieces (Nd:YVO4 laser and HeNe laser)
Variable telescope:3 pices (Nd:YVO4 laser, SOPO and HeNe laser)

 

Main optical unit

Optimized optics for spectral range:400 – 1100 nm
Polarization analyzer:Glan-Teylor prism
Laser beam attenuator:veriable neutral density filter 0 – 3D
Pinhole lens positioner:three-axis (X, Y, Z)
CARS, Raman, fluorescence filters positioner:five- position
Posotioner of dihroic mirrors:six- position

 

Imaging monochromator / spectrograph MS5004i

Focal length:520 mm
F number (input):9.8
Horizontal magnification:1.0
Vertical magnification:1.0
Vertical spatial resolution:< 20 μm
Flat field size:28 х 5 mm
Stray light ( 20 nm from 633 nm laser line)1 х 10-5
Difraction gratings unit:4-position motorized grating turret
Spectral resolution (wavelength 633 nm, CCD pixel size: 12×12 μm)0.01 nm (Echelle grating 75 l/mm) 0.025 nm (grating 1800 l/mm)
Entrance spectral slitmotorized confocal pinhole, continuously adjustable from 0 to 1.5 mm
Output spectral slit:motorized, width from 0 to 2 mm (continuously adjustable)
Ports:1 input, 2 output
Detector (Raman, E-CARS, fast measurements)Hamamatsu Photosensor module H7844 (with TE cooling)

 

Monochromator for fluorescence measurements (integrated with MS5004i)

Focal length:100 mm
Input spectral slit:motorized confocal pinhole
Output spectral slit:fixed, width 4.2 mm
Difraction grating:600 l/mm
Spectral range:400 – 920 nm
Linear reciprocal dispersion:13 nm/mm
DetectorHamamatsu Photosensor module H7844 (with TE cooling)

 

Spectral CCD camera (Raman, E-CARS)

Type:digital CCD camera
Photosensor:back-thinned CCD sensor 2048 х 122 pixels
Pixel size:12 х 12 μm
Photosensitive area:24.576 х 1.464 mm (length х heigh)
Spectral sensitivity range:from 200 to 1100 nm
Cooling with temperature stabilization:two-stage Peltier (TE) cooling down to – 45 °С
ADC digitization:16 bit
Sensitivity:1 photon for 1 ADC count (at 650 nm)
Dinamic range:at least 10 000

 

Fast scanning unit (X, Y)

Scanners:Galvano-mirror scanners (X, Y)
Scanning model:raster high speed and start-stop mode
Positioning accuracy:< 30 μm
Scanning area:225 μm х 225 μm (objective lens 60Х)
Full frame scanning speed:4 s/frame: 1000 х 1000 points

 

Laser confocal microscope unit (Reflected)

Prepinhole lens positioner:three -axis (X, Y, Z)
Confocal pinhole:motorized confocal pinhole, continuously adjustable from 0 to 1.5 mm
Detector:Hamamatsu Photosensor module H6780-01

 

F-CARS and transmitted signal registration system

Polarization alanyzer:Glan-Teylor prism
F-CARS filters positioner:4-position, motorized
F-CARS signal registration detector:Hamamatsu Photosensor module H7844 (with TE cooling)
Transmitted signal registration detector:Hamamatsu Photosensor module H6780-01

 

Registration and processing control unit

Number of registration channels:5
Number of simultaneously registered channels:up to 5
PC connection interface:Ethernet 100 Base-T (TCP/IP protocol)
Categories: , , , , ,

Description

Multifunctional – RAMOS CARS combines:

  • CARS scanning microscope
  • Raman / luminescent scanning confocal microscope
  • Conventional scanning confocal laser microscope

 

Multi-channel – five channels for simultaneous high-speed measurements:

  • F-CARS
  • E-CARS & Raman
  • reflected laser radiation
  • transmitted laser radiation
  • luminescent laser radiation
  Polysterene balls of various diameter (F-CARS, 3045 cm-1)

 

 

Advantages of CARS method

  • high sensitivity: CARS generates more intensive and directed signals in comparison to spontaneous Raman microscopy;
  • anti-Stokes CARS signal has frequency exceeding pumping waves frequencies and is detected in a spectral range free from the stray light of Stokes luminescence;
  • CARS signal is registrated only in a focus where excitation intensity is the highest. It allows imaging with a high spatial resolution using non-confocal pinholes and also performs 3D layer-by-layer scanning with minimal neighboring layers influence on measuring results;
  • spectral resolution of CARS signal is defined only by the width of pumping lasers lines, what simplifies spectral measurements, as detection of CARS signals can be performed without any spectral instrument;
  • CARS signal is proportional to the squared molecule concentration, it allows using CARS (along with the selectivity and noninvasivity of the method) for quantitative measurements of chemical substance concentration in a sample;
  • minimal invasive (nondestructive) CARS method for biological samples. Due to the high sensitivity of CARS method molecules in living cells can be detected without fluorescent markers.

