4.1.1 Raman micro-spectrometer for skin tissue spectral imaging
For spectral imaging of skin tissue samples a Raman micro-spectrometer using an inverted optical microscope and a near-infrared laser was developed (see Fi-gure 4.1) [164]. The inverted microscope (IX71, Olympus, Japan) was equip-ped with an automated XYZ translation stage (H117, Prior Ltd., UK), a deep-depletion back-illuminated CCD (DU401A-BR-DD, Andor Ltd., UK) and spec-trograph (SR-303i, Andor Ltd.), and a 785-nm continuous wave GaAs diode laser (XTRA, Toptica Photonics, Germany). The laser power was set to 50 mW at the
sample to avoid sample damage and ensure repeatable measurements. The objec-tive lens of the microscope had a numerical aperture of 0.75 and a magnification of 50×. A microscopic camera (2-1C Infinity, Lumenera, Canada) was used to record images of the tissue sections.
Figure 4.1: Schematic of the Raman micro-spectrometer. L785- laser; PF- plasmaline filter;
m-mirror; M-microscope; ST-stage; s-tissue sample; NF- notch filter; CL- collecting lens; SP-Czerny-Turner Spectrograph; CCD- detector. Courtesy of Dr. Zoladek.
4.1.2 Wide-Field Fourier transform Raman spectroscopic imaging instrument
A schematic of the instrument is included in Figure 4.2. A continuous wavelength Nd:YAG (neodymium-doped yttrium aluminium garnet) laser (JPM-X-3, Laser Vision, USA), frequency doubled to emit light of 532 nm-wavelength, was used to irradiate the sample which was placed on a microscope stage (Prior Scientific, UK). The inverted microscope (Diaphot 300, Nikon Corp., Japan) was equipped with a 50× microscope objective of 0.75 numerical aperture (UIS2, Olympus). A microscope digital camera (DCM35, Nikon Corp.) was attached to the microscope.
A 45 degree dichroic beamsplitter (RazorEdge, Semrock) was placed on the cube of the microscope, in the optical path. The microscope was coupled to a Michelson interferometer and a low-noise, cooled CCD (DV420-OE, Andor Ltd.). The CCD was connected to a personal computer by a graphics card.
St
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Figure 4.2: Wide-field Fourier-transform (WF-FT) Raman set-up. Abbreviations: Beam ex-pander and collimator (Be and Co), dichroic mirror (Dm), mirrors (M1, M2, M3), lenses(L1, L2), retroreflectors (R1, R2), step-motor (Nm), stage (St), photodiode (Pd), beamsplitter (Bs), triggering box (Trigg. Box), and charged coupled device (CCD).
The beam of light coming from the laser was expanded and collimated in order to illuminate a wide area of the sample in the so-called wide-field sample illumination regime. The diameter of the laser beam on the sample was ≈50 µm.
Interaction between the monochromatic light and the molecules of the sample resulted in elastically (Rayleigh) and inelastically (Raman) scattered light. This combined signal was collected by the microscope objective, the Rayleigh light being blocked by a dichroic beamsplitter. In practice, a weak beam of 532 nm wavelength passed through and travelled along with the Raman light. The collimated beam was reflected in an internal mirror of the microscope (M1), which directed the beam towards the exit port of the microscope. The beam entered the interferometer by reflection in mirrors M2 and M3. These mirrors allowed the collimated beam to be aligned along the optical axis of the interferometer at the correct height and angle.
The Michelson interferometer described in Figure 4.2 used a 50:50 non-polarizing N-BK7 cube beamsplitter (J49-682 Techspec, Edmund Optics, USA). As explained in Chapter 3 in a classical Michelson interferometer two beams of equal intensity named B1 and B2 exit the beamsplitter. In this system each one of these beams followed one of the arms of the interferometer, being reflected in either the fixed or moving corner cube retroreflectors (UBBR2.5-1I, Newport, USA). The moving
corner cube retroreflector (labelled R1 in Figure 4.2) was attached to a step-motor of 10 nm of spatial resolution and minimum speed of 5000 nms−1(Nanomotion II, Melles Griot, USA).
After exiting the beamsplitter for a second time, B1 and B2 passed through a 45 degree dichroic beamsplitter for visible light labelled Dm in Figure 4.2. This component let the Raman signal pass through reflecting the remaining laser light.
