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Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti:Sa laser

Iestyn Pope, Wolfgang Langbein, Peter Watson, and Paola Borri

Open Access Open Access

Abstract

We have developed a multimodal multiphoton laser-scanning microscope for cell imaging featuring simultaneous acquisition of differential Coherent Antistokes Raman Scattering (D-CARS), two-photon fluorescence (TPF) and second harmonic generation (SHG) using a single 5 fs Ti:Sa broadband (660–970 nm) laser. The spectral and temporal pulse requirements of these modalities were optimized independently by splitting the laser spectrum into three parts: TPF/SHG excitation (> 900 nm), CARS Pump excitation (< 730 nm), and CARS Stokes excitation (730–900 nm). In particular, by applying an equal linear chirp to pump and Stokes pulses using glass dispersion we achieved a CARS spectral resolution of 10 cm−1, and acquired CARS images over the 1200–3800 cm−1 vibrational range selected by the time delay between pump and Stokes. A prism pulse compressor in the TPF/SHG excitation was used to achieve Fourier limited 30 fs pulses at the sample for optimum TPF and SHG. D-CARS was implemented with few passive optical elements and enabled simultaneous excitation and detection of two vibrational frequencies with a separation adjustable from 20 cm−1 to 150 cm−1 for selective chemical contrast and background suppression. The excitation/detection set-up using beam-scanning was built around a commercial inverted microscope stand providing conventional bright-field, differential interference contrast and epi-fluorescence for user-friendly characterization of biological samples. Examples of CARS hyperspectral images and simultaneous acquisition of D-CARS, TPF and SHG images in both forward and epi-direction are shown on HeLa cells, stem-cell derived human adipocytes and mouse tissues.

© 2013 Optical Society of America

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Figures (6)
Fig. 1
Fig. 1 Sketch of the microscope set-up. M: mirror; DM: dichroic mirror; SF57: glass blocks; R: reflecting prism; λ/2: half-wave plate; BE: beam expander; PBS: polarizing beam splitter; F: filter. The side view of the optics between by the two indicated arrows shows the beam height difference. Graph: typical spectra of the laser, pump, Stokes and TPE beams.

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Fig. 2
Fig. 2 Top: Spectrum of the CARS intensity ratio of polystyrene relative to glass (solid line) and spontaneous Raman (grey area, taken from http://www.sigmaaldrich.com/). F1#1 (F1#3) are the detection filters (see Table 1) used below (above) 2450cm−1, respectively. Bottom: CARS ratio of 4-Nitrophenylacetonitrile (left) and polystyrene (right), with insets showing the CARS linewidths (full width at half maximum) of the 1340cm−1 (left) and 3054cm−1 (right) resonance as a function of the Stokes glass length L1. Dashed lines in the insets indicate the Raman linewidths.

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Fig. 3
Fig. 3 Simultaneously acquired epi-TPF (left), and forward CARS (middle) images (on a linear grey scale) of live Hela cells transfected with a GFP-Golgi label using the 60× 1.27 NA objective. Images cover a xyz range of 80 μm×80 μm×10 μm with 800×800×50 pixels, and are shown as maximum intensity projections along z. CARS was acquired with only Π1 at 2850 cm−1. Excitation powers at the sample were TPE 6 mW, Pump 27 mW, and Stokes 13 mW. The false color image (right) overlays the CARS (red) and TPF (green) images. Pixel dwell time = 0.01 ms; 6.4 seconds per xy image.

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Fig. 4
Fig. 4 Mouse tail tissue section fluorescently labeled with FITC-Concanavalin A. Left: Stitched epi-fluorescence image, scale bar = 300 μm. Middle: Single epi-fluorecence image cropped to 150 × 150 μm of the boxed region shown on the left. Right: False colour TPF (green), CARS from subcutaneous lipid deposits at 2850 cm−1 (red) and SHG from collagen (blue), scale bar = 20 μm. Excitation powers at the sample were TPE 7 mW, Pump 14 mW, and Stokes 7 mW. 20× 0.75 NA objective. 150×150 μm, 501×501 pixels, 0.01 ms pixel dwell time (2.5 s total image acquisition).

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Fig. 5
Fig. 5 Simultaneously acquired images for TPE (top row), pump and Stokes excitation at 2850 cm−1 (middle row), and CARS with TPE (bottom row) of the mouse tail section in Fig. 4. Linear grey scale over the range shown at the top of each column in photoelectrons/sec at the PMT cathode. Scale bar 20 μm.

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Fig. 6
Fig. 6 CARS and D-CARS hyperspectral imaging of differentiated human adipose derived stem cells using the 60× 1.27 NA objective. Top left: CARS spectrum in the CH-stretch range of the lipid droplet indicated by the white arrow on the right using a single pulse pair Π1 (a), and two pulse pairs Π1 and Π2 exciting two vibrational frequencies separated by 65 cm−1 giving rise to (b) the sum-CARS spectrum (SCARS) and (c) the difference CARS spectrum (DCARS). Top right: Extracts of sum-CARS and D-CARS xy images from the hyperspectral movie ( Media 1 for D-CARS) at three instantaneous frequency differences IFD1= 2850 cm−1, IFD2= 2898 cm−1, IFD3= 2915 cm−1 as indicated by the dashed lines in a). The grey and color scales are given in b), c) and refer to the y-axis. Power of pump 15 mW and Stokes 7 mW, 80 × 80 μm, 801 × 801 pixels, 0.01 ms pixel dwell time. Scale bars 10 μm. Bottom left: as top left but over the fingerprint spectral range and with Π1 and Π2 exciting two vibrational frequencies separated by 38 cm−1. Bottom right: Extracts of sum-CARS and D-CARS xy images from the hyperspectral movie ( Media 2 for D-CARS) at three instantaneous frequency differences IFD4= 1650 cm−1, IFD5= 1682 cm−1, IFD6= 1713 cm−1. Power of pump 22 mW and Stokes 10 mW.

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Tables (1)

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Table 1 Filters in forward and epi detection, from Semrock or †CVI Melles Griot.

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