 

 

Features

High spatial resolution:

CARSXYZ < 0.7 μм
RamanXY < 300 nm Z < 700 nm

Wide spectral range:

CARS985 – 5000 cm-1
Raman75 – 6000 cm-1

High spectral resolution:

CARS7 – 8 cm-1
Raman0.25 cm-1
3D CARS image of liquid crystal 8CB structure on resonant frequency 2236 cm-1

Simultaneous / multifunctional analysis:

  • high-speed imaging by CARS signal (no preliminary additions to the samples)
  • confocal Raman imaging
  • confocal fluorescence imaging including two-photon (or multi-photon) emission
  • confocal imaging in the reflected laser radiation
  • high-contrast imaging in the transmitted laser radiation
  • visualization of a surface profile by means of the second harmonics generation signal

 

Three types of CARS measurements:

  • F-CARS
  • Е-CARS
  • P-CARS
Multimodal CARS/TPEF image of growing cancer cells HeLa: DNA/RNA/Proteins/Lipids colored

 

Five independent speed channels for simultaneous detection up to four 2D and 3D images:

  • F-CARS: CARS signal in forward direction
  • E-CARS & Raman: CARS signal in backward (EPI) direction/ Raman signal
  • Reflected: reflected laser radiation signal
  • Transmitted: transmitted laser radiation signal
  • Luminescent: transmitted laser radiation signal
  • Polarization control over the excitation and detection
  • Mono-block laser system for CARS signal excitation
  • Additional laser 633nm for excitation of a conventional one-photon fluorescence and spontaneous Raman scattering
  • PC-controlled: switching of modes of measurement is performed by automatic switching of the components inside the system

 

Three scanning modes:

  • Laser beam scanning over fixed sample surface with XY scanner
  • Sample transfer by means of XY automated stage relative to the fixed laser beam
  • Multiplexed mode for panorama imaging with a high speed and high spatial resolution: XY scanner + automated stage

Highly-precise calibration over the wavelengths: better than ±0,002nm due to an embedded calibration lamp as a source of reference lines for automated operative calibration of the monochromator-spectrograph. Block, rigid framing provides a high temporal and thermal stability.

Intuitive software NanoSP®:

  • Online control over the system parameters
  • All-round automation : measuring modes switching by automated switching of the components inside the system; control over the shutters for wavelength selection of the excitation laser; control over the polarization in the excitation / registration channel; selection of a grating, setting of a central wavelength and selection of an output port of the monochromator-spectrograph, adjustment of a pinhole position , etc.
  • Confocal 2D and 3D imaging: scanning, accumulation and data storage
  • CARS image, Raman and luminescence spectra
  • Various calibration methods of spectra, also by means of the embedded calibration lamp as a source of reference lines
  • 2D / 3D image of data
  • Image processing: – image correction – metric and statistic image processing, free sections – digital filtration, image resize and rotation
  • Spectra processing: – background correction (background subtraction) – mathematics operations: addition, subtraction, division, multiplication ,etc. – smoothing with several techniques – searching and definition of spectral lines peaks
  • Scanning modes: – Point – XY – XZ – YZ – XYZ
  • Scanning techniques: – galvanoscanner: 2D high-speed images – galvanoscanner + Piezo-Z scanner: 3D high-speed images – automated stage: 2D images – automated stage + Piezo-Z scanner: 3D images – galvanoscanner + automated stage: 2D panorama images – galvanoscanner + automated stage + Piezo-Z scanner: 3D panorama images

 

Application

  • Nanobiotechnology: real-time noninvasive analysis of biological samples (cells and components of living cells) with high spatial resolution
  • Micro- and nanotechnology investigations of properties of non-biological microstructures: semiconductors, liquid crystals, polymers, pharmaceutical components, micro- and nanoparticles
         Selective imaging of MIA-PaCa pancreatic human cancer cell for resonance 2845 cm -1 C-H bound (C-H aliphatic stretch). Selective lipids visualization. 3D image of a cell References: Dr. A. V. Kachynski, The Institute for Lasers, Photonics, and Bio-photonics, State University of New York at Buffalo

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