Extensive research in biomedical optics essentially requires the determination of optical properties of various biological tissues. Quantitative characterization of biological tissues in terms of optical properties is achieved with an integrating sphere. However, samples having significantly higher scattering and absorption coefficients such as malignant tissues potentially reduce the signal-to-noise ratio (SNR) and the accuracy of an integrating sphere. We describe the design, implementation and characterization of a modified sample holder (path length of up to 1 mm) for an integration sphere. Experiments conducted with various phantoms reveal significant improvement of the SNR for a wide range of optical properties.The alternative approach opens up potential applications in the measurement of optical properties of highly diffusive biological samples. For 20% intralipid µ a = 0.112 ± 0.046 cm −1 and µ s = 392.299 ± 10.090 cm −1 at 632.8 nm. For 1.0% Indian ink µ a = 9.808 ± 0.490 cm −1 and µ s = 1.258 ± 0.063 cm −1 at the same wavelength. The system shows good repeatability and reproducibility within a 4.9% error.

Recently, the pump-probe technique has found highly valued developments and applications in optical microscopy to study both labeled (fluorescence) and non-labeled (vibrational) for observation of cells and tissues. In this work, we use the mechanism of stimulated emission (SE) with dual frequency modulation to remove the large background signal that is originated from spontaneous emission. We have applied a high performance digital signal processing based lock-in detection for stimulated emission fluorescence microscopy. Critically, the pump beam is modulated at f1 and the probe beam is at a different frequency, f2, with the demodulation carried out at the sum of the two frequencies, (f1 + f2). In this way, the DC background is effectively removed. Note that the background is mainly attributed to the spontaneous emission of the pump beam, with modulated frequency f1.

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We have implemented polarization-resolved fluorescence lifetime measurement through stimulated emission based pump-probe technique, which promises much higher temporal resolution (∼4 ps) than conventional time-correlated single-photon counting (TCSPC). The depolarization of ATTO 647N fluorescent dye is resolved through anisotropy fluorescence lifetime measurements, with variable time delay introduced between the pump and the probe beams. Importantly, the polarization anisotropy measurement and the corresponding rotational correlation time characterization of the fluorescent dye are carried out at various temperatures. We have also demonstrated the need of high temporal resolution via hetero Förster energy transfer (Hetero-FRET) through the interaction between the gold nanorods (GNRs) and the fluorescent dye ATTO 647N. Notably, our results compare highly favorably with conventional TCSPC method, which is rather limited in temporal resolution, for the above characterization. Additionally, this technique is applicable even under ambient light while being very cost-effective and robust.