The Raman light was focused by lens L1 on a CCD sensor acquiring images for each position of the movable retroreflector R1. The CCD sensor consisted of 1024×512 26 µm-pixels. Each one of these pixels recorded the total number of Raman photons that reached the surface of the CCD chip for a certain acquisition time at a fixed position of the retrorreflector R1. The values of the Raman photon counts, in addition to the position of R1, and the spatial coordinates of the pixel within the CCD sensor was the information needed to create Raman spectral images of the sample.
The Rayleigh light reflected by the dichroic beamsplitter was focused by lens L2
on a silicon pin photodiode (Pd)(S5973-02, Hamamatsu Photonics, Japan). The signal detected on the Pd was the result of the interference between the laser light carried by beams B1 and B2. Due to the monochromatic nature of the two interfering beams the signal recorded in the Pd was sinusoidal. This sinusoidal signal was amplified and converted to a TTL pulse by a customised electronic box. This signal was monitored on an oscilloscope, and data was recorded on a SD memory card for later processing with MATLAB software. The TTL signal was used to externally trigger the CCD.
4.1.3 Integrated SERS-AFM system for lipid characteri-sation
An integrated AFM-Raman microscope has been developed for characterisation of nanomaterials [165]. The system consists of a Raman micro-spectroscopic instru-ment coupled to a commercial AFM (Nanowizard, JPK Instruinstru-ments, Germany) with a piezoelectric XY-stage fitted to the inverted Raman spectroscopic micro-scope. Figure 4.3 is a schematic of the instrument. For the AFM measurements in air, silicon probes of 70 (50-90) kHz resonant frequency and 2 (0.5-4.4) Nm−1 spring constant were used (AC240TS, Asylum Research, UK). For the AFM in liquid mode silicon nitride cantilevers were preferred, gold coated, with a spring constant of 0.12 (0.06-0.24) Nm−1, and a resonant frequency of 23 (16-28) kHz (NPG, Veeco, USA). AFM data were processed with several software packages
(commercial JPK image analyser processor and open source softwares WSxM 5.0 [166] and Gwyddion [167]).
The Raman micro-spectroscopic instrument consists of a continuous wave laser of 532 nm wavelength and maximum power 20 mW (MSL532, Laser 2000, UK). The laser was directed through 100 µm diameter optical fibres (Ocean Optics, USA) towards the inverted microscope (IX71, Olympus), crossing a dichroic 45 degree beamsplitter (Razor Edge, Semrock Inc., USA) which is optimised for transmission of light of 532 nm wavelength with a negligible reduction in transmitted intensity.
The beam arrives at the microscope and is focused onto the sample by a water-immersion 1.2 NA 60× objective (UPLSAPO, Olympus) producing a maximum laser spot at full laser power of 500 nm in diameter at the focal plane of the illuminated sample. The scattered light is collected in transmission mode and sent back to the beamsplitter, which directs half of the beam intensity to a photodiode detector and the other half towards the spectrometer.
The dichroic beamsplitter-photodiode system was added to improve the lateral alignment between the Raman laser and the AFM tip. The alignment was carried out using the tip-assisted optics (TAO) module by scanning the tip through the laser spot and then locking it at the position of maximum scattering intensity (see Figure 4.4). The second beam existing the beamsplitter was directed via fibre optics towards the entrance of the spectrometer. A notch filter (Semrock Inc.) was placed in front of the spectrometer to filter the Rayleigh signal. The Czerny-Turner Raman spectrometer (SR-303i, Andor Ltd.) used incorporates a back-illuminated CCD (DV-model, Andor Ltd.). Raman spectra recorded with commercial software (Solis, Andor Ltd.) were pre-processed in MATLAB (MathWorks, USA) following the procedure described in section 4.3.1, optimising the number of points in the smoothing algorithm.
Figure 4.3: The combined AFM-Raman micro-sprectroscopic instrument. Courtesy of Dr. Swee-tenham.
Figure 4.4: Measured intensity of the laser light reflected on the AFM tip and detected by the photodiode for an XY laser scan at a fixed focal plane. (a) The microscope is focused far from the plane where the tip is located. The shadow corresponds to the region closer to the tip, according to the tip geometry. (b) The microscope is focused on the tip. Interference fringes are detected at this plane.